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Coffalyser.NET analysis manual

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1. 19 P335 A2 lot 0809 BMP ID 1 sample al wl Oss O Y F P235 A2 lot 0809 BMP ID 1 sample all wll 51 51 P3 Channel 3 NED not used ae a all 5151 16 Olv Channel 4 PET not used z Sora ee il wl 551 6 Ov Channel 5 LIZ size marker channel GS500 250 23 P335 A2 lot 0809 Promega reference al ul 551 7 XM B 24 P335 A2 lot 0809 Promega reference al al O 551 O V Xx 25 P335 A2 lot 0809 Promega reference al dl O 551 Ov X u 26 P335 A2 lot 0809 Promega reference al l O 551 O J XF 27 P225 A2 lot 0809 Promega reference al al 551 OS X m 28 P335 A2 lot 0809 Promega reference wl wil 551 O Y VY 5 29 P325 A2 lot 0809 REF A H sample wl al O51 OVX om 30 P335 A2 lot 0809 REF B H sample wll al O55 O Y Xa 31 P335 A2 lot 0809 REF C H sample al al 551 O Y X cz 32 P335 A2 lot 0809 REF D H sample E al al O 551 Y X r 33 P335 A2 lot 0809 REF E H sample wll al O55 OY Xm 4 P225 A2 lot 0809 REF F H sample all wl O55 O Y Xe 35 P335 A2 lot 0809 REF H H sample al l O 55 O Y Xa 36 P335 A2 lot 0809 REF LHY sample ull ll 551 OS X 05 37 P335 A2 lot 0809 REF J H sample dl wl 551 Y Xo 38 P335 A2 lot 0809 TE HYI D sample al il O 351 OXX 3
2. Minimum peak amplitude RFU Maximum peak amplitude RFU Minimum peak area to total fluorescence Maximum peak area to total fluorescence Minmum peak width datapoints Maximum peak width datapoints Minimum peak amplitude to median signal Maximum peak amplitude to median signal Detect fake peaks reset peak start datapoints Detect stutter peaks Minimal peak stutter distance datapoints Restore Default Settings Figure 14 Peak detection settings tab Program administrators can modulate the peak detection algorithm thresholds for size marker channels and probe channels by clicking on the second tab of the CE devices properties form figure 14 which make use of the following criteria 1 Detection Intensity threshold This threshold is used to filter out small peaks in flat regions The minimal and maximal peak amplitudes are arbitrary units and default values are provided for each different capillary system These value are called the minimum and maximum peak amplitude RFU 2 Peak area ratio percentage Peak area is computed as the area under the curve within the distance of a peak candidate Peak area ratio percentage is computed as the peak area divided by the total amount of fluorescence times one hundred The peak area ratio percentage of a peak must be larger than the minimum threshold and lower
3. experiment reference probe std dev filter medium high extend reference probe collection only use equal called reference probes probe minimal reference samples reference sample filter lt median Z score extend reference sample collection Figure 35 Iteration normalization analysis settings On default settings the number of iteration round is set to 1 which means a single round of analysis without further adjustment of settings To use the iteration the number of rounds need to be at least 2 and in most cases when using just 3 rounds the iterations is optimal You can furthermore change several settings in order to optimize the iteration procedure 74 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 1 Title Normalization cycles the normalization cycles refer to optional experimental iteration of results Iterative normalization means that all samples will be completely analyzed where after the results will be automatically interpreted and a new normalization starts with new parameters based on the results of the previous normalization This method allows a number of methods which are discussed more extensively in the advanced analysis section In short each sample may obtain sample related reference probes and reference samples which were found to be normal or equal
4. Found Control Fragments Control Fragment Results D fragment 96 nt ratio 0000000000000000 068000000 B 8 Figure 29 Fragment results explorer sample overview screen Other available screens are 1 Fragment analysis QC overview This grid contains the results of all earlier discusses quality control factors In case there is a problem with one of the separate factors the reason behind this can be found by hovering above the specific cell These factors are also separately tested against thresholds and thereby also give the quality scores color indications in order to easily spot which factors were considered to be bad 2 Raw data displays the signals of the dye data streams as your capillary electrophoresis device measures them This screen can be used to 60 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 evaluate the quality of the fragment separation and to see if the dye filters are working correct Fragment profile displays the baseline corrected signals of each dye data streams that was set as a probes or size marker channel Displayed signals of channels set as probe mix content will also show which signals were identified as peaks and what their relative length in nucleotides is In this chart black triangle markers represent the position of the start of a peak red circle markers represent the peak top and
5. First we need to determine what type of experiment we are analyzing There are basically 3 types of experiments these being 1 Copy number analysis DNA MLPA default These are experiments that are performed using standard MLPA probes or custom probes that are designed according to the same rules The used probes can only produce signals that are proportional to the amount of the DNA target sequences present in each sample These experiments furthermore require data obtained from reference samples that were performed in the same experiment This reference sample is usually performed on a sample that has a normal diploid DNA copy number for all target sequences 2 Copy number methylation status analysis DNA MS MLPA These experiment are combined experiment where both the copy number and methylation status of the probe target sequences are calculated in a single analysis While the copy number part is equal to that described at point 1 the methylation status analysis requires a digested sample result together with each standard MLPA sample result For MLPA probes that contain HHA1 sites the methylation status can then be determined by comparing the signal that is proportional to the amount of the DNA target 35 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sequences present in each sample after digestion to the signal of the same ta
6. 7 4 Using the fragment results explorer sssssesssssessrnesssrnesrnnnesrnnnesnnnnennnnnnnnnnnnnnnnnnnnnnneennnne 8 About the comparative analysis copy NUMbEL ccceseeneeseeeeeeeseeeeeeseeenenseeees 8 1 Setting up the comparative analysis ccccccceseceeeeeceeeeeeeeeeeeeeceeeeeseaeeesaeeeeneeeeeeeeeas 8 2 About the comparative analysis quality SCOPES ececeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaeeeteeeaees 8 3 About the comparative experiment results explorer 02 eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaees 8 4 About the comparative sample results EXxplOrer eeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeaees 9 Methylation specific MLPA analysis cccccseeceeeseeeeeeeseeeeeeeseeeeeenseeneeenseeneeenneeneees 9 1 Introduction to MS MLPA analysis ccccccceeeeeeeeeeeeeeeeeeeaeeeeeaeeeeeeesaeeesaeeeaaeeeeeeeeaas 9 2 Setting up the MS MLPA analysis cccccceceee cesses eeeeee cee eeeeaeeeeneeseeeeesaeeeeeaeeeeeeeeaas 9 3 Comparative analysis experiment explorer with MS Data ccccesseeesseseeeeeseeees 9 4 9 4 Comparative analysis sample explorer with MS Data ccceecceeeeeeeeeteeeeeeeeees 10 Fc E ee ca cca cuted capac te cute A E cued weacestehreuuaceucuacteetg 10 1 What is the different when the analysis method is set to RNA cceeeeeseeeeeeeteeeeeees 11 Referents aiiora aeron EEA EEEE Eaa EA E AEAEE EEE 5 Title Coffalyser NET analysis manual beta version Status Release
7. 8 FMRS Bake 4 A All Samples Suitable For Analysis ye E All Samples With Detected Y Control Fagment i al y x i All Samples Without Detected Y Control Fagment gt Start Comparative Analysis Figure 32 Comparative analysis sample selection menu The first thing that needs to be done at the comparative analysis tab is the selection of samples that will be included in the normalization To make the selection easier you may use the right click menu to make a pre selection of samples based on their FRMS score Right click anywhere in the grid select the option Select samples for comparative analysis Next select a level of quality you which to apply for the comparative analysis Dependent on the setting of the study e g research or diagnostic a higher quality level may be desired You can further adjust the selection of samples by using the option box in column analyze After finishing your selection of samples click on the button Start comparative analysis blue arrow figure 32 which will open the comparative basic analysis settings form figure 33 Basic normalization settings On this form you can adjust some of the settings that will influence some of the most basic analysis settings On default all settings are set to auto resulting in a multistep analysis where the best settings are chosen dependent on the number of samples and their sample types the MLPA mix presence of reference probes and results obta
8. If all samples were imported correct you can close this window to make the sample specific settings Figure 23 File folder import form 7 2 Starting the fragment analysis After importing your samples and you have closed the file folder import form the fragment analysis sample setup window will appear figure 24 This form allows you to adjust the sample types that you have used in your experiment You can set 4 different sample types either by using the key shortcuts or by changing the combo box by double clicking on the cells in the second column called sample type We distinguish the following types 1 Samples or test samples key s which will be normalized against the reference and are considered to be the unknown samples of which we want to know the copy number status of the test probes For these samples we assume that the target sequences of the reference probes are normal or diploid for all autosomes or have an equal copy number as compared to the reference samples In case no reference samples are defined in the experiment each sample will be used as a reference The data for each test probe of each sample will be compared to each other sample producing as many dosage quotients as there are samples The final ratio will then estimated by calculating the median over these dosage quotients 2 Reference samples key r are used to display the balance of the measured signal intensities between
9. P335 A2 lot 0809 BMP ID 264 HYI DNA P335 A2 lot 0809 Promega F HYI DNA P335 A2 lot 0809 Promega F HYI DNA P335 A2 lot 0809 Promega F HYI DNA P335 A2 lot 0809 Promega F HYI DNA P335 A2 lot 0809 Promega F HYI DNA P335 A2 lot 0809 Promega M HYI DNA P335 A2 lot 0809 REF A HYI DNA R P335 A2 lot 0809 REF B HYI DNA R P335 A2 lot 0809 REF C HYI DNA R P335 A2 lot 0808 REF D HYI DNA R P335 A2 lot 0809 REF E HYI DNA R P335 A2 lot 0809 REF F HYI DNA R P335 A2 lot 0809 REF H HYI DNA R P335 A2 lot 0809 REF I HYI DNA R P335 A2 lot 0809 REF J HYI DNA Ratio hg18 location Figure 44 Sample explorer ratio chart Each black red or purple circular marker points indicate the result of a single probe in the selected sample On default the X axis loads with the hg18 track map view locations and the labels display a probe design length probe gene name probe gene exon number notation The found whiskers at each probe marker ratio indicate the estimated 95 confidence range for that signal These confidence ranges are estimated by combining the found discrepancies of the estimated dosage quotients by the used reference 90 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 probes and or reference samples The estimated variability of each probe in the used reference collection may thus provide information if that probe was found to be re
10. 009 BMP ID TA HYLDNA P335 A2 lo 0809 BMAD B1 HYLDNA P235 A2 lo 0809 BMP ID 24 HY DNA P235 A2 lo 0809 BMP ID 91 HYLDNA P235 A2 lot 0009 BMP ID 92 HYLDNA 205A lot 0009 BMF ID 95A HY DHA P295 A2 fo 0009 BMP D 97 HY DNA P395 A2 lot 0009 BMP ID 177B1 HY DNA 205 2 lei 0809 BMP ID 17601 HV DNA P335 A2 lo 0809 BMP ID 198B1 HY DNA P335 A2 lo 0809 BMP ID 2408 HY DNA P335 A2 lo 0809 BMP D 26 HYI DNA P335 A2 lot 0809 Promega F HYI DNA R P335 A2 lot 0809 Promega F HYI DNA IR P335 A2 lot 0809 Promega F HYI DNA R P335 A2 lot 0809 Promega F HYI DNA R P335 A2 let 0809 Promega F HY DNA R P335 A2 lot 0809 Promega M HY DNA R P335 A2 lot 0809 REF A HYI DNA P335 A2 lot 0809 REF B HYI DNA P335 A2 lot 0809 REF C HYI DNA P335 A2 lot 0809 REF D HYI DNA P335 A2 lo 0809 REF E HYI DNA P335 A2 lot 0809 REF F HYI DNA P335 A2 lot 0809 REF H HYI DNA P335 A2 lot 0809 REF I HYI DNA P335 A2 lot 0809 REF J HYI DNA P335 A2 lot 0809 TE HYI DNA P335 A2 lot 0809 TE HYI DNA vy 163 ta IKETE 20 2m EM a 2 28 cee 2a an eo aay Idk 1 241 26 eteence 218 azt oP A an Katey ea a es a8 ta eters A ETET Keteenen 121 400 eto Crama m mi CURRY 204 SHUR Aten 8 009373 02 242 25600 02782 102724 1 05q31 1 05 132 037600 00787 L00463 4oqz2 xom amza 13089115427 5 orpizz 07 050 417760 13872115390 dow X22 000 770580 05648 L06218 1 08p132 09 037 024270 12501 113551 vp 12021 33 12 090 30548
11. 1946 22529 22529 105 098 099 002 005 004 50 231301 Average Medan Sicey Min Max Cahan i ee 180k Region analysis 034 042 025 040 039 ETE 142 13047821213 0178201346 1900 20475 20475 080 075 075 00r O03 003 lt lt lt lt S 2109 00 310 RB1 14 139142 13047851432 01789 01353 975 12043 12043 086 082 08t 002 005 Q04 lt lt 48 309800 355 RBI 19 13q14 2 13047928373 01792 15022 1115 13537 13537 077 073 073 001 004 003 lt lt lt e 45 3547 00 418 RB1 24 134142 13 047945205 01797 01360 684 9239 9239 075 071 072 001 004 003 lt lt lt lt S0 415502 445 RB1 26 3g142 13047949488 01799 01362 773 10665 10865 081 077 O77 002 004 003 lt lt lt lt 50 441601 SEE Average Macan Sissy tn Max Che bend 12914 2 Size 128275 nt 1284 Region analysis 076 075 003 072 081 Figure 50 Dual page sample extended pdf report page 2 97 Title Status Classification Versie Coffalyser NET analysis manual beta version Release candidate Confidential 0 1 9 Methylation specific MLPA analysis 9 1 Introduction to MS MLPA analysis MS MLPA analysis can be applied as an extension on the normal copy number DNA MLPA analysis In Coffalyser NET copy number and methylations status analysis always occurs in a single analysis Results of copy number and methylations status are then displayed together making data interpretation easier Interpreting MS MLPA data with the copy number status is crucial since only relative methylation percentages of target sequence
12. 8 9 18 58y RSE PY Se opgi e ed a 8 4 2 3 5 PA sah Hae s 8 133 8 2 138 RES S82 ESS BRRFy d ET 22004 amp j22 ex S ING SONS DN Fo 5 daetou 85 828 Go s gt ES E E ses t SB Sse sgass pe 2000 fs S 5 8 8 exe e Se RSe Seseoes F ge S kd ela BS ses ki OW S rR ear 3s 2 1800 R IN fo 2x on S 5 gorl gga amp gs q a 8 8 ou B 3 Oo oe ee oe G g 2 1600 gs 2 11 18 S32 T2588 z y 140044 a amp R R a g9 8 S AESi 1200 a aE 10004 38 8 8004 3 600 400 i a W UO O J UU ral MAN 8583 S3 8J E38 FEBRABE IBNR SS BBOAV ES 2 2BSPBSEL2 AS BB25 RE BERRB 85 3 83 832538 CSRR3SSZEIRSR RBBB ESE ZRSS SELES 3z 2yg Z BERR He RFE BRR SPPA INI REEE 82s SIRS SSS RR FP SIGE 2 FHF B Length nt et a ee Sa 5000 3H 3 fo T gya lt 5 g gt 6 Siw ns g r kd gi t 4500 RS CG sot m Test probe Se Qi Pea St Gene 5 40004 8jao 883 TSEZ 8S _ ae 5 Szar3 LESEN Hg18 Location 09 21957822 a 3500 Ss 28 PR sss o Chromosome band 09p21 3 2 RSq ote z Probe length 256 flo s 3000 can ive 152 gt Ejj Ma alex os 22g sh 8 2 am ze 2 Sq of Final ratio 0 558 BER ee ee 2 Dg S S4 Standard deviation 0 029 B tSean BEN EES Sg 2000 4 a es Sos Opent ong S ae a 2eGmaetyak roy S 5z e OG Intra normalized ratio 0 583 DE south eu 0b oS a al g e on Sg25880 ye kee 1 38 Z Fg Pre normalized ratio 0 599 Ga TFS Ute sees d a a i og wos os Ss 03 7 118 Slope co
13. Coffalyser MLPA extended sample report P335 A2 lot 0809 BMP ID 55 HYI DNA Sample type Sample Project p335 Expenment p325 Performed by Administrator Machine AB Report date 3 10 2077 Run date 4 8 2017 Probe Nr Height Area T Area Pre Chr band Hg18 loc i ee rt he ld ee T 3 Ratio IRatio RSQ RPQ Stdev REF Sam Width E nt d nt 59 Q6Ont 66 Q68nt 73 Q 4nt 79 Qrt 85 DD88n 91 Qrt 96 DOn 100 Xt00nt 105 _ 105nt 7 0 0 0 00 000 0 00 000 000 000 0 0 0 0 0 0 00 0 00 0 00 0 00 000 000 0 0 0 o te 0 0 00 0 00 000 000 000 O00 e 0 0 o 0 o 0 00 0 00 000 0 00 000 O00 0 0 692131 06 035528120 0027 01229 1285 12957 12957 000 000 163 000 000 000 252 0 0 02913 02 113307442 0005 00509 789 8014 801d 000 000 160 000 000 000 5 01 01p36 32 01 003559038 0029 02789 746 7663 7663 000 000 095 000 000 O00 6 0 1 Xq23 X 111945319 0384 12717 733 7344 7344 000 0 00 0 93 0 00 0 00 000 100 1 0 1 621 31 06 035528120 0369 12720 937 9527 9527 0 00 0 00 779 000 000 000 104 6 0 0 a 05q33 3 05 158071810 13868 15386 1088 436 EBFI 14 14704 14794 107 1 01 1 07 001 005 O04 s0 432 7 0 5 364 EBFt 10 059333 05158137017 14059 15657 1672 21110 21110 107 102 102 001 005 Q04 5 32900 228 EBF1 1 059333 05158459187 12509 14269 1404 14904 14004 105 098 096 002 005 004 43 226300 Ri i Q Average Madan Sicev Min Max Cnt bana alsa 3 Size 401358 nt 5
14. In the right mouse click menu you may find options allowing you to export print save zoom and adjust the 93 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 labels of the chart The option Lock current sample in the right click menu will split the chart area in two The upper part of the chart area will then show the results of the sample that is displayed at the moment of locking while the lower part listens to the original functionality figure 47 Automatic zooming allows 3 levels of zoom this being show all detected peaks show all recognized peaks and show all peaks recognized as MLPA test probes You may furthermore use manual zooming by clicking anywhere in the chart and dragging the mouse over the area you wish to zoom into Zooming in double view modus always automatically perform a zoom on the both parts of the chart It should be noted that due to differences in separation speed between different channels peaks might appear to be a slightly different positions At full automatic zoom methods there will be corrected for these differences uy 8 7 8 5 P338 A2 It 0809 REF A HYI DNA P335 A2 lot 0809 BMP ID 05 HYI DNA Su cee g 2 Sa eE e 83 ones g O83 Se se E 8 555 30005 S gs ey eug Ss 2 as o gt Se gs feg A T Tz S 2 ked 2800 T BE SZU pT S53 38 ms SA TPST OJo EE o gt p so 26004 o SS8_ th cs2fass Sag ON ce 252 aaa
15. NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 7 About the fragment analysis 7 1 Importing the data files After you have set the settings on the details page you can go to the next tab where you can import your sample files Right click anywhere on the screen and select Add from file from the right click menu figure 22 Figure 22 Fragment analysis window for importing samples After selecting Add from file the file folder import window will appear figure 23 Here you can import files or complete folders into the database and automatically link them to the current experiment For ABl devices ABIF files from all series can be imported fsa extensions for CEQ devices Beckman data from the CEQ 2000 CEQ8000 and CEQ8000 can be imported SCF or esd extensions for Megabace devices data of all series can be imported rsd extensions and for Agilent devices data of the Bioanalyzer can be imported xml extensions Select the Add files or Add folder blue arrow figure 23 and then select the files you wish to import in the explorer window At this point the files are not stored in the database yet click on Import red arrow figure 23 and to decode the 38 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 binary files and save them in the database
16. PCR artifacts irregular stutter bands and incomplete fragment separations a typical MLPA project requires manual examination of almost all sample data Our software was designed to eliminate this bottleneck by substantially minimizing the need to review data By creating a series of quality scores to the different processes users can easily pinpoint the basis for the failed analysis These scores include quality assessment related to the sample DNA MLPA reaction capillary separation and normalization steps figure 28 Each collective quality score or score that summarizes a number of aspects or factors starts with 100 points which can be correlated with high quality or green Depending on the importance and found severity of abnormality of each factor a number of penalty points are being given for each measured quality factor The quality of each step can fall roughly into three categories 1 High quality or green The results of these analysis steps can be accepted without reviewing 2 Low quality or red These steps represent samples with contamination and other failures which render the resulted data unsuitable to continue with This data can quickly be rejected without reviewing recommendations can be reviewed in Coffalyser NET and used for troubleshooting 3 Intermediate quality or yellow The results of these steps fall between high and low quality The related data and additional recommendations can be reviewed in Coffalyser N
17. REF H HYI DNA reference ul xX E ull Start Comparative Analysis Figure 36 Comparative analysis quality score screen 8 3 About the comparative experiment results explorer Coffalyser NET provides two ways to evaluate the results exploration of the results of the complete experiment or exploration of results of a single sample To open the experiment explorer right mouse click on the grid showing the quality scores and select from the right click menu Open experiment results The comparative analysis experiment explorer has three tabs allowing getting a quick overview of the results of the complete experiment 8 3 1 Comparative analysis experiment explorer statistical overview chart The last tab shows a statistical overview chart which loads with the statistical results found over all samples that were set with the sample type sample figure 37 All probe results are displayed as ratios on the Y axis the X axis will on default load on displaying the map view locations of the target sequences of the probes obtained by the hg18 tracks generated by UCSC and collaborators worldwide The labels above the probes on default load with a text field containing probe length gene name of target sequence exon number within gene of target sequence e g 126 DMD 01 which thus suggests that this probe had a design length of 126 nucleotides and was targeted to exon 1 of the DMD dystrophy gene The diff
18. Secondly the 95 2 standard deviations confidence range of the probe did not overlap with the 95 confidence range of that probe in the reference sample population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol gt gt e Blue one tint lighter or gt If found results did not meet the criteria of 1 then 2 new criteria are tested which if realized will color the cells one tint lighter blue First the magnitude of the probe ratio exceeded the upper set arbitrary border value on default 1 3 Secondly the 68 1 1 standard deviation confidence range of the probe did not overlap with the 68 1 confidence range of that probe in the reference sample 83 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol gt Blue two tint lighter or gt gt If found results did not meet the criteria of 1 or 2 then we will check if found probe results has a significant different to the reference sample population without employing the magnitude of the probe ratio Cells will be color blue 2 tint lighter blue if the 95 confidence range of the probe did not overlap with the 95 confidence range of that probe in the reference sample population Probe results
19. a Experiments E j A owe E CEQ Test p01 dd Experiment Colon data path Refresh MS MLPA p004 ERBB1 lot1210 p017 QT A lot0711 p033b2 lot 0410 p034 p050 cah 0410 p245 p245 QT p335 PWS lot0608 ray wide peaks X0039 X 035 Test X 039 a Settings 5 amp p33 bmp a Experiments Sa Settings m CE Devices Settings A Sheet Library DEUD D UD DD E u T A na aA ww Figure 19 Adding a new experiment in the database exploration window 6 3 Experiment settings Directly after you create an experiment you will be able to adjust the experiment settings and give the newly created experiment a name and description figure 20 The capillary electrophoresis device should already be filled in to be the default machine for that project You may however choose to also include different machines within one project After you click ok the experiment will be created in the database allowing you to continue to define the content of each channel 34 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 created by modified by CE device ABI 3130XL schooltje title description Figure 20 Experiment settings window 6 4 Setting the experiment type After adding a name and description to your experiment you may define the settings required to start the fragment analysis in the next form figure 20
20. a final length reference vector or bin is constructed for each probe This final bin set can be used directly for data filtering but may also be edited manually in case the automatically created bin set may not suffice To edit the manual bin set right click on the fragments analysis experiment explorer and choose edit manual bin set from the context menu and then select the probe mix channel you wish to edit see also figure 28 Selecting this option will open the Coffalyser work sheet editor for manual bin sets allowing you to edit both the design an Coffalyser length that are used for the auto binning procedure but it also allows you to edit the manual bin set for that mix The manual bin set allows you to set the start and end value in which a probe will be sought during data filtering The manual filter values are loaded on default with the values of the Coffalyser length 2 nt for the upper and lower bound By selecting any sample in the left list box the sample will be loaded together with the detected peaks In case a peaks fall within a bin and the signal of that peak met the criteria of the probe data filtering settings then the bin will be colored green in case no peak was found on the peak did not match the criteria the bin will be red This coloring method allows you to easily spot which bins should be changed By selecting or changing any of the displayed bins the displayed set in the chart will directly change into the set manual bi
21. a signal to size drop Excess DNA may also result in lower signal strength since it will compete with the labeled DNA for injection leading to poor resolution again leading to unreliable results Overloading and more specifically truncated peaks will result in a complete fail of the quantification of the fragment since only part of the product will be measured Capillary system usually use CCD camera s to measure the amount of fluorescence so over or under loading of sample can be a problem for signal quantification The optimal range for peak quantification is quite limited as compared to the dynamic range of most devices and it is thus crucial that most MLPA probe signal are in the optimal range when using it for copy number assessment Problems are indicated when for most devices we give warning in case the median probe signal is below 4 of the maximum intensity or above 60 of the maximum signal intensity resulting in a subtraction of 20 points from the FMRS In case the signals are between the 4 5 or 50 60 of the absolute maximum the penalty is only 10 points In case of an ABI 3130XL the minimum signal intensity of the median probe signal is 300 units and the maximum intensity is 5000 units Recommendation with problems low raw data signal can be caused by a variety of issues One of the most common causes is lack of sufficient DNA template in the cycle reaction It is vital to the success of fragment analysis to have the correct amount of
22. algorithm based on the Smith and Waterman method adapted for sample names Each undigested sample in the first column will be matched against a digested sample in the collection which will afterwards appear in the column digested Because matching may not always be 100 successful users may adapt the matched sample by double clicking on any of the cells in the column digested and change it into the corrected sample Please note that each undigested sample can only be matched against one unique digested sample After making all the correct matches click on start comparative analysis All the settings can be made exactly as described earlier for the DNA MLPA at chapter 8 1 Methylation specific normalization occurs always in the same way and the methodology cannot be adapted the available settings thus only influence the analysis of the DNA MLPA normalization This method normalized each target test probe of each test sample directly against its undigested counterpart by making use of the set reference probes This method does not require any slope correction since the sample is the same on both side of the equation and a difference in sloping between the two is not expected 99 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sample nami sample type FMRS X Y analy digested cas PSLP FSLP RSQ RPQ LAURA mPa 2097113 sarpie all LAUR
23. and the statistical significance of that result within the experiment 8 4 1 Comparative analysis sample explorer statistical sample chart The first tab opens shows a sample chart displaying the ratios results of the last normalization step figure 44 On the Y axis the probe ratios are displayed of the sample that was selected in the left list box You may switch samples by either using the cursor keys or by selecting a sample from the list use a mouse click After each sample of the type reference sample you will find the tag r and sample of the type positive reference will have an added tag p P335 A2 lot 0809 BMP ID 05 HYF DNA Electropherograms P335 A2 lot 0809 BMP ID 27 HYI DNA _ rstectert Sample report P335 A2 lot 0809 BMP ID 28 HYI DNA P335 A2 lot 0809 BMP ID 31 HYI DNA P335 A2 lot 0809 BMP ID 55 HYI DNA PositiveReference P335 A2 lot 0809 BMP ID H P335 A2 lot 0809 BMP ID 4 P335 A2 lot 0809 BMP ID 47 HYI P335 A2 lot 0809 BMP ID 55 HYI ONA P P335 A2 lot 0809 BMP ID 57 HYI DNA P335 A2 lot 0809 BMP ID 74 HYI DNA P335 A2 lot 0809 BMP ID 81 HYI DNA P335 A2 lot 0809 BMP ID 84 HYI DNA P335 A2 lot 0809 BMP ID 91 HYI DNA P335 A2 lot 0809 BMP ID 92 HYI DNA P335 A2 lot 0809 BMP ID 95A HYI DNA P335 A2 lot 0809 BMP ID 97A HYI DNA P335 A2 lot 0809 BMP ID 17781 HY DNA P335 A2 lot 0809 BMP ID 17881 HY DNA P335 A2 lot 0809 BMP ID 19981 HYI DNA P335 A2 lot 0809 BMP ID 2408 HYI DNA
24. as compared to all probes is computed as the peak area divided by the total amount of fluorescence of all probes added times one hundred The peak area ratio percentage of a peak must be larger than the minimum threshold and lower than the maximum set threshold to compete in the binning procedure These values are called the minimum and maximum peak amplitude to fluorescence all probes 3 Search range nt The search range determines the size in which the binning procedure will look for probes If the minimal distance between probes is 6 nucleotides the search range of each probe is 3 nucleotides plus minus The smaller this 29 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 search range the set the more difficult it will be for the binning procedure to relate detected peaks with certainty to a probe 4 5 4 Data filtering settings Data filtering is the actual process where the detected fragments of each sample are linked with gene information to a probe target or control fragment The binning procedure is thus only used to create common probe length reference vector and not for filtering The binning procedure may for instance only be applied on the sample that were set as reference samples while the filtering procedure will be applied on all selected samples Our algorithm assumes that peaks within each sample that fall within the same provided window or bin and have
25. available data for that result yellow arrow figure 40 For more information about these different normalized ratios and distribution comparisons values also see the FAQ in the end of this document or published articles about the methodology behind Cofaflyser NET J Coffa 2011 J Coffa 2008 82 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 5 ProbetargetInfo 5 y E Allsamples a E P335 A2 lot 0809 BMP ID 05 HYI DNA g a a Heen eS aa Lo aai Ones io Preratio Ratio wo Finalratio Stdev RefPop _ SamPop PosRefp_ 2 P3395 P335 E 45 EBF1 16 _ o5a33 3 _ o5s 158057 ass oor los joss loss 0 04 e p 5 joss 43 EBF _ osa333 05158071 436 looz loso E oss oos k E E ps E EBF 1 10 059332 3 J05 158137_ 264 003 0 1 05 104 0 04 l l l 1 03 0 99 16 jesti osa333 os 158459 228 oos io E oss oos E E J E hor Toz 1o loos 5 k 5 ry 037 06 p z 3 hag hor mi0 ose ose pos E s 5 E 2 23 izri 1 07p122 13 ixzF1 2 07122 a iKzri3s 07p122 22 izri 4 070122 3 keris ompi22 46 iKzF16 07p122 32 iKzri7 07p122 26 izri s 07p122 21 CDKN2A 5 090213 013 20 CDKN2A 2 09p21 3 love 18 coKN2e 2 0921 3 os o21s95_ 238 ors 36 PAxS 10 fo9p132 _ os o36830 373 lore 24 paxss ospt32_ _ os oze872_ 275 lov7 12 Pass osp132 09036956 202 ois 25 PAX65 _ oapia2 _ os
26. behind the channel type In case you have indicated that you are using a probes channel type you also need to set an analysis method for the probe mix The default method that will appear in most cases is block default Block analysis means that the available reference probes are used to normalize the samples against the reference samples Normalization in this case referrers to the division of multiple sets of data by a common variable in order to cancel out that variable s effect on the data Reference probe are usually targeted to chromosomal regions that are assumed to remain normal diploid in DNA of applicable samples In case a MLPA kit does not contain any reference probes users may define their own reference set see section 3 4 amp 3 5 or use population method instead In population analysis mode all probes are used for normalization this method is therefore only recommended in case the number of aberrations in each sample is expected to be very low e g 1 2 aberrant probes target sequences in each sample To change the analysis method click on the little arrow row define as a probes channel type green arrow figure 21 The last two columns DNA type and marker will automatically be set for your and require no more adjusting If you chance to work with more than 2 channels in one capillary sample runs please see our advanced analysis section for more information about this 37 Title Coffalyser
27. configuration at 11 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 the login screen is set to Client in Multi PC Networked as noted in figure COFFALYSER configuration Networked server 192 168 82 15 user name yco password reset close all other sessions for this user Figure 3 Coffalyser NET login form for clients in multi PC networked environments To get instruction on how to set up a computer as a Coffalyser NET server want to refer to the Coffalyser NET installation manual The server name should be the IP address of the computer where the Coffalyser NET server is installed and the port number should be 1231 If you don t know the IP address of your server computer start command prompt on the server computer click start menu click run type cmd press enter and type in command prompt Ipconfig all Your IP address should turn up in the list 12 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 3 About the Coffalyser NET sheet manager 3 1 What is the Coffalyser NET sheet manager Coffalyser NET is equipped with MLPA sheet manager software allowing users to obtain information about commercial MLPA kits and size markers directly from the MRC Holland database Next to this the sheet manager als
28. fragments is between the 2 3 Recommendation with problems in case there is a clear problem with the DNA concentration reaction should be repeated using higher DNA concentrations If no higher concentrations are available samples may be concentrated by alcohol precipitation or vacuum drying 7 3 2 9 FMRS Check 9 DNA denaturation check Background incomplete DNA denaturation will not provide reliable results We assume the DNA denaturation was incomplete if the ratio of the signal BB Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 intensity of the 96 fragment divided by the 92 fragment is lower than 03 and if the signal intensity intensity of the 88 fragment divided by the 92 fragment is lower than 0 5 We assume that the DNA denaturation is partially incomplete if the ratio of the signal intensity of or the 96 or the 88 divided by the 92 fragment is smaller than 0 5 If the ratio of the signal intensity of the 88 or 96 fragment is higher than 1 5 a warning is also given Problems are indicated when in case the ratio of 88 control fragment and the 96 control fragment as opposed to the 92 fragment are both lower than 0 5 we assume that the denaturation completely failed and a warning is given In case the denaturation failed 60 points are subtracted from the FMRS In case the ratio of only the 96 or 88 as opposed to the 92 fragment is lower than 0 5 or higher than 2 5 a
29. from samples with mosaic cell population may often be contaminated with normal cells which may cause the magnitude of the probe ratio to be within the set of arbitrary border while the result may still be significantly different from the reference population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol gt gt Region color or white with black text or figure 45a The color of the cells will not change if the result was found to be equal to the reference sample population Results are assumed to be equal if 2 criteria are met First the magnitude of the probe ratio falls within the lower and upper set arbitrary border values Secondly the probe result falls within the 95 confidence range of that probe in the reference sample population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol White with red text or figure 45e amp 39f The cells will become white with red text if the result was found to be ambiguous Results are assumed to be ambiguous if the magnitude of the probe ratio falls does not fall within the lower and upper set arbitrary border values The result was however also found to fall within the 68 1 confidence range of that probe in the reference sample population This indicates that this probe was found to be very variable in the reference sam
30. left top of the column headers You may for instance click on the plus sign of the column header or double click anywhere in the header that states probe target info which will then open the columns probe name chromosomal position hg18 track position probe length and the recommended order blue arrow figure 39 Each of these columns can be used to sort the whole grid by clicking on the column header cells 81 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sci ii a E All samples a EP335 EP335 EP335 E P335 E P335 agaca 2325 E P335 E P3365 EP335 EP335 EP335 EP335 2P335 E P335 E P335 a p los2 oss 1 01 1 00 lose 195 1 03 loss os7 1 01 1 06 jos7 1 02 1 06 hor ose Joss pa oso Joss ose 1 02 f1 00 ose 101 joss h10 E 1 00 1 02 1 00 1 04 1 03 ose joss ho ose 1 02 39 oss 101 101 ps oss 1 04 1 01 1 03 ps 37 E loa I ftor 34 38 192 7 00 ose jior os7 03 199 1 03 joss jos7 103 103 1 02 100 los7 loss hor loss hor los7 119 tor os7 losz os7 095 tor hor 1o tos jos7 oss fot lose E ose ros jos os7 ioo 33 ioo roo ce oss o7 s oz hos joss ose oss lose jor oz os 1 02 jos7 loss joo bss foss ho p fost ho ps ps fo a TE jose
31. log transformed pre normalized signal to its predicted signal 5 Normalization of signal to size corrected data in the user selected mode usually block method using reference probes and determination of significance of the found results Even though each of these different steps has default settings for analysis most steps may be adapted in order to provide the possibility to optimize for specialized data types 8 1 Setting up the comparative analysis After you have analyzed and explored your fragment data you can navigate to the next step which is the sample dependent comparative analysis Since the fragment analysis is sample independent you may select any combinations of samples in the fragment analysis Please note that leaving out samples may influence the normalization of all samples and thus the probe ratios of all samples 68 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sample name sampletype FMRS X Y anaiy cas PSLP FSLP RSQ RPQ fy a o 6 8 O wll V y 9 8 idl V y O Q O fy a o O 8 y4 a o 0O O O il e e w o O 8 O i f f B OF vga Open Sample Results f f a Open Experiment Results vv a i Select Samples For Analysis gt 1 FMRS Bar f s E ted Samples For MS MLPA ill gt 2FMRS Bars 4 a Export grid data to image aij 2 2 FES Bas fva fh Copy to clipboard all
32. requirements of the bin settings as described in the CE devices chapter and falls between the start and end length of a bin then that peak signal is assumed to originate from the probe product related to that bin and that signal will be called as that probe On the X axis right underneath each bin the probe name and rounded bin start end length is displayed In case a signal was related to a bin then this bin will be colored green in case no signals were related to that bin it will be colored red By hovering over each bin the gene name design length bin start center and end can be viewed in a tool tip Next to this the median average and standard deviation which are the result of the auto bin procedure are also displayed in the tool tip Our algorithm is also able to link more than one peak to a probe within one sample The amount of fluorescence of each Title 61 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 probe product may then be expresses the peak height peak area of the main peak and the summarized peak area of all peaks in a bin 7 File details on this page you may view any file details that are added mostly to ABIF to files Here you may view encoded data from each file allowing you to view details about the used capillary device and run settings For example ABI gel type may be viewed by GTYP 1 Machine type by HCFG 1 to 3 injection time in seconds
33. sample and reference The data for each test probe of each sample will be compared to each available 39 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Title reference sample producing as many dosage quotients as there are reference samples The final ratio will then estimated by calculating the average over these dosage quotients In case no reference samples are set each sample will be used as reference and the median over the ratios be calculated Next to this reference samples are used to estimate the effect of sample to sample variation on probe ratios of test probes by calculating the reproducibility of these probes in the reference sample population These calculations may be more accurate under circumstances where reference samples are randomly distributed across the performed experiment Positive reference samples key p are used to make an estimation of the behavior of a probe within a sample population with a known aberration We can do this by calculating the distribution statistics for each probe over all sample ratio results of the same type Next each unknown test sample result can be tested against several variables of that distribution such as the average median standard deviation CV and 95 confidence range in order to calculate the probability that an unknown sample is equal of different to the distribution results of that sample type No DN
34. sufficient fluorescence intensity are the same probe Our algorithm is also able to link more than one peak to a probe within one sample The amount of fluorescence of each probe product may then be expresses the peak height peak area of the main peak and the summarized peak area of all peaks in a bin An algorithm can then be used to compare these metrics and decide which should optimally be used alternatively users may set a default metric Filtering can be optimized by adjusting the settings shown in figure 16 these properties responds as equal to their values as the equally named properties do in the binning procedure described in 4 5 3 general base line detection peak detection binning fitering probes control frag Minimum peak amplitude RFU 70 Maximum peak amplitude RFU 0 Minimum peak area to fluorescence all probes Maximum peak area to fluorescence all probes p 00 Restore Default Settings Figure 16 Filtering settings tab 30 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 5 About projects 5 1 What does a project contain Our database setup contains a large number of subtraction levels not only allowing users to efficiently store and review experimental sample data but also allowing users to get integrative view on comprehensive data collections as well as supplying an integrated platform for comparative ge
35. tet oo tee Breeton teat upper genbank info position direction Lee HHAT Hi 09 58 00 60 00 a 066 65 00 67 00 C ic 073 72 00 74 00 a ca 079 78 00 80 00 B ica 085 24 00 86 00 a ost 90 00 92 00 a a 096 95 00 97 00 mf A so wa E lt 2 16 104 00 106 00 Ba 8024373 0 02 a 15 1234 121 40 125 40 NM_013325 4 67 exon forward no ic 5 08q24 22 0 00 08 ical 130 128 1 126 10 130 10 NM_003882 3 11 exon forward no a 101391211 13 019 499380 13 ica 1 148 132 80 136 80 NM_003453 3 21 undetermined undetermined no C OE a 12 5 1850 14350NM 054173 7 undetermined no a 308q24 21 08 128 08 ca 15 1521 150 10 154 10 NM_002467 4 1 no a 2 20q11 21 20 029 20 160 158 3 156 90 160 90 NM_138578 1 3 no C u a 16 1667 18270 16770NM_0080477 ro mi 3 a 15 1742 17220 176 20 NM_000059 3 47 ur no E probe MAPRET 20 a wm 190 8 178 20 182 80 NM_012225 2 15 ur no E robe co 3 a 106 19 4290 18790NM_O0I2653 77 u ha a probe EVA o8 193 1328 130 80 194 80 NM_000503 4 16 ur no C probe TPD52 9 08421 13 08 081 08 ic 202 218 499 80 203 80 NM_091025252 1 un no a probe CYP24A1 620q132 2 20 ic 2m 2m1 208 10 213 10NM_000782 4 11 undetermined undetermined no a probe PSPCT 209 2181 216 10 220 10 NM_01042414 1 exc no a probe SCNIA 29 225 226 50 230 50 NM_006920 4 57 un no E ebe ATPT ataman 12 051 aaneen 13 mi oR vars ZRWANNM MNRAS 17 h et CEE Figure 9 Coffalyser NET sheet library product lot version probes
36. the database is better protected and both client and server will always have the same version number In case an older client will try to connect to a server that has a newer version number the client needs to be updated first A client does not share any of its resources but requests a server s content or service function Clients therefore initiate communication sessions with servers that await incoming requests When a new client is installed on a computer it will implement a discovery protocol in order to search for a server by means of broadcasting The server application will then answer with its dynamic address that resolves any issues with dynamic IP addresses 2 2 User Access In addition to serving as a common data archive the database provides user authentication robust and scalable data management and flexible archive capabilities via the utilities provided within Software Our database model acts in accordance with a simple legal system linking users to one or multiple organizations Each user receives a certain role within each organization to which certain right are linked These rights may for instance include denial of access to certain data but may also be used to deny access to certain parts of the program These same levels may also be applied on project level Projects will have project administrators and project members The initial project creators will also be the project administrators who are responsible for user manag
37. thereby allowing some of the reference probes to be altered lt 40 without having an effect on the final results User may however also choose to use the average minimum of maximum of the collected ratios Minimum and maximum should be avoided unless you are choosing this for a special kind of analysis Normalization inter in the presence of multiple reference samples each test sample will be compared to each reference sample thus generating as many dosage quotient or ratios as there are references samples for each probe In order to obtain a single result for each sample probe a for these dosage quotients the normalization factor inter will be taken over these ratios When this option is set to auto the average will be taken when there are more than 2 reference samples present if no reference samples are available all samples will be used as a reference and the median will be taken User may however also choose to use the minimum or maximum which should only be chosen if you are choosing this for a special kind of analysis Arbitrary ratio border low high the arbitrary borders are the set borders where we expect normal results to fall in between In figure 32 a delta of ratio 0 3 is set as opposed to the reference which is always 1 resulting in a normal range of ratio 0 7 1 3 for results that appear to be normal or equal to the signals found in the reference samples 70 Coffalyser NET analysis manual beta version Stat
38. version Status Release candidate Classification Confidential Versie 0 1 The right click menu enables you to customize the chart The exact same options can be found in this menu as earlier described at 8 3 1 for the comparative analysis experiment explorer statistical overview chart a b c d Figure 45 Different probe ratio results stages versus the reference sample populations Result a displays a result that was found to be equal to the reference sample population Result b was found to be significantly different from the reference population but did not fall outside the arbitrary borders Such cases are often seen when samples have mosaic cell populations Result c is significantly increased as opposed to the reference population and the result is also higher than the set arbitrary borders Result d is significantly decreased as opposed to the reference population and the result is also lower than the set arbitrary borders Results e and f are both half ambiguous Result e is lower than the set arbitrary border but the reference probes for this sample were variable the result was therefore found to be different from only 68 of the reference population and not 95 Results f on the other hand shows a very wide 95 confidence range for that probe in the reference sample collection indicating low reproducibility for that probe in the experiment Again this result was found to be different f
39. warning will be given and only 15 points are subtracted from the FMRS Recommendation with problems in case there is a clear problem with the DNA denaturation the reaction should be repeated and DNA should be denatured for at least 10 minutes at 96 degrees In case the sample may contains high salt concentration it is advisable to desalt samples before repeating the reaction or diluting the sample by using lower sample volumes 7 3 2 X and Y control fragments Displayed as X amp Y Checks If the X and Y control fragments were detected and if the signal intensity as opposed to the 92 control fragment was in the expected range If the ratio of the signal of control fragment X as opposed to the 92 fragment signal is between ratios 0 2 3 the fragment will be marked green In case the signal of X or Y control fragment was zero the fragment will be marked red or as not present In case the ratio is between 0 0 2 or in case in of the X fragment higher than 3 and in case of the Y fragments a higher than 2 a warning will be given The Y control fragment is furthermore used to estimate the expected gender of each sample Runs that have a Y control fragment with a ratio higher than 0 15 as opposed to the 92 fragment are expected to be males 7 5 Using the fragment results explorer By selecting Open from the right click menu on the fragments analysis settings window while hovering above a sample row you can open the fragment results e
40. 0 14116 15717 16X022 X001 38386 14052 115650 2 07p122 07 050 397790 12536 113586 dom p22 000 755205 06239 106219 Zilospae 2 03 058 591790 07815 07545 8 12p132 12 071 935360 14054 115652 note 6090132 006 sR 55 L15653 206 e E E E A Gamay E O j 09000000000000 00000000 PEPEPE ERE E ES Figure 31 Coffalyser work sheet editor manual bin set 65 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 8 About the comparative analysis copy number Signals of MLPA oligo nucleotide probes are directly proportional to the amount of the target sequences present in a sample Since these measurements have little meaning on itself the signals of an unknown sample need to be compared to a reference in order to assess the copy number For MLPA in order to assess the copy number signals of unknown samples can be compared to reference data by normalization Normalization refers to the division of multiple sets of data by a common variable in order to negate that variable s effect on the data thus allowing underlying characteristics of the data sets to be compared this allows data on different scales to be compared by bringing them to a common scale The common variable or normalization constant thus needs to be derived from a factor that remains constant in each sample To make the normalization more robust when normalizing the signal of a probe
41. 1 0 36 0 98 0 03 11 02 0 02 1 00 1 00 0 38 1 03 0 02 0 02 0 90 1 00 0 87 los7 072 oss los7 loos 101 002 r00 foo jose foz looz oor froo 1100 gt 0 98 0 09 0 98 099 0 03 0 02 0 99 0 02 1 00 11 00 0 97 1 03 0 02 0 02 284 1 00 48 09 037010 0 92 0 10 0 38 11 00 0 04 0 94 0 02 1 00 1 00 0 98 11 02 0 02 0 01 2 50 1 00 5 09037024 1 02 0 05 1 02 1 02 0 05 0 03 1 00 0 02 1 00 0 99 jo 98 1 03 0 02 0 01 1 00 1 00 28 12 011694 0 98 0 14 0 98 1 02 0 02 1 00 0 01 1 00 1 00 0 98 1 03 0 02 0 00 1 05 1 00 1 05 37_ iz o1t6 4_ 097 014 joss for ooz or 002 1 00 froo jose fros poz joor 0 v 38 12 011796_ 0 95 0 15 0 96 1 02 0 02 0 99 0 01 1 00 1 00 0 98 1 01 0 01 0 01 19 12 011883_ 0 95 0 15 10 94 11 00 0 03 1 01 0 02 1 00 11 00 0 98 1 02 0 02 0 02 Dye 224 47 iz 011973_ 093 015 oss ose jos jose 0 02 100 ioo 036 hos aos 003 11 12 0711935 0 97 0 15 0 98 1 04 10 05 0 99 0 01 1 00 11 00 0 98 1 02 10 01 0 07 1 01 1 00 1 01 40 12 090903_ 0 98 0 09 0 99 1 00 0 08 0 02 0 99 0 01 1 00 0 99 0 99 102 0 01 0 00 0 41 1 00 6 12 090905 0 96 0 09 10 97 0 97 0 09 0 03 1 00 0 00 1 00 11 00 0 99 1 01 0 01 0 00 0 41 1 00 31 12 091061_ 1 00 0 11 0 36 0 96 0 03 1 04 0 02 1 00 1 01 0 97 1 02 0 02 0 07 0 44 1 00 m Figure 43 Experiment explorer stati
42. 15004 15004 094 089 088 002 005 004 274 00 09p122 09 036956622 14055 15653 2158 23552 23552 106 099 097 001 005 004 50 21 00 09p142 09 036092609 13870 15020 3261 40183 40183 297 281 289 005 018 0 13 gt gt gt 50 280 01 09p13 2 09 03700669 13876 15024 1448 21743 21743 279 263 265 007 O14 012 gt gt 49 4798 05 8132 09037024272 1201 13581 1719 17812 17812 108 099 099 00 005 Q04 So 18234 i Averages Mesen Sidey Min 154 Region analysis 190 097 998 017 289 a Ener a eA asane E O O a OE E O EE SE T 382 ETVE 1 12p132 12011694328 14060 15658 1367 17495 17495 107 102 103 001 005 004 5 381600 391 ETVE 2 12p132 12 011796704 13875 15393 1171 15572 15572 108 102 703 002 006 004 389301 246 ETVE 3 1213 2 12 011883341 13874 15015 1803 19440 19460 111 104 703 002 005 004 49 245100 474 ETVES 12p132 12011912651 13871 15389 1291 18192 18192 113 1 07 108 003 006 005 50 4698 01 196 ETVES 132 12011995363 14054 15652 2557 27619 27619 107 099 100 002 006 005 50 1047 61 a Mesan isev Mn Max Chr bend 122123 2 Size 241152 mt 241 kb Region analysis 103 403 002 100 1 06 200 BTG AREA up 1232133 12 000903053 14117 15778 4900 s 102 002 lt lt lt lt 44 160 BTGIAREA up 12921 33 12 090005485 14116 15717 1071 11192 11192 045 041 040 001 002 002 lt e lt e 45 1876 01 328 BTG1 2 12q2133 12 001061700 12553 13603 862 10513 10513 045 0 43 042 0 01 003 002 lt lt lt lt 0 3279 02 232 ma 1292133 12 001063286 12542 15013
43. 40138 Region analysis 100 1 01 002 098 1 02 68 ZFA Dipi22 07 05015024 13877 15018 Tees 20201 20201 701 086 O08 OO O06 DOs 50 cera 00 208 iKZFt 2 07p122 07 050320206 14056 15654 2219 22098 22006 107 099 099 000 006 006 50 206800 177 IKZFI3 O7p122 07 050337794 12536 13586 1879 19141 19141 106 098 O98 002 005 006 48 176200 263 iKZFi4 079122 07 050411707 13873 15017 1806 21431 21431 105 099 099 O01 006 004 0 21700 142 IKZF1 5 079122 07 050417749 13872 15390 2400 24318 24318 109 099 Q95 002 005 Q04 50 1388 01 485 IKZFIS 07p122 07 050422507 14061 15023 678 9006 9006 104 098 095 002 008 004 50 462902 337 iKZFt7 079122 07 050426050 13869 15387 2217 26815 26815 104 099 097 002 005 004 50 3366 01 B8 l 18 079122 07 0504360 12549 4 4 1537 18376 18376 105 100 707 001 005 004 50 2866 0 0 OFp Average Macar Sisav Min Max Chr bend O7p12 2 Sire 127001 nt 121 kb Region analysis 59 098 002 095 101 CDKN24 28 CDKN2B 2 PAXS 10 PAXS 8 PAXS 6 PAXS 5 PAXS 2 PAXS 1 251 238 373 275 202 282 483 154 Chr band 09p13 2 09p21 3 Size 15066 kb 12 A6 325 015 017 017 OOO OO Cot lt lt lt lt e asa 00 00p21 3 09021064058 10333 15016 207 2734 2734 023 021 021 002 001 002 lt lt lt e 2 249201 09p21 3 09 02199583 10337 15014 336 3298 3298 019 018 0 18 000 001 001 lt e lt lt 33 2377 00 09p132 09036830475 12521 13571 1130 14355 14355 102 096 098 002 005 004 50 372100 09p132 09 036872086 14057 15019 1378
44. 6 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 correct as determined by your filter set If your channels are not set correct then click on the option box show all channels you will be able to select which channel you are using by ticking the option boxes in the first column called nr blue arrow figure 21 The name of each dye should appear in the next column You will not be able to change the dye names since they are related directly to your filter set Now you will be able to set the content type or channel type for each of your used channels by clicking on the dots or on the little arrow on the left side of the combo box in the channel content column red arrow figure 21 Channels are either set to probes indicating that in this channel peaks that can be related to a MLPA probe mix can be found or the channel type can be set to size marker indicating that this channel contains a size standard which can be used to compare the detected peaks against and give them a length in nucleotides also see our FAQ If you have set the content of a channel to be a probe mix then you also need to define the products lot and version number by using the probe mix selection form which will appear after selecting the dots red arrow figure 21 6 6 Setting the channel settings After settings the channel contents you will find some other settings
45. 7828 12527 13577 436 EBF1 14 05q33 3 05 158071810 13868 15386 364 EBF1 10 0533 3 05 158137017 14059 15657 228 EBF1 1 05q33 3 05 158459187 12509 14269 TK2F 1 1 pie 13877 1597 208 IKZF1 2 07p12 2 07 050329206 14056 15654 177 IKZF1 3 07p12 2 07 050337794 12536 13586 263 IKZF1 4 07p12 2 07 050411797 13873 15917 142 IKZF1 5 07p12 2 07 050417749 13872 15390 465 IKZF1 6 07p12 2 07 050422507 14061 15923 337 IKZF1 7 07p12 2 07 050426950 13869 15387 288 ___ IKZF1 8 07p12 2 07 050436025 _ 12549 15921 256 CDKN2A 5 09p21 3 09 021957822 10334 10871 251 CDKN2A 2a 09p21 3 09 021964958 10333 15916 238 CDKN2B 2 09p21 3 09 021995813 10337 15914 373 PAX5 10 09p13 2 09 036830475 12521 13571 275 PAX5 8 09p13 2 09 036872066 14057 15919 202 PAX5 6 09p13 2 09 036956622 14055 15653 282 PAX5 5 09p13 2 09 036992699 13870 15920 483 PAX5 2 09p13 2 09 037010669 13876 15924 154 PAX5 1 09p13 2 09 037024272 12501 13551 301 ETV6 7 HIEN 72 011694211 14058 15656 382 ETV6 1 12p13 2 12 011694328 14060 15658 391 ETV6 2 12p13 2 12 011796704 13875 15393 246 ETV6 3 12p13 2 12 011883341 13874 15915 474 ETV6 5 12p13 2 12 011913651 13871 15389 196 ETV6 8 12p13 2__12 011935363 14054 15652 409 BTG1 AREA up 12921 33 12 090903053 14117 15718 160 BTG1t AREA up 12q21 33 12 090905485 14116 15717 328 BTGI 2 12921 33 12 091061700 12553 13603 232 BTG1 1 12921 33_12 091063286 12542 15913 Figure 42 Part of the experiment pdf report ratio overview fg S oF SS Fe if Be P
46. 9 P335 A2 lot 0809 TE HYI D sample dl oll 255 X MK ts oe Figure 28 Fragment analysis quality scores and right click menu Based on the quality scores you may use the right click menu to open the fragment analysis results explorer add or remove samples and include sample for the comparative analysis 7 3 1 FRSS FRSS Fragment Run Separation Score displays the quality of the fragment separation and peak sizing quality by evaluating the quality of the peaks in the size marker channel To get to a final score several different criteria are evaluated that each have a penalty weight which is subtracted from 100 start points or 100 ok Each score that is dependent on the measurement of signal intensities has adjusted criteria that are dependent on the machine type The method of quality assessment may thus different between 47 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 machines to find the exact criteria for each machine for the different quality control checks please check the tables in the appendix 7 3 1 1 FRSS check 1 Correlation of the size marker curve Background the techniques described in this section are used to investigate relationships between two variables x and y Is a change in one of these variables associated with a change in the other For MLPA the size call correlation refers to the correlation between the relative mi
47. A MLPA 2027113 s TICE pean ull LAURA MLPA 2027119 C LAURA MLP 1U sample ull x E g amg ull LAURA MLP sample ull LAURA MLP sample ail LAURA MLP sample ull LAURA MLP ols wlr 38 S ai LAURA MLP eference ull LAURA MLP e ull LAURA MLP eference all IHRE EHSL XKRXKXKKXKKKKX 44 44444444 K3 Select Samples For Comparative Analysis gt Digested Samples For MS MLPA gt Remove All Match Sample Automaticly Start Comparative Analysis 0 Figure 51 Comparative analysis settings screen in DNA MS MLPA modus 9 3 Comparative analysis experiment explorer with MS Data After the analysis is finished you can reopen the comparative analysis experiment explorer as earlier described in chapter 8 3 When you select distribution type you will find extra distribution for each sample type separately for the DNA and MS MLPA analysis You may for instance display the results of the reference samples MS you can easily evaluate the reproducibility of each probe in that experiment for the methylation status assuming the selected reference samples were genetically equal and the reference samples were properly dispersed through the experiment In the heat map grid each digested sample MS result will be loaded directly next to its undigested sample DNA result In figure 52 you may for instance view the DNA MS MLPA results of the ME028 Prader Willi mix In the left column you m
48. A or blank controls key b are analyzed MLPA experiments that do not contain any DNA They are used to make sure not contamination has occurred during the performance of the experiment Digested sample are all samples that were digested key d during the experiments and are used only to estimate the methylation status of each target sequence 40 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 details fragment analysis comparative analysis sample name E sampletype FRss FMRS probes ona oo x y P335 A2 lot 0809 BMP ID 05 HYI DNA sample P235 A2 lot 0809 BMP ID 27 HY DNA sample P335 A2 lot 0809 BMP ID 28 HYI DNA sample Pj w P335 A2 lot 0809 BMP ID 31 HYI DNA sample P335 A2 lot 0809 BMP ID 32 HYI DNA sample ACIES P335 A2 lot 0809 BMP ID 43 HYI DNA sample P235 A2 lot 0809 BMP ID 47 HYI DNA sample P335 A2 lot 0809 BMP ID 55 HYI DNA sample P335 A2 lot 0809 Promega F HYI DNA reference P335 A2 lot 0809 Promega F HYI DNA reference P335 A2 lot 0809 Promega F HYI DNA reference P335 A2 lot 0809 Promega F HYI DNA reference P335 A2 lot 0809 Promega F HYI DNA reference P335 A2 lot 0809 Promega M HYI DNA bample sampe reference Jofa positive reference blank digested sample Figure 24 Fragment analysis sample setup window When you are finished adjusting all the sample types click on t
49. BS gS 4 2 amp x g oF SF oF oF amp 0 96 0 92 1 03 0 99 OS 098 0 95 1 00 096 1 1 04 1 03 0 99 096 1 0 93 0 97 094 098 Of 1 0 35 1 0 Ki i 0 97 1 19 097 095 1 098 1 21 097 100 Os 1 02 1 37 0 99 098 16 095 1 20 094 1 02 Of 098 1 39 0 99 103 1 088 1 15 094 1 01 Os 1 00 1 26 0 98 094 Of 0 56 0 89 0 16 0 93 Of 0 66 1 04 0 11 0 87 OE 0 13 0 98 0 15 0 95 Of 1 17 0 93 0 55 0 90 16 1 00 1 03 0 56 0 97 Of 0 96 0 83 0 89 106 Of 1 05 0 95 1 08 0 99 Of 1 11 0 89 1 02 105 16 1 03 0 98 1 02 095 16 0 52 0 96 fa 0 A 0 58 0 92 104 101 1 6 0 62 0 97 105 103 1 0 56 0 98 106 097 Of 0 57 1 03 1 10 094 1 1 05 0 93 099 106 11 1 07 1 00 1 02 1 01 i 094 1 07 1 00 095 Of 106 1 13 1 01 0 96 Of 092 0 96 094 102 Of 88 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 8 3 3 Comparative analysis experiment explorer statistical overview grid The third tab displays the same data as described at the 8 3 1 but now in grid format figure 43 g FAll samples E Reference Samples E Positive Reference Jal Probe E ia Enter i E na Enter e no Enter Mean Median Min Maximum Stdev
50. Coffalyser NET analysis manual Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Version management Version 0 1 Status Concept Date 22 July 2013 Classification Confidential Version history Version Date Status Auteur Owner Comments 0 1 09 03 12 Concept Jordy Coffa Jordy Coffa Initial version 2 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Foreword This document contains the analysis manual for Coffalyser NET This document is created as a release candidate matching the first beta version v120316 1250 Amsterdam 22 July 2013 Jordy Coffa 3 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Contents 1 BACK e KoE y To GEA E a A E sete tone tees OE secede cpa A AE A E E EA A 1 1 Introduction to MLPA normalization sene senenanian ena 2 LOCUM UND PARN A A E E E A E E E E ante 2 1 Organizations and user accounts ceecceeeeeeeeeneeeeeeeeceaeeeeeaeeeeeeeseeeesaeeeeaeeneneeseeeees 2a USer ACCESS a a a anita al aia E E OEE 2 3 Connecting to a local databDase ceccccecsececeeeeeeeeeeeeeeeeeeeseaeeeeeeeseeeesaeeeaeeeeaeeseeeees 2 4 Connecting to a Server database 1 cccccceeeeeeeeeeeeeeceeeeeseaeeeeeaeeeeeeeseaeeesaeeeeeeeseenees 3 About the Cof
51. DNA 5 Relerence otal 3500 3400 amp E 330044 s D 32004 pa P 2 300044 E 5 32 8 2 2900 4 B65 g iai z 2800 a 8 g a 85 Lock current sample d se 2700 4 amp z 3 32 Serie label gt ey amp Si 8 g 2 2500 4 m2 5 3 2 Copy to clipboard 2400 5 gy n 2300 g D g a 25 Save as u 8 T elg 2 as see s_ amp AEA Sa a E8 Save all al isa 5 eg 2 ic a M 3 N 6 Page setup 20004 Se zig amp lt gt uia d Pu rg 6B bal B Print preview 1900 2 z PAGE E 5 pi 8 lt fe Fra Sup a 3 gt 18004 AE S re g efx Print 170 is e A z gag 2118 Jelg 5 Bus 16004 Bile g ES ne 5 c T brd N T 5 s 150 slitz i gog a a 8 5 A Z EER 3 es 14004 8 z 5 D z 35 Ben 2 13004 G x g 5 Sa TS te gs i fo a B 7 29 S S 1200 S11 5 2 52 Saji 11004 Fs 3 Sy BE 3 S 5 BES 5z Ed 8 3 900 er g 7004 600 500 400 300 200 1004 I 8583 Q2 SJL E233 LERLABY AEX VJBONDSS2II0Z9 EONS IZIN RAE BE BBB 8583 a3 SIF EBB TSRS JAY BBLBESS 229z SEL yg 232388 8 BS BBB Es ABBSE GES TEBPARAING QASRI RnS SES RR 2252S SREB RB ith Figure 46 Comparative analysis sample explorer electropherogram tab By hovering above this marker you may view different information about this detection peak including probe target information and probe ratio at the different stages of analysis figure 47
52. E jroo joo oss fos os oo Ey los7 jose o tor jose 115 loss loss joz loss kor oss jose joe oo oe lost jos 1 00 2 foe 1 00 E 89 joss lose tos 33 loss 3 jose hbe jos7 faz loss 10 los7 jos fioo 1o E joz E 0 96 fos joes 0 96 0 26 fros loss jor jos los7 jin jos9 00 jtor j0 jo lose o tor or 28 12011694 ose h 03 o6 jos 37 fiz o11694_ losz tos oz los7 38 1201179 037 toz tor jos 19 12011883 oss kor loss fos Dye 224 47 i2 011913 ios i10 jos jas 11 12011935 1 05 loss los7 lose roo 40 12 090903 1 07 too jroo lose E Figure 39 Comparative analysis experiment explorer heat map grid overview The top levels of the columns contain a maximum of 4 entrees probe target info all samples reference samples and positive samples Each sample type group then contains all samples underneath it which levels are already opened on default Each sample can be furthermore opened to display separate information about each detected probe peak figure 40 The levels underneath each sample are closed on default and can contain the following levels peak signal intra normalized ratio pre normalized ratio ratio without iteration final ratio standard deviation and distribution comparison results against the collection of test samples reference samples and positive samples This information may also be summoned by hovering above a cell in the grid a tool tip control will then provide all
53. ET and used to optimize the obtained results 46 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 l om E sample name sampletype bin smpi FRSS FMRS probes pna pp x Y 3 P335 A2 lot 0809 BMP ID 2_ sample al wl O 5551 O Y X 4 P335 A2 lot 0809 BMP ID 3 sample al wl O55 OY ym 5 P335 A2 lot 0809 BMP ID 3_ sample ull al O 551 O Y f m 6 P335 A2 lot 0809 BMP ID 4 sample ul wl O 551 O Y f ez 7 P335 A2 lot 0809 BMP ID 4 sample al wl 551 Y f n 8 P335 A2 lot 0809 BMP ID 5 sample B al al O55 O Y ye 9 P335 A2 lot 0809 BMP ID 5 positive reference al all 551 w X a 10 P335 A2 lot 0809 BMP ID 6 positive reference all 1 P335 A2 lot 0809 BMP ID 7_ sample all L 12 P335 A2 lot 0809 BMP ID 8 sample ull 13 P335 A2 lot 0809 BMP ID 8_ sample ull 14 P335 A2 lot 0809 BMP ID 9 sample ull 15 P335 A2 lot 0809 BMP ID 9 sample all 16 P335 A2 lot 0809 BMP ID 9 sample 7 P335 A2 lot 0809 BMP ID 9 sample 7 PRE 18 P335 A2 lot 0809 BMP ID 1 sample ull g 3 Remove Include In Comparative Analysis Export grid to image Channel 1 6 FAM probe channel P335 ALL IKZF1 Channel 2 VIC not used
54. MAD Mean Median Min Maximum Stdev MaD Mean a 45 o5 158057_ 0 98 0 04 i 00 1 00 jos2 h o joos loos Jos9 001 i 00 1 00 oss 01 joo joo 98 1 00 099 43 05158071 s8 003 joss 0 98 1 93 105 0 03 0 03 1 99 0 01 1 00 1 00 Joss 1 02 0 01 0 00 11 00 1 00 1 00 35 05 158137_ 1 01 0 03 ose 33 0 92 w 0 03 0 01 1 02 0 01 1 00 1 00 17 00 01 0 00 0 00 1 03 1 00 1 01 16 o5 158489_ 0 97 0 09 0 36 0 97 0 09 0 03 11 02 0 02 1 00 0 99 097 1 03 0 02 0 02 1 99 1 00 0 97 23 1 01 0 12 1 00 11 07 10 02 1 01 0 01 1 00 11 00 0 99 101 0 01 0 00 10 98 1 00 0 96 13 329_ 1 02 0 11 0 99 11 00 10 02 11 00 1 00 100 0 00 0 00 1 01 1 00 0 99 8 7 37 1 00 0 13 1 00 11 00 0 04 1 00 0 98 1 03 0 02 0 02 0 98 1 00 0 97 22 50411 1 01 0 12 1 00 1 02 0 02 1 00 0 99 1 01 10 07 0 01 1 zl 3 7 1 00 0 11 0 98 0 99 0 03 11 00 0 99 1 0 10 07 0 00 46 2_ 03640 12 jose oss ooz oo o jose hon or loot 32 foo 011 ose To 0 05 02 002 1 00 joss joss ho 02 ooo jos7 071 joss jose ooz ror 002 1 00 froo jose hoz loor loor Ge E ps7 028 foes loss 3 loos 1o00 100 fioo joss foz oor joor x 0 83 0 28 079 0 86 za 0 05 11 06 0 06 1 00 1 02 0 92 1 07 0 06 0 04 21 j 1 00 osr 030 foes los 2 loos 02 001 1 00 foo joss fror loor ooo lore 100 0 91 0 11 0 34 0 96 0 05 0 97 0 02 1 00 1 01 0 96 1 02 0 02 0 01 0 95 1 00 10 98 2 0 98 0 1
55. NTPs All ligated probes have identical end sequences permitting simultaneous PCR amplification using only one primer pair In the PCR reaction one of the two primers is fluorescently labeled enabling the detection and quantification of the probe products The different length of every probe in the MLPA kit then allows these products to be separated and measured using standard capillary fragment electrophoresis The unique length of every probe in the probe mix is used to associate the detected signals back to the original probe sequences These probe product measurements are proportional to the amount of the target sequences present in a sample but cannot simply be translated to copy numbers or methylation percentages To make the data intelligible data of a probe originating from an unknown sample needs to be compared with a reference sample This reference sample is usually performed on a sample that has a normal diploid DNA copy number for all target sequences In case the signal strengths of the probes are compared with those obtained from a reference DNA sample known to have two copies of the chromosome the signals are expected to be 1 5 times the intensities of the respective probes from the reference if an extra copy is present If only one 6 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 copy is present the proportion is expected to be 0 5 If the sample has two cop
56. RER cown Xpz SHOX AREA down X22 CRLF24 Xp22 343P CSF2RA 18 Xp2233P X X02233P X00141570 1 ara 02242284733 2 IL3RA 1 Fisference Refer 03 038591703 05 0128763 1 05 09005646 05 132037612 1705 55115 Reference Reference 10 Reference 205 Median value all probe value Figure 49 Single page sample pdf report The dual page contains all relevant quality control information on the first page together with a larger sample chart and electropherogram The second page figure 50 contains a report of all probes and their target information Next to this the peak height peak area total peak area in the probe bin population normalized ratio slope corrected ratio final ratio reference sample quality standard deviation reference probe standard deviation final standard deviation distribution comparison values peak width expected peak length and delta to that expected length are also added in the report In case any of the columns contain values that were found to differ from the rest they will become bold Note that the expected lengths are the lengths of the peak that were used as the center for data filtering These values are commonly based on the entire data set and peaks are not expected to differ much from their expected length lt 0 5 nt 96 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 COFFALYS D nt Gene Exon
57. RS Recommendation with problems lf the peaks are too high the simplest solution is to rerun the same sample using a shorter injection time for example 7 5 seconds instead of 15 seconds Use less template DNA or less thermal cycles to decrease the amount of fluorescence signal generated by the sequencing reaction 7 3 2 3 FMRS check 3 Baseline Intensity of the probe dye Background high baselines decrease the dynamic range of detection of that channel Optimal performance of the capillary system is achieved when the baselines for all channels are below 5 of the maximum detectable intensity In most cases we expect that problems with baselines are also visible in the size marker however it may also be apparent that background fluorescence may be caused by the injected fluids itself and it is therefore impossible to resolve this issue with methods other that data analysis corrections Problems are indicated when in case the measured average baseline or signal intensity of a probe specific dye stream without running fluorescent products is above 10 of the maximum intensity of the machine a warning will be given and 15 points subtraction on the FMRS total In case the baseline is between the 7 and 10 only a notification will be given and 10 points will be subtracted from the FMRS total For an ABI 3130x for example the maximum baseline intensity for the marker is set at 700 units for a warning and 560 units for a notification Recomm
58. Sample hgt8 location Figure 55 Sample explorer result chart of digested sample showing a complete methylation of all target sequences except for the digestion controls LAURA MLPA 2037153 U Sample Ratio 024 es SE a3 Figure 56 Sample explorer result chart of undigested sample showing a deletion of all test probes matched undigested sample to the result of figure 55 104 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 10 FAQ 10 1 What is the different when the analysis method is set to RNA RNA MLPA is very similar to the DNA MLPA analysis method except that the used normalization factor will always be comprised out of the reference probes Next to this slope correction methods are not allowed since the probe signals can never define the amount of sloping on signals originate from such different numbers of targets sequences as is found with RNA sequences You may however set reference samples which in case of RNA serve for instance as a zero time point while all samples are measurements on later time points In case you wish to only investigate the intra normalized signals against a reference e g B2M you need to adjust the Y values or probe ratios to the intra normalized ratios in the results screens 105 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 11 Re
59. ZF1 5 07p12 2 07 050 142 009 3458 0 99 1 09 0 99 0 96 0 02 10 06 0 04 46 IKZF1 6 07p12 2 07 050 465 010 744 0 98 10 93 1 0 99 0 02 0 06 0 05 32 IKZF1 7 07p12 2 07 050 337 011 2719 0 91 0 9 10 9 0 88 0 03 0 05 0 04 26 IKZF1 8 07p12 2 07 050 288 012 2019 1 02 1 03 1 01 1 0 01 0 07 0 05 21 CDKN2 09p21 3 09 021 256 013 871 0 58 0 6 20 CDKN2_ 09p21 3 03 021 251 014 1088 0 71 0 74 18 CDKN2_ 09p21 3 0S 021_ 238 015 346 0 13 10 14 36 PAX5 10 09p13 2 09 036 373 016 1769 117 1 14 24 PAXS 8 09p13 2 09 036 275 017 1914 1 04 1 06 12 PAX5 6 09p13 2 09 036 202 018 3119 0 99 1 05 25 PAX55 09p13 2 09 036 282 019 1561 1 07 1 08 48 PAX5 2 09p13 2 0S 037_ 483 020 J657 1 07 1 02 5 PAX5 1 09p13 2 09 037 154 021 2565 1 04 1 14 28 ETV6 1 12p13 2 12 011 301 022 1121 0 53 10 53 37 ETV6 1 12p13 2 12 011 382 023 365 0 59 0 57 38 JETV6 2 12p13 2 12 011 391 024 1905 0 62 0 6 19 JETV6 3 12p13 2 12 011_ 246 025 1293 0 56 0 58 Figure 48 Comparative analysis sample report grid 95 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Authorization Dats Height Ares Ratio Stdev REF Sem Width dint 11908 aos y sarod he Coffalyser MLPA sample report P335 A2 lot 0809 BMP ID 55 HYI DNA COFFALYSER aes eet zr Me 5 3a SHOX A
60. a administrative user to be able to change the CE device settings 4 5 1 Baseline settings When performing detection of fluorescence in capillary electrophoresis devices it is sometimes the case that spectra can be contaminated by fluorescence Baseline curvature and offset are generally caused by the sample itself and little can be designed in an instrument to avoid these interferences Nancy T Kawai 2000 Non specific fluorescence or background auto fluorescence should be subtracted from the fluorescence 23 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 obtained from the probe products to obtain the relative fluorescence as a result of the incorporation of the fluorophore The baseline wander of the fluorescence signals may cause problems in the detection of peaks and should be removed before starting peak detection Our software corrects for this baseline by applying two times a median signal filter on the raw signals First the signals of the first 200 default is 80 for the size marker channel see figure 19 Baseline moving median point marker and Baseline moving median points probes rough data points of each dye channel were extracted and its median was calculated Then for every 200 subsequent data points till the end of the data stream the same procedure was carried out These median values are then subtracted from the signal of the original data stream t
61. a MLPA mix is expected to have 40 probe signals more than 40 primer will result in a warning and 40 points will be subtracted In case this percentage is between the 20 40 a notification will be given and 15 points will be subtracted Smaller mixes are allowed to have larger primer percentages for mixes with 15 30 probes primer percentage criteria are increase with 10 and for mixes with less than 15 probes percentages may be 20 higher Recommendation with problems either use different primers or make sure that the PCR is started with a hot start MRC Holland decided to use special primer blockers in combination with the PCR primers which circumvent the need for a hot start 7 3 2 6 FMRS Check 6 Probes to peaks noise percentage Background the percentage of peaks that were detected that were not recognized as MLPA fragments or probes is considered as noise Large amount of background peaks may disturb the quantification of fluorescence of other probe related peaks Large amounts of shoulder peaks may furthermore be caused by too large DNA concentrations or too high polymerase concentrations Problems are indicated when if more than 70 peak signals are detected that were not recognized as probe signals a warning will be given and 20 points will be subtracted from the FMRS In case the percentage of noise peaks is between the 40 70 a notification will be given and 10 points will be subtracted from the FMRS Recommendation with problems
62. age length over all real peaks of all used reference samples If no reference samples exist the median length over all collected real peak from all samples will be used Since some probes may have a large difference between their original and detected length the previously created results may often not suffice We therefore check if the length that we have related to each probe is applicable in our sample set We do this by calculating how much variation exists over collected peaks length in each of the previous bins If the variation was too large standard deviation gt 0 2 or no peak at all was found in any of the bins the expected peak length for that probe will be estimated by prediction The expected probe peak lengths may be predicted by using a second order polynomial regression using the available data of the probes for which reproducible data was found Even though a full collection of bins is now available the lengths of the probe products that were predicted may not be very accurate The set of bins for each probe in the selected MLPA mix will therefore be improved by iteration of the previous steps The lengths provided for the bins are now based on the previously detected or predicted probe product lengths allowing a more accurate 63 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 detection of the real probe peaks Probes that still were not found are predicted and
63. ain erson nieren tinaaa ea aa aa aaa aaia AE ES ian as 5 2 Greating a NeW Projets nseto ineat epee a a aaar aira eana naasar Bid Proe seting or n a a E en EET AR AAEREN EAE AENEA E AEE 6 About CXPeCriMe nt eeeecceceeeeeceeeeeeeeneneeee ee seenenseseeneeseseenenseseenenseseenensasesneeseseeneesneenes 6 1 What does an experiment contain ccecececeeeeeseeeeeeeeeceeeeeeeaeeseaeeseeeesaeeesaeeeeeeesaas 6 2 Creating a NEW experiment 2 2 cee ce ceeeeeeeeeceeeeeeeaeeeeeeeceeeeeceaeeeeaaeseeeeseeesaeeeeaeseneeeseas 4 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 6 3 Experiment Settings Sr aa R chien aaah ites nh A tare a En 6 4 Setting the experiment type cccceeeeeececeeeeeceeeeeeeaeeeeeeeceaeeeseaeeseaeeseeeeescaeeeeaeseeeeeeaas 6 5 Setting the channel CONteNtS ccccceeeceeeeeeeeeeeeeeeaeeeeeeeceaeeesaaeeeeaeeseeeescaeeeeaeeeeeeeeeas 6 6 Setting the channel SettingS ccccccecesceceeeeeeeeeeeeeeeceeeeceaeeesaaeseeeeeseeeesaeeeeaaeseeeeeeeas 7 About the fragment analySis ccceseeeeseseeeeeeeeeeeeeneeneeeeseenensesesneeseseenensesesnenenseenes Pi Importing the data Mes nosa ain a o T E TEE EE ERE 7 2 Starting the fragment analysis cceescceceeeeeeeeeeeeeeeceeeeeceaeeeenaeseaneeseaeesaeseeaaeeseneeseas 7 3 About the fragment analysis quality scores sssessesssessssrnsserresrrnssrirnssrirnssrnnesrnnns
64. andard deviation is estimated by combining these two 2 factors as explained at 8 4 1 By using the right mouse menu this grid may be exported to a file in csv HTML XML document or XML spreadsheet format More important by using the right mouse click sample pdf reports may be generated Coffalyser NET allows the generation of two types of pdf sample reports A single page report where all data of the three sample explorer tabs are put together in landscape modus figure 49 and a two page report which also contains extended information ratio chart Electropherograms Semple report Name Chrband hg18 Length Order 7 Signal Intraratio Preratio Ratiowoiteration Finalratio RSQ RPQ Stdev RefPop SamPop 45 EBF1 16 059333 158_ 454 001 0 96 198 37 0 01 7 0 05 j 43 EBF1 14 05q33 3 158 436 002 1246 0 99 94 1 99 0 01 0 06 0 04 j 35 EBF1 10 05q33 3 158 364 003 2271 1 07 1 04 11 06 1 03 0 06 0 04 16 J EBF1 1 05933 3 158 228 004 2000 0 96 t 95 0 92 0 02 06 0 05 j 23 IKZF1 1 07p12 2 07 050 269 005 2514 1 04 1 06 1 02 1 01 0 02 0 06 0 05 13 IKZF1 2 07p122 07 050 208 J006 3229 1 01 1 08 1 0 38 0 0 08 0 06 8 IKZF1 3 07p12 2 07 050 177 007 2784 1 01 1 09 1 0 99 0 02 0 07 0 05 22 IKZF1 4 07p12 2 07 050 263 008 2632 1 04 1 06 1 02 1 01 0 01 0 07 0 05 3 IK
65. andidate Classification Confidential Versie 0 1 2 Measure of the relative amount of signal to size drop If the relative drop is less than 10 a direct normalization will suffice any larger drop will automatically be corrected by means of regression analysis step 4 5 3 Before correction of the actual amount of signal to size drop samples are corrected for the MLPA mix specific probe signal bias This can be done by calculating the extent of this bias in each reference run by regressing the probe signals and probe lengths using a least squares method Correction factors for these probe specific biases are then computed by dividing the actual probe signal through its predicted signal The final probe wise correction factor is then determined by taking a median of the calculated values over all reference runs This correction factor is then applied to all runs to reduce the effect of probe bias due to particular probe properties on the forthcoming regression normalization 4 Next we calculate the amount of signal to size drop for every sample by using a function where the log transformed probe bias corrected signals are regressed with the probe lengths using a specialized local median least squares method Signals from aberrant targets are left out of this function by applying an outlier detection method that makes use of the results found at step 2 The signal to size corrected values can then be obtained by calculating the distance of each
66. apillary system and diffusion of the MLPA products within the capillaries You can change several settings in order to optimize the slope correction procedure 71 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Title Slope correction slope correction aims to correct the drop in fragment signal intensity to the length of each fragment that is unrelated to the number of target sequences available in each sample When this option is set to auto if gt 10 default slope correction will only occur if the difference in sloping between reference and samples is more than 10 If this difference is less than 12 sloping correction may not be required because the normalization itself will then resolve this issue You can furthermore choose to always do the slope correction or never X metric main metric that will be used for the regression analysis s on the X axis For each probe signal we can apply either the lengths or data points related to the probes Y metric by changing the Y metric you can influence whether the raw signals will be corrected or the pre normalized ratios Instead of the correcting the signals the pre normalized ratios calculated in the first normalization of all data in population mode may also be corrected and normalized afterwards Log correction of signals determines whether or not the signals used for regression analysis are first converted
67. ay find the reference samples which are normal ratio 1 for the DNA MLPA results while the SNRPN have a normal methylation status of 50 in these samples displayed by a red cell ratio 0 5 Other probes also known as digestion control probes will not have a signal at all The tab of the comparative analysis experiment explorer containing the statistical overview grid will also automatically be extended with a separate level for each sample type for all methylation results 100 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 i feo po fi i x bo Figure 52 Comparative analysis sample report grid on dual modus DNA and MS MLPA results are organized next to each other 9 4 9 4 Comparative analysis sample explorer with MS Data After the analysis is finished you can open the comparative analysis sample explorer as earlier described in chapter 8 4 We recommend viewing both DNA MLPA results and MS MLPA together to make this process easier results of coupled samples undigested to digested are listed right underneath each other Each normalized digested methylation sample result will be placed directly under the DNA MLPA results with an added tag d figure 53 101 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 AURA MLPA 227ITBU e isp roca Bernt Sente
68. ayed as red round markers figure 45c amp 45d Finally we may find results that fall within the 95 confidence range of the reference sample population but fall outside of the set arbitrary border Such contrary results are marked by a salmon colored circular marker and are also called ambiguous figure 45e amp 45f Please note that these different result stages accord to the population comparison values as described at 8 3 2 The displayed regions listen to the same functionality as described in 8 3 1 at the comparative analysis experiment explorer statistical overview chart The tool tip controls display the basic statistics for all the probes that fall within that region based on their final estimated ratios Chromosomal aberrations often span larger regions M Hermsen 2002 which allow probes targeted to that region to cluster together by sorting This data may aid in determination if all signals of the probes that fall in one region are either or decreases as opposed to a certain population In figure 49 for instance shows a case where all signals of the probes targeted to 13q14 2 are decreased with 25 The median ratio of that region was 0 75 with a standard deviation of 0 03 indicating that this sample probably contains a mixed cell population where 50 of the cells harbor a heterozygous deletion for 13q14 2 while the other 50 of the cells originate from cells that are diploid for 13q14 2 91 Title Coffalyser NET analysis manual beta
69. binning is to create a common probe length reference vector or bin While this procedure occurs completely automatically some aspects may be adjusted in the CE devices properties window under the binning tab figure 15 28 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 general base line detection peak detection binning filtering probes control frag Minimum peak amplitude RFU 70 po Maximum peak amplitude RFU 10000 J Minimum peak area to fluorescence all probes poi por Maximum peak area to fluorescence all probes poo poo Search range probes nt 3 00 2 00 Restore Default Settings OK Cancel Figure 15 Binning settings tab Specific settings can be applied for control fragments and probe fragments such as 1 Detection Intensity threshold This threshold is used to filter out peaks which may be detected in the first step described at 4 5 1 but will compete in the automatic binning procedure The minimal and maximal peak amplitudes are arbitrary units and default values are provided for each different capillary system These values are called the minimum and maximum peak amplitude RFU at the binning tab 2 Peak area ratio percentage to all probe fluorescence Peak area is computed as the area under the curve within the distance of a peak candidate Peak area ratio percentage
70. btracted in case baseline curvature is between the 30 50 only a notification is given and 15 points are subtracted Recommendation with problems in most cases baseline curvature is likely caused by a high concentration of part of the injection products which are of similar size This can be caused by inadequate mixing of the injection mixture or an injection bias e g by a too high injection voltage 7 3 2 8 FMRS Check 8 DNA concentration check Background If the DNA concentration during the MLPA hybridization was insufficient for a reliable MLPA reaction unreliable results may be produced MLPA reactions can be performed in a concentration range between the 20 500ng We assume the DNA concentration is about 10ng if the median signal intensity of the Q fragments is higher than the signal intensity of the 92 fragment 3 We furthermore assume the DNA concentration was about 5 ng if the median signal intensity of the Q fragments is higher than the signal intensity of the 92 fragment 2 In case Problems are indicated when in case the ratio of 92 ligation fragment as opposed to the median signal of the Q fragments is lower than 2 the DNA concentration was assumed to be too low and a warning will be given Even though the DNA concentration is evaluated separately it will also affect the FMRS warnings will minimize the FMRS with 60 points A warning will be given if the ratio of the 92 ligation fragment as opposed to the median signal of the Q
71. by INSC 1 injection voltage by INVT 1 capillary length by LNTD 1 run voltage by LSAP 1 capillary number by LANE 1 plate size by 96 Well run protocol by RPRN 1 used size standard by STDF 1 run temperature by TMPR 1 tube position by TUBE 7 and user name by USER 1 overview raw data fragment profile genomic profile sizing curve binning fragment data instrument data P335 A2 lot 0809 Promega F HYI DNA 7 SS7AKZF 1 Save as Copy to Clipboard Page Setup Print Preview Print Auto Zoom Reset Zoom 310 RB1 14 SMA A ONAL 2235 23528 IERES IONS SgnIr 9e 2 D9 se BE 22 220 908 27929 2358 3229388822733238 3538 3 5335883683737252 FFF DataPoint Figure 30 Fragment results explorer genomic profile tab Each tab of the fragment results explorer has several options that can be found in the right click menu figure 30 You can for instance view each channel independent show or hide legends save images in a wide variety of formats copy to clipboard print and make a print setup and use automatic zooming functions Automatic zooming allows 3 levels of zoom this being show all detected peaks show all recognized peaks and show all peaks 62 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 recognized as MLPA test probes You may furthermore use manual zooming by clicking anywhere in the chart and
72. candidate Classification Confidential Versie 0 1 1 Background 1 1 Introduction to MLPA normalization MLPA kits generally contain about 40 50 oligo nucleotide probes targeted to mainly the exonic regions of a single or multiple genes The number of genes that each kit contains is dependent on the purpose of the designed kit Each oligo probe consists of two hemi probes which after denaturation of the sample DNA hybridize to adjacent sites of the target sequence during an overnight incubation For each probe oligo nucleotide in a MLPA kit there are about 600 000 000 copies present during the overnight incubation An average MLPA reaction contains 60 ng of human DNA sample which correlates to about 20 000 haploid genomes This abundance of probes as compared to the sample DNA allows all target sequences in the sample to be covered After the overnight hybridization adjacent hybridized hemi probe oligo nucleotides are then ligated using a ligase enzyme and the ligase cofactor NAD at a slightly lower temperature than the hybridization reaction 54 instead of 60 The ligase enzyme used L igase 65 is heat inactivated after the ligation reaction Afterwards the non ligated probe oligonucleotides do not have to be removed since the ionic conditions during the ligation reaction resemble those of an ordinary 1x PCR buffer The PCR reaction can therefore be started directly after the ligation reaction by adding the PCR primers polymerase and d
73. change these settings figure 13 24 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Baseline moving median points marker Baseline moving median points probes rough Baseline moving median points probes fine Baseline maximum signal for correction fine Figure 13 Baseline settings 4 5 2 Peak detection settings In capillary based MLPA data analysis peak detection is an essential step for subsequent analysis Even though various peak detection algorithms for capillary electrophoresis data exist most of them are designed for detection of peaks in sequencing profiles While peak detection and peak size calling are very important processes for sequencing applications peak quantification is not so important Due to the relatively nature of the MLPA data peak quantification is particularly important and has a large influence on the final results Our peak detection algorithm exists of two separate steps the first step exists of peak detection by comparison of the intensities of fluorescent units to set arbitrary thresholds and shape recognition the second step exist of filtering of the generated peak list by relative comparison 25 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 are tase tne aeecin kane tee Auto detect parameters E marker
74. description with the available products in the sheet manager Each product developed by MRC Holland has a P number for copy number products a M number for products that can be used for detection of methylation status or a R number indicating products that can be used for quantification of RNA sequences To view the list with all available products right click on the on Sheet library in the database explorer and select Open from the right click menu figure 4 Next the currently active Coffalyser Work Sheets will open By selecting Ada from the right click context menu you can create a new 15 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Coffalyser Work Sheet based on an existing MRC Holland MLPA product or create an empty work sheet starting from scratch figure 7 LOTO410 A2 2012 03 14 12 34 LOT0410 A2 2012 03 14 12 34 LOTO711 X2 2012 03 16 11 49 LOT1010 A1 2012 03 14 17 10 LOTO811 2012 03 15 14 01 LOT1010 X1 2012 03 15 19 22 Figure 7 Coffalyser NET sheet library product overview window 3 4 Viewing the available products lots and active sheets To view a product first add it to the Coffalyser Work Sheet library by using the right click context menu After you add a product the Coffalyser Work Sheet Editor will automatically open allowing you to make the necessary changes or view if the content are as expected At the moment
75. dragging the mouse over the area you wish to zoom into Exact details about control over charts and grid is described in the chapter Context Menus 7 6 Creating a manual bin set for data filtering Once all peaks have been size called the profiles must be aligned to compare the fluorescence of the different targets across samples an operation that is perhaps the single most difficult task in raw data analysis Peaks corresponding to similar lengths of nucleotides may still be reported with slight differences or drifts due to secondary structures or bound dye compounds These shifts in length make a direct numerical alignment based on the original probe lengths all but impossible Our software uses an algorithm that automatically considers what the same peaks are between different samples allowing easy peak to probe linkage this procedure is called Auto binning Our software algorithm follows four steps reference profile analysis applying and prediction of new probe lengths reiteration of profile analysis and data filtering of all samples The crucial task in data binning is to create a common probe length reference vector or bin In the first step our algorithm applies a bin set that searches for all peaks with a length closely resembling that of the design length of that probe Next the largest peak in each temporary bin is assumed to be the real peak descending from the related probe product To create a stable bin we calculate the aver
76. e maximum a warning will be given and 15 points will be subtracted from the FRSS Notifications will be given in case the maximum peak signal intensity is between the 70 and 87 5 of the machines absolute maximum and 10 points will be subtracted from the FRSS total Fr an ABI 3130xl the maximum peak intensity is set at 5600 for a notification and at 7000 units for a warning Recommendation with problems adjust the concentration of the size marker in the injection mixture to increase or decrease the signal intensities of the marker 7 3 1 4 FRSS Check 4 Signal drop of the internal run of the size marker fragments Background size markers are usually developed having fragments concentrations in equal amounts of all peaks the reproducibility of the separation method may thus be examined by evaluating the intensity of the size marker fragments In addition the presence of the same multiple bands in several lanes in different regions of the gel provides information regarding possible lane to lane variation in the electrophoresis migration of sample material Most markers gs 500 600 CEQ are designed to give equal signal intensities over all fragments A drop in signal of the fragments is thus probably introduced by the capillary electrophoresis and will also have a similar effect on the MLPA probes Problems are indicated when To make sure that the signal drop is caused by a problem during the separation we combine a check on the drop of signals
77. e samples the analysis settings and the composition of your computer the analysis may take as little as 10 seconds while large experiment may take several minutes 8 2 About the comparative analysis quality scores After the analysis is finished you will be confronted with a number of quality scores that may indicate the quality of the normalization slope correction and overall analysis quality of each sample figure 36 Evaluation of the comparative analysis quality scores should be done as described in 7 3 Here we describe the meaning of the different displayed scores 76 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 8 2 1 PSLP Check 1 Pre normalization signal sloping probes displays the relative amount of signal to size drop of the probe fragments of a sample as opposed to the reference See also 7 3 4 FMRS Check 4 Signal drop of the internal run of the probe fragments 8 2 2 FSLP Check 2 Final normalization signal sloping probes displays the relative amount of signal to size drop of the probe fragments of a sample as opposed to the reference after the signal have been corrected for signal to size sloping effects See also 7 3 4 FMRS Check 4 Signal drop of the internal run of the probe fragments This measurement checks if performed slope correction method was successful 8 2 3 RSQ Check 3 Reference sample quality displays if relative probe signal
78. e sloping is accompanied by peak widening of more than 50 another penalty will be given to the FRSS total In case of a severe warning another 30 penalty points will be given in case of a warning 20 extra penalty points will be given and in case of a notification in combination with peak widening 10 extra penalty points are subtracted from the FMRS Recommendation with problems rerun with alternative injection settings If the size to signal drop is clearly not linear and not visible in the size marker redoing the MLPA reaction with a lower sample volume or cleaned up sample may provide better results 7 3 2 5 FMRS Check 5 Percentage of unused primer Background in a successful MLPA reaction more than 70 of the added primer should be incorporated in probes Often a lot of the available primer is caught away before the start of the MLPA reaction either by contaminants or DNA fragments that have complimentary sequences to one or both used 56 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 primers If more than 30 of the primer is unused it may cause a drop in signal and thus can give unreliable results A large primer complex peak should be seen in the shorter length region of the profile Problems are indicated when to measure if there is too much primer left we compare the fluorescence of the primer to the total amount of fluorescence of the probe peaks In case
79. ected you should select the correct filter set that matches the chemistry you are using figure 12 The filter sets defines what fluorescent dyes certain channels in the machine recognize Table 1 contains the most common filter sets used by ABI If for instance you are using a FAM label for the probes and you are using LIZ for a size marker then you need to select filter set G5 base line detection peak detection binning filtering created by modified by CE device ABI 3130XL CE device fiter lt select a CE device fiter gt Restore Default Settings OK Cancel Figure 12 Choosing your filter set 22 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Table 1 Filter sets used by ABI capillary devices Ds 29 5 FAM ko Ds 34 FAMIN TAMRA DS 30 ed FAM O LIZ DS 02 dR110 dR6G dTAMRA dROX 4 5 Changing the CE devices settings By going through the different tabs of the CE Device properties window you will be able to change the different device specific analysis settings There are four types of settings that you may change which are baseline settings peak detection settings binning settings and filter settings When you are working in a specific organization CE device settings will be applied for all users that are working in that organization In that case you need to be
80. eir own characteristics and the level of increase or decrease that a probe ratio displays that was targeted to a region that contains a heterozygous gain or loss may differ for each probe Interpretation of normalized data may even be more complicated due to shifts in ratios caused by sample to sample variation such as dissimilarities in PCR efficiency and size to signal sloping Other reasons for fluctuations in probe ratios may be poor amplification misinterpretation of an artifact peak band as a true probe signal incorrect interpretation of stutter patterns or artifact peaks contamination mislabeling or data entry errors Bonin et al 2004 To make result interpretation more reliable our software combines effect size statistics and statistical interference allowing users to evaluate the magnitude of each probe ratio in combination with it s significance in the population The significance of each ratio can be estimated by the quality of the performed normalization which can be assessed two factors the robustness of the normalization factor and the reproducibility of the sample reactions In this document we show the features and integrated analysis methods of our novel MLPA analysis software called Coffalyser NET Our software uses an analysis strategy that can adapt to fit the researcher objectives while considering both the biological context and the technical limitations of the overall study We use statistical parameters appropriate to
81. ement of that project 10 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 2 3 Connecting to a local database After installing Coffalyser NET on a computer in a standalone configuration the first screen you will find each time you start the program in the user login screen If you did not install Coffalyser NET yet please find installation instruction in the Coffalyser NET installation manual To login to your local database make sure that the configuration is set to Single PC Standalone next fill in the user name of the account you have created during the installation and the password and click on Login configuration Stand alone user name Admin password paa reset close all other sessions for this user Figure 2 Coffalyser NET login form for single PC standalone configurations If your login fails please check the configuration settings of your database To change or check your configuration click on the Windows Start button then navigate to All programs and search the entree Coffalyser NET From the Coffalyser NET program menu click on Configure Coffalyser NET 2 4 Connecting to a server database If you have installed Coffalyser NET on a computer in a client server configuration the login procedure will be similar to that described at 2 3 the configuration settings however will differ Make sure that the
82. endation with problems Remove the capillary array at the manifold end and clean the capillary window Use sterile water DO NOT USE METHANOL Clean in one direction only Use Direct Control from the Run application to purge the manifold and to fill the capillaries with new gel and then clean the capillaries 7 3 2 4 FMRS Check 4 Signal drop of the internal run of the probe fragments Background An effect that is commonly seen with MLPA data is a drop of signal intensity that is proportional with the length of the MLPA product fragments This signal to size drop is caused by a decreasing efficiency of amplification of the larger MLPA probes and may be intensified by sample contaminants or evaporation during the hybridization reaction Chemical remnants from the DNA extraction procedure and other treatments sample tissue was subjected to may allot to impurities that influence the Taq DNA 55 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 polymerase fidelity Alternatively target DNA sequences may have been modified by external factors e g by aggressive chemical reactants and or UV irradiation which may result in differences in amplification rate or extensive secondary structures of the template DNA that may prevent access to region of the target DNA by the polymerase enzyme Elizatbeth van Pelt Verkuil 2008 Signal to size drop may further be influenced by in
83. eotides To add a new product start at the sheet library product overview window right click anywhere in the window and then select Add product from the right click menu This will open product lot details window allowing you to adjust the organization this sheet belongs to the control fragments this mix contains and the default analysis method The second tab probes can then be used to add the probes to this mix and add the necessary details about these probes 19 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 4 About CE devices 4 1 What are the CE devices CE devices within Coffalyser NET are considered to be the capillary electrophoresis devices you are using to separate the MLPA products Coffalyser NET fragment analysis settings are specific for that machine and optimized settings for each known device are provided by defaults Since detection of fluorescent units occurs on arbitrary scales and the measured intensities may also differ even from device to device even of the same type peak detection settings may often require empiric optimization Each organization can therefore create a CE devices within the software that can be related to an actual device in your laboratory When such a device is created it will be loaded with the default fragment analysis properties provided by MRC Holland These settings will suffice in most cases but in some cas
84. erence samples Sort Data gt Region analysis Save as Copy to clipboard 05 158071810 05 158057828 Page setup Print preview 2116943284 2 11796704 4 755205 X 770580 X 13883864 X 1415701 4 02 2422547394 09 219958134 2 11694211 4 X 12812744 03 38591793 4 05 13876361 05 90056461 4 05 1320376124 07 727551154 11 1536655 11 20606070 15 46584530 20 25197773 4 09 36830475 09 36872066 09 36956622 07 50315024 4 07 50329206 4 07 50411797 4 07 50417749 07 50422507 07 50426950 07 50436025 4 09 21957822 09 21964958 09 36992699 09 37010669 09 37024272 07 50337794 05 158137017 05 158459187 hg18 location Figure 38 Comparative analysis experiment explorer statistical overview chart showing descriptive statistics of the reference samples The following features may be selected 1 Distribution type changing the distribution type results in the display of the descriptive statistics of all samples of a sample type By displaying results of the reference samples you can easily evaluate the reproducibility of each probe in that experiment assuming the reference samples were genetically equal and the reference samples were properly dispersed through the experiment figure 32 Title Status Classification Versie 80 Coffalyser NET analysis manual beta version Release candidate Confidential 0 1 2 X axis by selecting a different field for t
85. erent vertical stripes or color bands indicate which probes fall within a certain region On default the chart will load placing all probes within one region that are located on the same chromosome arm Other regions include chromosome 78 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 chromosome band or user defined regions On default information on user defined regions are filled in by MRC Holland All results are furthermore organized according to the MRC Holland recommended order In practice this often means that test probes and reference probes are separated and sorted by the hg18 tracks if no recommended order exists results will automatically organized by the hg18 tracks All samples s D 3 i ajia eee e ee eo e ee Figure 37 Comparative analysis experiment explorer statistical overview chart Results of all samples of the same sample type for each probe are displayed by a box plot also known as a box and whisker diagram or plot graphically depicting the results in groups of numerical data through their five number summaries the smallest observation theoretical minimum lower quartile Q1 median Q2 upper quartile Q3 and largest observation theoretical maximum A box plot may also indicate which observations if any might be considered outliers The quartiles of a set of values are the three p
86. es manual optimization of these settings is required 4 2 Adding a new machine Our software is compatible with binary data files produced by all major capillary electrophoresis systems including ABIF files FSA AB1 ABI produced by Applied Biosystems devices SCF and RSD files produced by Megabace systems Amersham and SCF and ESD files produced by CEQ systems Beckman Before any data import can be performed we first need to define what machines are being used in your laboratory Right click in the Coffalyser NET database exploration window on the CE Devices and select Add CE Device from the right click menu 20 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Figure 10 Adding a new CE device to Coffalyser NET 4 3 Choosing the correct machine After selecting Add device a new window will open allowing you to define which capillary electrophoresis device your are using figure 11 Next to CE device choose the machine you wish to use form the dropdown list If you are unsure what machine you are using please contact your provider Figure 11 Choosing your machine type 21 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 4 4 Choosing the correct filter set After the correct machine was sel
87. esent in the raw data can usually be resolved by adjusting the peak detection settings of the marker 7 3 1 2 FRSS check 2 Baseline of the size marker channel 48 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Background high baseline levels can lead to erroneous base calling and short read lengths High baselines furthermore decrease the dynamic range of detection of that channel Optimal performance of the capillary system is achieved when the baselines for all channels are below 5 of the maximum detectable intensity Problems are indicated when in case the measured average baseline or signal intensity of the size marker specific dye stream without running fluorescent products is above 10 of the maximum intensity of the machine a warning will be given and 15 points subtraction on the FRSS total In case the baseline is between the 7 and 10 only a notification will be given and 10 points will be subtracted from the FRSS total For an ABI 3130xlI for example the maximum baseline intensity for the marker is set at 700 units for a warning and 560 units for a notification Recommendation with problems Remove the capillary array at the manifold end and clean the capillary window Use sterile water DO NOT USE METHANOL Clean in one direction only Use Direct Control from the Run application to purge the manifold and to fill the capillaries with new gel and
88. etermined over the reference probes is determined during the MS MLPA normalization It should also be noted that Coffalyser NET allows a separate reference probe selection for DNA MLPA normalization and MS MLPA normalization For more information about how to change the reference probes used for the different normalization please see chapter 3 about the MRC MLPA sheet manager 9 2 Setting up the MS MLPA analysis 98 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 To perform a MS MLPA analysis first set the experiment type to DNA MS MLPA as explained earlier at 6 3 Next import all your samples as you would normally do however when settings the sample types set every digested sample to the sample type digested as explained in chapter 6 4 Perform the fragment analysis and explore the fragment data as described earlier in chapter 7 and 8 Please note during data exploration that digested target sequences are expected to be absent and that these sample runs are therefore expected to have a lower normal number of peaks as compared to their undigested counterparts Following the fragment analysis you need to match the digested and undigested samples in the comparative analysis settings screen Right click to open the context menu and select digested samples for MS MLPA and then select match samples automatically figure 51 This will enable a matching
89. ex ligation dependent probe amplification MLPA BMC bioinformatics 9 261 10 Hermsen M Postma C 2002 Colorectal adenoma to carcinoma progression follows multiple pathways of chromosomal instability Gastroenterology 123 1109 1119 11 Holtzman NA Murphy PD Watson MS Barr PA 1997 Predictive genetic testing from basic research to clinical practice Science journal 278 5338 602 5 12 Huang C H Chang Y Y Chen C H Kuo Y S Hwu W L Gerdes T and Ko T M 2007 Copy number analysis of survival motor neuron genes by multiplex ligation dependent probe amplification Genet Med 4 241 248 106 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 13 Janssen B Hartmann C Scholz V Jauch A and Zschocke J 2005 MLPA analysis for the detection of deletions duplications and complex rearrangements in the dystrophin gene potential and pitfalls Neurogenetics 1 29 35 14 Kluwe L Nygren A O Errami A Heinrich B Matthies C Tatagiba M and Mautner V 2005 Screening for large mutations of the NF2 gene Genes Chromosomes Cancer 42 384 391 15 Michils G Tejpar S Thoelen R van Cutsem E Vermeesch J R Fryns J P Legius E and Matthijs G 2005 Large deletions of the APC gene in 15 of mutation negative patients with classical polyposis FAP a Belgian study Hum Mutat 2 125 34 16 Nakagawa Shinich
90. falyser NET sheet manager ccssccssecceseeeseseeseeeseseeeeneeeeeeeeeeneeeeeas 3 1 What is the Coffalyser NET sheet Managel ccccceceeeeeeeeeeeeneeteeeeeteaeeeeaeeeeeeeseeeeees 3 2 Downloading new sheets cccceeeeeeeceeeneeeeeeeeeeeeeeeeaeeeeeneaeeeeeeaaeeeeeaaeeeseeaaeeeeseaaeeeeeeaas 3 3 About products lots and VErSiOn cccccccecceseeeecceceeeeeeeeecaaeaeceeeeesesecaaeaeeeeeeeeesiaeeeeess 3 4 Viewing the available products lots and versions eeeeeceeeeeeeeeeeeneeeeeeeaeeeeeeaeeeeeeaas 3 5 Adjusting a sheet lote ciasaeisct etd di eee ELE ind aie 3 6 Adding a new custom product to the sheet manager c ccccceeeseceeceeeeeeestteeeeees 4 ADOUUCE deV eE esc co cc ceeecs a araa ce raea teed cuca ah Aa eo ea aa eaaa Rea aaaeeeaa 4 1 What are the CE devices neeseesseeseessseessesriesrietrintrintrtnttnnttnntnsennsnnntnstnnsensnnns 4 2 Addng anew Machines ses Audit ada nt aaa nl as aes 4 3 Choosing the Correct machine ccccceeeceeeeeeeeeeeeeeeee certs sae eeeaaeseeeeeseaeeesaeeteaeeseeneees 4 4 Choosing the Correct filter set ceeececeeeceeeee cesses eeeeeeceaeeeeaaeeeeeeeseeeesaeeesaaeseeaeeeeeeeess 4 5 Changing the CE devices settings cc cccceeeeeeeeceeeeeeeeeeeeaeeeeeeeseeeeseaeeeeaeeeeeeseeeeess 5 About projects n22 cccaccctccteccsecccceectecet cc tectedes stcececedecucdvectccdteesscacees stcacecghetccetesdteceeedsecces 5 1 What does a project com
91. ferences Most information has been directly copied from the following book chapter which can be viewed freely online Coffa J 2011 Analysis of MLPA data using novel software by MRC Holland Coffalyser NET Intech open acces publishing http www intechweb org 1 Ahn J W 2007 Detection of subtelomere imbalance using MLPA validation development of an analysis protocol and application in a diagnostic centre BMC Medical Genetics 8 9 2 Albert J 2007 Bayesian Computation with R Springer New York 3 Applied Biosystems 1988 AmpFf STR Profiler Plus PCR Amplification Kit user s manual 4 Bickel Peter J Doksum et al 2001 Mathematical statistics Basic and selected topics 1 5 Clark J M 1988 Novel non templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases Nucleic Acids Res 16 20 9677 86 6 Coffa J 2008 MLPAnalyzer data analysis tool for reliable automated normalization of MLPA fragment data Cellular oncology 30 4 323 35 7 Ellis Paul D 2010 The Essential Guide to Effect Sizes An Introduction to Statistical Power Meta Analysis and the Interpretation of Research Results United Kingdom Cambridge University Press 8 Elizatbeth van Pelt Verkuil Alex Van Belkum John P Hays 2008 Principles and technical aspects of PCR amplification 9 Gonzalez J 2008 Probe specific mixed model approach to detect copy number differences using multipl
92. figure 45b If found results did not meet the criteria of 6 or 7 then we will check if found probe results has a significant different to the reference sample population without employing the magnitude of the probe ratio Cells will be colored 2 tint lighter red if the 95 confidence range of the probe did not overlap with the 95 confidence range of that probe in the reference sample population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol lt lt Yellow with red text or lt lt if a probe could not be related to any peak then the cell will be colored bright yellow It s always recommended to confirm these kind of results by evaluation of the raw electropherogram to ensure that the signal is actually gone By using the right mouse click menu you may find the following options 1 2 Title Open sample results this will open the sample explorer explained in the next chapter allows a more detailed exploration of all data on this sample Region analysis changing the region analysis allows you to change what probes are sorted together and obtain the same default cell color Applicable region settings are chromosome chromosome arm chromosome band or custom region numbering Show data type this option allows you to change the data of the main grid You can select RNA to display the intra normalized ratios It should be noted that this op
93. gnal 2 Depurination of the sample DNA in the rest of the sequence detected by the probe can result in destabilization of the binding of the probe oligonucleotide to the sample DNA resulting in a lower signal 7 3 2 2 FMRS check 2 maximum probe signal Intensity of the sample Background high probe signal intensity in a few cases the signal strength can be so high that it saturates the detector This can lead to an erroneous base call where the software will artificially estimate peak height and position In this case the software inserts extra bases into the base sequence By setting the raw data to full scale CEQ 137 000 counts and looking at the peak shapes the user can determine if peaks are over ranged If the peaks are squared off at the top then the detector is saturated and the peaks are over ranged Problems are indicated when next to the median probe signal intensity we also check the signal intensity of the highest probe signal A probe signal that is completely off scale may give a wrong ratio as compared to the reference and should thus be treated with caution In case the highest probe signal surpasses 95 a warning is given and 15 points are subtracted from 54 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 the FMR S In case this signal is between the 90 95 only a notification is given and 10 points are subtracted from the FM
94. grations of the size marker fragments in data point or time with the expected fragment lengths of the used size marker in nucleotides We can use the technique of correlation to test the statistical significance of the association In other cases we use regression analysis to describe the relationship precisely by means of an equation that has predictive value We deal separately with these two types of analysis correlation and regression because they have different roles The Correlation can be calculated by D a 2 u 9 ae n 1 sa5y Problems are indicated when without a good correlation no proper length estimation of unknown peak fragments can be performed We therefore require a correlation of minimal 0 999 to continue with size calling If case the correlation is lower than 0 999 size calling may still be successful but probe lengths may deviate more than 0 5 nt from their corresponding partners in other capillaries For correlations lower than 0 999 a subtraction will therefore be executed of 80 points resulting in a direct fail of this run Recommendation with problems check the detected size marker peaks pattern and the peak detection settings for the marker If peaks are not present or similar peaks exist in the pattern that are undistinguishable from the original size marker peaks samples need to be rerun Problems with peaks that are detected and should not have been detected or peaks that are not detected but are pr
95. green asterisk markers represent the peak end Above each peak top the estimated size called length is also displayed By hovering over the peak top markers the tool tip information will appear showing the exact peak start top and end data points the peak height and the peak area To make optimization of peak detection settings easier the set minimal maximum RFU and peak area of the probes channels are displayed as line series Genomic profile displays the baseline corrected signals of each dye data streams that was set as a probes or size marker channel Displayed patterns will also show which peak signals were identified as a probes labels furthermore show the design length of each probe gene name and exon number The coordinates of the peak top of the peak that was recognized as the main peak related to a probe contains a green circular marker in case the probe is a reference probe and a purple circular maker in case it is a test probes Fragment analysis steps on this page you view each of the fragment analysis steps by using the right mouse context menu You can also navigate through the steps by using the key combination ctrl shift number key 1 10 Binning displays the peak heights on the y axis and the estimated peak length of each detected peak on the x axis Next to this using green and red stripes the bin set that was used for data filtering is displayed In case a peak signal was found that meets the
96. gure 25 Fragment analysis settings As discussed earlier but also in the coming chapter about manual binning Coffalyser a window or panel based approach to link peaks with comparable lengths to the same probe In order to define these panels or bins we need to compare the peak information we have with what is expected This is done automatically with an auto bin procedure The more the lengths are comparable to the found lengths the more chance that the procedure will find all probes successfully Coffalyser allows two types of probe length to be used for the auto bin procedure the probe design length which are the real length of the fragments and the Coffalyser lengths Coffalyser lengths are lengths that are filled by MRC Holland to make the binning procedure more successful since they are based on the detected lengths found during the quality tests Finally you can also filter you data based on a manual bin set by selecting the manual option How to create a manual bin set is explained in the section creating a manual bin set for data filtering 43 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Fragment Analysis Settings peak recognition probe mix alignment auto bin minimal number minimal correlation of points start value found path 10 999 ho similarity matris 10 999 3 path retrieval 10 999 3 0 ig
97. he X axis you may display probe length gene name gene exon number gene name gene exon number hg18 track location chromosomal band or probe length Series label allows display of the same info described at point 2 4 Sort data change the order of the probes sort either by recommended order hg18 tracks chromosomal band or probe lengths Sorting on probe length allows you to see if there was a general trend of signal sloping within this data set 5 Region analysis changing the region analysis allows you to change what probes are harboring together in a stripe This requires some calculation since you may also find a statistical description of all probe results that fall in that region by hovering above any of the stripes or regions blue arrow figure 31 6 Other options include the option to save images in a wide variety of formats copy to clipboard print print setup and print preview 7 Channels enables or disables the results of the data found for each channel that was set as a probe mix channel This option works only in multichannel modus also see the advanced analysis section 8 3 2 Comparative analysis experiment explorer heat map grid The second tab shows the ratio results of all samples for each probe in a sorted grid The probes are displayed on the rows while the columns may contain a number of hierarchies Hierarchical levels allow you to hide or show information by clicking on the plus sign in the
98. he button called Start fragment analysis to perform the all necessary steps to qualify and quantify each of the probe signals The screen will automatically update and present the quality scores for each sample after the analysis is finished 7 3 Fragment analysis settings After you click on the fragment analysis button the fragment analysis settings screen will open figure 25 This screen will allow you to change the main advanced fragment analysis settings that are unrelated to the CE device settings The form consists of three pages related to different processes of the fragment analysis First tab contains all settings related to the peak recognition at the top you can set whether to use a basic baseline correction or an advanced baseline detection method The exact differences and effect can be viewed in the fragments explorer at the fragment analysis steps tab as discussed later in this chapter Enabling the advanced baseline method has to effect that the peak detection method is repeated twice First baseline correction and peaks detection are performed as discusses earlier following this the process is repeated the second baseline correction however will now be made based on all the signals that are known to be unrelated to 41 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 peaks When the baseline gets to an area known to be related to peak the underlyi
99. he data In MLPA kits 7 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 so called reference probes are usually added which may be used in multiple ways in order to comprise a common variable Reference probe are usually targeted to chromosomal regions that are assumed to remain normal diploid in DNA of applicable samples The results of data normalization are probe ratios which display the balance of the measured signal intensities between sample and reference In most MLPA studies comparing the calculated MLPA probe ratios to a set of arbitrary borders is used to recognize gains and losses Gonzalez 2008 Probe ratios of below 0 7 or above 1 3 are for instance regarded as indicative of a heterozygous deletion copy number change from two to one or duplication copy number change from two to three respectively A delta value of 0 3 is a commonly accepted empirically derived threshold value for genetic dosage quotient analysis Bunyan et al 2004 Since chromosomal aberrations often span larger regions ordering probe data by Map View locations NCBI Map view version 36 results in clustering of probes targeting the same region Aberrations can be recognized more easily this way and probes targeting the same region may confirm each other s result This criterion alone may often not provide the conclusive results required for diagnosing disease MLPA probes all have th
100. hod called size calling Size OT Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 calling is a method that compares the detected peaks of a MLPA sample channels against a selected size standard Lengths of unknown probe peaks can then be predicted using a regression curve between the data points and the expected fragment lengths of the used size standard resulting in a fragment profile Once all peaks have been size called the profiles must be aligned to compare the fluorescence of the different targets across samples an operation that is perhaps the single most difficult task in raw data analysis Peaks corresponding to similar lengths of nucleotides may still be reported with slight differences or drifts due to secondary structures or bound dye compounds These shifts in length make a direct numerical alignment based on the original probe lengths all but impossible Our software uses an algorithm that automatically considers what the same peaks are between different samples allowing easy peak to probe linkage This procedure follows a window based peak binning approach whereby all peaks within a given window across different samples are considered to be the same peak Our software algorithm follows four steps reference profile analysis applying and prediction of new probe lengths reiteration of profile analysis and data filtering of all samples The crucial task in data
101. i Cuthill Innes C 2007 Effect size confidence interval and statistical significance a practical guide for biologists Biological Reviews Cambridge Philosophical Society 82 4 591 605 17 NCBI Genes and Disease NIH National Center for Biotechnology Information 2008 18 Redeker E J de Visser A S Bergen A A and Mannens M M 2008 Multiplex ligation dependent probe amplification MLPA enhances the molecular diagnosis of aniridia and related disorders Mol Vis 14 836 840 19 Schouten J P 2002 Relative quantification of 40 nucleic acid sequences by multiplex ligation dependent probe amplification Nucleic Acids Research 20 12 e57 20 Scott R H Douglas J Baskcomb L Nygren A O Birch J M Cole T R Cormier Daire V Eastwood D M Garcia Minaur S Lupunzina P Tatton Brown K Bliek J Maher E R and Rahman N 2008 Methylation specific multiplex ligation dependent probe amplification MS MLPA robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation J Med Genet 45 106 13 21 Sequeiros Jorge Guimaraes Barbara 2008 Definitions of Genetic Testing EuroGentest Network of Excellence Project 22 Taylor C F Charlton R S Burn J Sheridan E and Taylor GR 2003 Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non polyposis colorectal cancer identification of novel and recurrent deletions by MLPA Hum M
102. ies the relative probe strengths are expected to be equal In some circumstances reliable results can be obtained by comparing unknown samples to reference samples simply by visual assessment This can be done best by overlaying two fragment profiles and comparing relative intensities of fragments figure 1 N Figure 1 MLPA fragment profile of a patient sample with Canavan disease top and that of a reference sample bottom Canavan disease is the result of a defect in the ASPA gene on chromosome 17p13 The fragment profile shows that the probe signals targeted to exon 1 6 of the ASPA gene have a 50 decrease as compared to the reference which may be the result of a heterozygous deletion It may however not be feasible to obtain reliable results out of such a visual comparison if 1 The DNA quality of the samples and references is incomparable 2 The MLPA kit contains probes targeted to a number of different genes or different chromosomal regions resulting in complex fragment profiles 3 The data set is very large making visual assessment very laborious 4 The DNA was isolated tumor tissue which often shows DNA profiles with altered reference probes To make complex MLPA data easier understandable unknown and reference samples have to be brought on a common scale This can be done by normalization the division of multiple sets of data by a common variable in order to cancel out that variable s effect on t
103. ill be colored based on their population comparison value as described earlier Results may however also be compared to the test sample population or positive sample population Resize column width by resizing the column widths you may fit more or less sample data on your screen Columns widths may be changed directly to 25 50 100 or 150 points or may be auto resized to fit the sample name of each column Alternatively the width can be changed gradually by increasing or decreasing with steps through the menu or by using the short cut CTRL SHIFT Plus or Minus key Resize row height by resizing the row height you may fit more or less probe data on your screen Row heights may be changed directly to 8 10 15 or 20 points Alternatively the height can be changed gradually by increasing or decreasing with steps through the menu or by using the short cut CTRL ALT Plus or Minus key Export grid exports the displayed grid data to a file in csv HTML XML document or XML spreadsheet format Export pdf overview creates a quality control list of each sample with their quality control scores see chapter 7 3 and 8 2 in pdf format figure 41 and creates a pdf overview of all samples final ratio results figure 42 In this document result cells become bold if they do not fall within the set arbitrary border and obtain one or two asterisk symbols behind the value is the result is different than 68 1 or 95 respectively different fro
104. in the previous analysis This method works best in case you have a large sample collection and no reference probes and you do not have any background information about the samples Experiment reference probe filter this filter adapt the reference probes influences the way the reference probes are selected in the next round of normalization The filter uses the statistical results as found over the combined samples of the types reference sample and test sample Depending on the type of filter the effect of each settings should be combined with the Experiment probe reference filter low high and the Experiment reference probe std dev filter medium high In case the filter is set to low medium high or incremental the probes that have an average ratio as calculated over the reference samples or over all test samples that is outside the Experiment probe reference filter will NOT be used as reference probes In case the filter is set to medium the probes that have a standard deviation as calculated over the reference samples or over all test samples that is higher than the Experiment reference probe std dev filter medium value 0 2 at default will NOT be used as reference probes In case the filter is set to high the same rule applies but now the standard deviation is compared the maximal values set under high 0 1 at default Extend reference probe collection in case this option is enabled the selection of reference probes is e
105. inconsistencies existed in the selected reference sample population The amount of variation is estimated by measuring the standard deviation over the calculated final normalized dosage quotients of each probe over all the reference samples 8 2 4 RPQ Check 4 Reference probe quality displays if relative reference probe inconsistencies existed in the complete sample population The amount of variation is estimated by measuring the standard deviation over the calculated ratios which are generated when a probe is normalized against each separate reference probe during each sample to reference normalization 8 2 5 CAS Check 5 Coffalyser analysis score displays the quality of the complete analysis of a sample that comprises all quality points calculated during the fragment and comparative analysis into a single score 77 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 FMRS X Y_ analy CAS PSLP FSLP RSQ RPQ ll F F 4 ull o O O O ill f f ull o O O O ill F Y F oil 6 O O ill F X A ull o 6 O O ill F X ull o 6 O O ill X ull o O O O d X ull 6 O O O il x E ull o O O il Y X a ull oOo 6 O O ll f y ull O O O ill ull o 0O O O dl F X a ull Open Sample Results 4 x E d Open Experiment Results il X a ull EAE FERJE F Select Samples For Comparative Analy ence ill x ull 6
106. increase the minimal peak detection thresholds If the peaks are clearly visible and may cause problems with the quantification of other probe related peaks then the product separation should be repeated or the MLPA reaction 7 3 2 7 FMRS Check 7 Baseline curvature Background next to normal baseline heightening baseline curving may also occur Baseline curving is a heightening baseline at a local spot most of the times directly below the probe signals Most of this signal originates from the probe products but are often not proportionally to the rest of the run Our baseline correction method resolved this issue by cutting the baseline through this curve which resolves the issue in most cases Baseline 57 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 curvature however may still influence the peak height and area of all probe signals in that area By measuring the fluorescence underneath the probes by a Zero Baseline which can be created by plotting a regression line through the data points in the beginning and end of each run and comparing this to the fluorescence underneath the probes with our actual baseline we may find how much curvature there exists This thus also indicated how much fluorescence we removed by not applying a straight baseline Problems are indicated when if more than 50 of baseline curvature exists a warning is given and 40 penalty points are su
107. ined from earlier steps during the analysis By using the different tabs users may influence the parameters for the different steps of the comparative analysis On the first tab we find the basic normalization settings 1 Normalization metric the normalization metric is the system of measurement of each detected probe that will be used during normalization If this option is set to Calculate best signal to noise 69 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Title default each possible probe metric will be compared to each other and the metric showing the highest signal as opposed the amount of noise will be used for normalization Users may furthermore choose if they want to use peak heights peak areas or peaks areas including their siblings are used for normalization Peak areas plus siblings means that all peaks that passes the minimal peak detection thresholds and fall within the bin set of a probe are summarized and used for normalization Normalization factor intra during normalization of a test sample against a reference sample each test probe will be normalized using each set reference probe thereby producing as much ratios as there are reference probes To create a final estimator dosage quotient for each test probe the normalization factor intra will be taken over these ratios On auto default this factor is set to median
108. inst outlier than regular linear regression By using the field regression local size the number of local points may be determines Polynomial regression polynomial regression is a form of linear regression in which the relationship between the independent variable x and the dependent variable y is modeled as an nth order polynomial Although polynomial regression fits a nonlinear model to the data as a statistical estimation problem it is linear in the sense that the regression function E y x is linear in the unknown parameters that are estimated from the data The order can be changed by changing the field regression polynomial degree Local linear regression this methods closely resembles the LS local median regression but instead of feeding the model median values of conditional distribution of y regression coefficient are determined locally by the actual data points This results in a regression line with a differential coefficient at different points and is in effect not a straight line as the LS local median regression line is 42 Coffalyser NET analysis manual beta version Release candidate Classification Confidential Versie 0 1 advanced baseline detection advanced baseline detection max degrees regression method for size calling local linear regression local size 3 regression polynomial degree 2 probe recognition method auto bin design length defa Fi
109. intensity over the run together with the measurement of the widening 50 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 of the peak signals In case there is a problem with the capillaries in most cases a signal drop is always accompanied by widening of the peaks First we measure the percentage of signal drop by comparing the median signal intensity of the first half of all size marker peaks to the median signal intensity of the last half of all size marker peaks Then we measure the amount of peak widening by taking the first quartile of all measured peak widths and comparing this with the widest peak in that run In case signal drop more than 60 a warning will be given and 30 point will be subtracted from the FRSS in case this percentage is 40 a notification will be given and 15 points will be subtracted In case this sloping is accompanied by peak widening of more than 50 another 35 penalty points will be given to the FRSS total Recommendation with problems rerun with alternative injection settings Run also a lane with only marker since contaminant introduced from the sample DNA may also have an effect 7 3 1 5 FRSS Check 5 Size marker complete incomplete Background we usually expect that all fragments of the used size marker will be visible and detectable In some cases however the marker may only be partially present or there is too much noise surrounding cer
110. jection bias of the capillary system and diffusion of the MLPA products within the capillaries Even though some signal to size drop is expected extreme drops may give problems due to the low signal intensity of the largest probes furthermore if there exists a difference in size to signal sloping between the samples and references the ratio results will also be affected Problems are indicated when to check if there are problems with probe signal to size drops we use a similar procedure as applied for the signal drop of the size marker Measurements are again combined with a check on the widening of the probe signals In case there is a problem with the capillaries in most cases a signal drop is always accompanied by widening of the peaks First we measure the percentage of probe signal drop by comparing the median signal intensity of the first half of all probe peaks to the median signal intensity of the last half of all probe peaks Then we measure the amount of peak widening by taking the median of the first half of all probes peak widths and comparing this with the widest peak in that run In case of a signal drop of more than 70 a severe warning will be given and 60 point will be subtracted from the FRSS in case this percentage is between the 60 70 a warning will be given and 35 point will be subtracted from the FRSS in case this percentage is between 40 60 a notification will be given and 15 points will be subtracted In case signal to siz
111. les with the discrepancies computed between the probe ratios per reference probes within the sample This works as following during normalization our algorithm makes use of each reference probe for normalization of each test probe thereby 66 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 producing as many dosage quotients DQ as there are references probes The median of these DQ s will then be used as the definite ratio The median of absolute deviations MAD between the computed dosage quotients may therefore reflects the introduced mathematical imprecision of the used normalization factor By combining the standard deviation found over the reference sample data with this MAD factor and mulitplying it by two we can estimate a 95 confidence range for a probe result By comparing each sample s test probe ratio and its 95 confidence range to the available data of sample groups in the experiment we can conclude if found results are significantly different from e g the reference sample population or equal to a positive sample population The algorithm then completes the analysis by evaluating these results in combination with the familiar set of arbitrary borders used to recognize gains and losses A probe signal in concluded to be aberrant to the reference samples if a probe signal is significantly different as from that reference sample populations and if the e
112. m the reference sample population Hide column by selecting this option you can hide any column in the grid When hiding columns the effect will be passed through on the export grid function but not on the PDF reports 10 Show all columns after hiding columns all invisible columns can be recovered by selecting this option 11 Channels enables or disables the results of the data found for each channel that was set as a probe mix channel This option works only in multichannel modus also see the advanced analysis section PDF experiment ratio overview Coffalyser NET allows a number of pdf report functions which create easy storable files that show a complete overview of either a single sample or the Title complete experiment By selecting the export PDF function from the 86 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 experiment results explorer a document will be created that has 3 subsections The first section contains an overview of all samples their analysis status and all other important quality control aspect that were measured during the analysis The second section contains a print of the ratio overview grid in PDF form here you can find each sample with their probe results In case the sample s quality was below standard the name will be bold The data in this grid will be sorted as set in the experiment results explorer Ratios of probes
113. minimal values for dynamic detection 12 regression method for probe bias correction least squares local median regression method for slope correction polynomial regression local size 8 regression polynomial degree 2 Figure 34 Sloping correction analysis settings Iteration settings The final tab contains settings concerning the iterations steps of the comparative analysis figure 35 Iteration means the act of repeating a process usually with the aim of approaching a desired goal or target or result Each repetition of the process is also called iteration and the results of one iteration are used as the starting point for the next iteration Coffalyser NET allows repeated normalization rounds where the used reference probes reference samples and slope correction methods may be optimized based on the results found in the previous rounds During these rounds of normalization we aim to make the results more normal or perform normalization more towards the original set normal image Results will thus always be normalized to the set median or average reference sample reference probe status 73 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 normalization slope correction normalization cycles experiment reference probe filter experiment reference probe ratio filter low high
114. n peak signal is calculated by the percentage of intensity of each peak as opposed to the median peak signal intensity of all detected peaks Since the minimum and maximum thresholds are dependent on detected peaks this filter will be applied after an initial peak detection procedure based on the criteria point 1 3 This value is called minimum and maximum peak amplitude to median signal 5 Peak width filter After peak end points have been identified the peak width is computed as the difference of right end point and left end point The peak width should be within a given range This filter is also applied after an initial peak detection procedure This value is called minimum and maximum peak width datapoints 6 Peak pattern recognition This method is only applied for the size marker channel and involves the calculation of the correlation between the data point of the peak top of the detected peak list based on the criteria point 1 5 and the expected lengths of the set size marker In case the correlation is less than 0 999 the previous thresholds will be automatically adapted and peak detected will be restarted These adaptations mainly include adjustment of minimal and maximal threshold values The minimal correlation quality is a default value and cannot be adjusted 4 5 3 Binning settings After each peak is detected it needs to obtain a size which in general will be quantified in a number of nucleotides by a met
115. n set the color will also change into purple By selecting a sample you can view the data filtering results of that sample again unless you enable the option box Always display the manual bin set while browsing through samples Enabling this option will hold on to the manual bin set as can be seen in the grid allowing you to make changes and directly view if a peak actually falls within that newly created bin In the context menu under right click you may find a few options to make editing of a manual bin set more easy from right mouse click menu select set manual bin This option will replace the manual bin set for either the selected row or for all rows with either the design length Coffalyser length auto bin results for currently selected samples or auto bin results for the current experiment The upper and lower bounds will be defined by taking the selected length and adding the set search range of probes as defined by the binning settings 64 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 P335 A2 lot 0809 BMP ID 27 HY DNA P335 A2 lot 0809 BMP ID 81 HYI DNA P335 A2 lot 0809 BMP ID 28 HYI DNA P335 A2 lot 0809 BMF ID 31 HYI DNA P335 A2 lot 0809 BMP ID 32 HYI DNA P335 A2 lot 0809 BMF ID 42 HY DNA P335 A2 lot 0809 BMP ID 47 HYI DNA P335 A2 lot 0809 BMF ID 55 HY DNA P335 A2 lot 0809 BMF ID 57 HYI DNA F P335 A2 lot 0809 BMP ID 62 HYI DNA FI 25 0 Io
116. nd give a title to your project figure 18 You can also fill in a short description about the project created by modified by CE device Figure 18 Project settings window 32 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 6 About experiments 6 1 What does an experiment contain After creating an initial project we can create experiment within this project In each experiment data files can then be imported to the database and linked to this experiment Users then need to define the experiment type and for each used channel or dye stream of each capillary sample run what the contents are Each detectable dye channel can be set as a sample MLPA kit or a size marker Samples may further be typed as MLPA test sample MLPA reference sample MLPA positive control or MLPA digested sample 6 2 Creating a new experiment To create a new experiment within a project right click on the project you wish to add the new experiment to and select Add experiment from the right click menu figure 19 33 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 COFFAL SER 2 ll COFFALYSER PC Coffalyser DB 1 0950 a Organisations E f t Test Organisation 01 S Projects amp Customer data amp MS MLPA B ps E
117. ng e brown contains the Q fragments for DNA concentration check Q92nt peak for ligation control DD88nt amp DD96nt for denaturation control X100nt and Y105nt for gender check e orange Q92nt only contains the Q92nt peak for ligation control probe e pink QD contains the Q fragments for DNA concentration check Q92nt peak for ligation control and the DD88nt amp DD96nt for denaturation control for control of contamination with DNA not for concentration estimations e purple MQD mouse equal to the pink QD but for mouse DNA e red Q fragments contain only the Q fragments this control mix is usually added to RNA products e yellow QDX an older version of the control mix brown contains the same fragment lengths but the DD88nt is less sensitive for higher salt concentrations than its equivalent in control mix brown e blue BQDX BIG equal to yellow QDX but with adapted concentrations for BIG MLPA mixes 18 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Please note that control mixes when using MLPA mixes bought from MRC Holland are already pre set to the proper control mix and should thus not be edited 3 6 Adding a new custom product to the sheet manager You may also add new custom product to the database These are products that are made from scratch in your own laboratory based on synthetic or cloned oligo nucl
118. ng baseline will be predicted towards the next point in the data streams that was unrelated to a probes The advanced baseline detection max degrees determines how much the degrees increase the predicted line may have In case the increase is more than the set percentage the line will be predicted to the next possible point that has a less steep increase This method ensures that the baseline will be corrected as close as possible but that peaks that are so close that they are shaped into one peak are not split Instead such peaks will be assumed to be split peaks and the fluorescence of these peaks will be divided over the two by splitting them in two on the lowest points in between the two peaks The peak recognition tab also allows you to change the way peaks are being size called Coffalyser NET allows 4 different regression types 1 Title Status Linear regression In statistics linear regression is an approach to modeling the relationship between a scalar variable y and one or more explanatory variables denoted X The case of one explanatory variable is called simple regression Linear regression refers to a model in which the median which the conditional mean of y given the value of X is an affine function of X Least squares Local median regression LS local median regression refers to a model in which the median of the conditional distribution of y given X is expressed as a linear function of X This makes the lines more robust aga
119. nhibitor whereas other 53 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 situations may require the use of commercial DNA preparation methods such as the Qiagen QiaQuick kit Low Raw Data Signal Caused by Poor Quality Mineral Oil the mineral oil supplied in the DTCS kit is high quality oil containing no detectable nuclease activity The use of other lower quality mineral oils can lead to sample degradation and hence low signal as shown below The red and black dyes are particularly susceptible to this problem Recent experiments at MRC Holland have shown that the use of patient or reference DNA samples with insufficient buffering capacity can result in abnormal MLPA results These experiments indicate that a minimum of 5 mM preferably 10 mM Tris HCI with a pH between 8 0 and 9 0 should be present in the 5 ul DNA sample before heating to 98 oC for DNA denaturation Depurination of DNA at low pH and elevated temperatures is well known e g PMID16412692 PMID10454625 http openwetware org wiki DNA_stability This depurination is more severe at low ionic strength DNA samples eluted from a purification column with water are therefore especially vulnerable This depurination of sample DNA can have two effects 1 No ligation of the MLPA probe oligonucleotides can occur when the sample DNA is depurinated at the ligation site of the MLPA probe resulting in a lower probe si
120. nomics and systems biology While all data normalization occurs per experiment experiments can be organized in a project allowing advanced data mining options enabling users to retrieve and review data in many different ways Users can for instance review multiple MLPA sample runs from a single patient in a single report view Multiple MLPA mix results may be clustered together allowing users gain more confidence on any found results The database can further handle an almost unlimited number of specimens for each patient and each specimen can additionally handle an almost unlimited number of MLPA sample runs By creating projects users can furthermore collect data of different experiments in one project collection In future updated version it will then be possible to create a summary of all data within one experiment or for instance compare all data in a project against all data in another project 5 2 Creating a new project After creating an empty solution users can add new or existing items to the empty solution by right clicking on the folder Projects in the organizations folder and selecting the option add project figure 17 31 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Figure 17 Filtering settings tab 5 3 Project settings After a new project is created you can define the default capillary electrophoresis device you wish to use a
121. nore missing fragments Figure 26 Fragment analysis size marker alignment settings On the second tab of the fragment analysis settings form you can find the size marker alignment matrix settings These settings are in general optimized for the user and do not require adaption This page describes the properties that are used to auto detect which detected peaks in the size marker stream can be related to the expected fragments To do this Coffalyser uses several matrix correlations calculations From the top down we first find the minimal correlation and number of local points that should be used for that correlation This is the minimal correlation between the data points of the found peaks with the lengths of the expected fragments using a local correlation estimator in other words we do not expect the complete line to be linear with a high correlation but each 10 consecutive points The similarity matrix refers to the method of peak probe selection this is done by creating a matrix of all peak data points versus all expected fragment lengths Then all local correlations of a number of local points 3 in figure 26 with the crossed lengths is calculated diagonally In case a correlation was found over position in the matrix each correlation that is higher than the minimal demand scores 1 In case some position have already a score of one and an overlapping correlation is found the score will be plus one This 44 Title C
122. nt for each sample and a minimal number of reference samples that should remain is recommended 6 Reference sample filter lt median Z score this option enables users to minimize the reference sample collection by decreasing the used reference sample signals each round by half If we for instance start with 10 samples and no reference samples the first analysis will use all samples as reference samples and the final estimator for each ratio will be estimated by taking the median By applying the Z scores the reference samples will be limited to the signals that had a Z scores that is lower than the median Z scores overall samples divided by two This basically minimizes the used signals to the 50 that are closest around the original reference set By increasing the number of cycles the number of used reference samples will be divided in two each round until the minimum number of reference samples is reached 7 Extend reference sample collection extending your reference sample collection means that we can use the data of all samples in order to create a new reference sample collection This option only has use in case you are already using a collection of reference samples but you want to increase this set automatically It should be noted that this option is OFF on default because it may skew the results After changing the desired settings click on OK to start the analyzed Dependent on the number of samples the number of referenc
123. o allows users to create custom MLPA mixes The sheet manager software can be used to check if updates to any of the MLPA mixes are available The sheet manager can further carry out automatic checks for updates at the frequency you choose or it can be used to make manual checks whenever you wish It can display scheduled update checks and can work completely in the background if you choose With just one click you can check to see if there are new versions of the program or updated MLPA mix sheets If updates are available you can download them quickly and easily In case some MLPA mixes are already in use users may choose to hold on to both the older version and updated versions of the mix or replace the older version 3 2 Downloading new sheets Each Coffalyser NET version starts with an empty database containing only information about the created organizations users within the database and standard capillary electrophoresis threshold needed for fragment analysis To obtain the necessary information for data analysis right click on Sheet manager in the database explorer and select Download updates from the right click menu as indicated by the arrow in figure 4 13 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 amp Server Administrator C dinsdag 20 sep 2011 17 18 06 Figure 4 Coffalyser NET start screen and location to download sheet upda
124. o remove the baseline wander resulting in baseline 1 For size marker channel no further correction is necessary since not much baseline wandering or shoulder peaks are expected For probe channels this corrected baseline 1 is then fed as input for a filter that calculates the median signal over every 50 subsequent data points figure 19 Baseline moving median points probes fine Alternatively an advanced secondary baseline can be used which follows the baseline more accurately as described at the fragment analysis settings chapter These median values are then subtracted from all the signals that are below 300 RFU see figure 19 Baseline maximum signal for correction fine on baseline 1 resulting in baseline 2 This second baseline is often necessary due to the relatively short distance between the peaks that derive from probe products with only a few nucleotides difference By applying this second baseline correction solely on the signals that are in the lower range of detection even peaks that reside close to each other may reside back to zero signal without subtracting too much fluorescence that originates from the probe products Program administrators can modulate the default baseline correction settings and also may store different defaults for each used capillary system In general it is not recommended to adjust the baseline settings however if one may notice that certain peaks are not being detected it may be necessary to
125. offalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 is a Supporting matrix for the path retrieval method After this similarity matrix is made path retrieval is performed by starting at the corner with the largest data point and length From this point a path will be tried to find follow a number of rules Basically these rules involve the movement of a peak in the size marker stream and relating it to the expected length of a size marker peak Going through the matrix you may then find the next peak at a position 1 1 of the previous peak but we will only move that way in case the correlation meets the set requirement of path retrieval together with the number of points Using this method we may skip both background peaks but also ignore expected size marker fragments if required After a complete path is found back we measure the correlation between the peak data points and the expected size marker lengths to make sure that the quality is ok This result will then be stored in a new matrix and the path retrieval method is repeat for all position in the similarity matrix or for all positions in the similarity matrix for which a minimal start value was found as set by the path retrieval minimal start value Finally the path that has the same length as the size marker and for which a high correlation was found for the found path will be used for the size calling procedure peak recogniti
126. oints that divide the data set into four equal groups each representing a fourth of the population being sampled IQR is the distance of Q1 to Q3 thus containing 50 of all values which is depicted in the chart by the yellow box The theoretical minimum is then estimated by Q1 minus 1 5xlIQR and the theoretical maximum by Q3 plus 1 5xlIQR which are displayed respectively as the lower and upper whiskers If results exist that fall outside the range of the theoretical minimum and maximum they will be displayed as by black 79 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 round markers for the minimum and black triangle markers for the maximum values The average is depicted in the chart by a blue cross and the median by a red stripe Whenever the mouse hovers above any of the displayed symbols extra information will be displayed by a tooltip box This allows you to quick find the exact result numbers and additional information about the probe and its target sequence The right click context menu enables you to customize the chart completely figure 38 Ratio Reference samples F E a Sg ee 2 penne ronresr2ge82eeuscs cago geeas SE re PLEEEEE EES SERGRERSSE STILE ETE CEEEE RBBB HUEN EESRSEGRRERESSHS Saga EES EER SESSSRSEES AES SS T 5 eh place zen pepe oases is ony Distribution type gt All samples Serie label Positive ref
127. on size marker alignment minimal number minimal correlation of points start value found path 0 999 10 similarity matrix 10 999 3 path retrieval 10 990 3 Figure 27 Fragment analysis probe alignment auto bin settings 45 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 On the final tab of the fragment analysis settings form you can find the probe mix alignment auto bin settings These settings are in general optimized for the user and do not require adaption This page describes the properties that are used to auto detect which detected peaks in the probe streams can be related to the expected fragments The method is essentially equal to the earlier described method for recognition of the size marker we correlate data points of all detected peaks in the probe channels against the probe design length or coffalyser lengths Note that the path retrieval correlation is less high than the same settings for the size marker This is done because the migration patterns of MLPA probes are not yet optimized and the mobility to length correlation tends to deviate a little This will be optimized in the future by completing all Coffalyser length and then the correlation may also be increased 7 4 About the fragment analysis quality scores Because of problems arising from poor sample preparations presence of
128. overview window 17 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 3 5 Adjusting a sheet lot You can now adjust to the organization to which this version belongs the control mix that is present in this mix and the default analysis method For more details about these fields please check the chapter about control fragments and analysis settings in this manual The second tab probes can be used to adjust the separate details of each probe in the mix use a single click in each cell to adjust each field Most fields will automatically be checked if they contain valid values Use the right click menu to add or remove probes select add from the menu followed by the selection of the number of probes you wish to add Please note that after adding a series of probes all fields need to be filled in and validated Right click on any probe and select Validate Sheet Data from the right click menu to check if all added data can be used by the software In case a probe has invalid information the related problems can be viewed by double clicking on the warning icon You may also change the control mix that is related to the probe mix by either changing the control fragments type fragments on the first page or by changing its in the probe editing grid in the right mouse click context menu In the Coffalyser NET 7 different control mixes are recognized these bei
129. oze992_ 282 ois 48 Pas osp132 _ os o37010_ 483 ozo 5 pact o132 _los os7oz4_ fis oat 28 etve1 fiz132 i2011694 207 fozz 37 Eea i132 _ fizomess baz oz3 38 etve2 129132 _ 120117 1 ozs 19 Eves _fiapt32__ iz 011883_ fase 025 Dye 224 47 Eves i2132 _fizoma fera loz 11 leves i2132 12 01935196 027 Figure 40 Comparative analysis experiment explorer heat map grid overview with probe target info columned opened and sample result lower levels also opened The probes in the grid are on default sorted by the recommended order if this does not exist the hg18 tracks will be used for data sorting Each row or probe is related to a certain region which depending on the settings will group probes together by giving them a certain color On default probes that have their target sequence to the same chromosomal arm are grouped together Cells that contain probe ratio results can be colored in different ways depending on the set conditional format On default cells will be colored if they were found to be different from the used reference sample collection e Blue or gt gt figure 45c Cells will be color blue if found results meets 2 criteria First the magnitude of the probe ratio exceeded the upper set arbitrary border value on default 1 3
130. ple collection and no unequivocal conclusion can be taken from this result In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol Red or lt lt figure 45d Cells will be colored red if the found result meets 2 criteria First the magnitude of the probe ratio is lower than the lower set arbitrary border value on default 0 7 Secondly the 95 2 standard deviations confidence range of the probe did not overlap with the 95 confidence range of that probe in the reference sample population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol lt lt Red one tint lighter or lt If found results did not meet the criteria of 6 then 2 new criteria are tested which if realized will color the cells one tint 84 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 lighter red First the magnitude of the probe ratio exceeded the upper set arbitrary border value on default 0 7 Secondly the 68 1 1 standard deviation confidence range of the probe did not overlap with the 68 1 confidence range of that probe in the reference sample population In the lower levels of the distribution comparison to the reference sample population this result will be noted by the symbol lt Red two tint lighter or lt lt
131. producible in the performed experiment and the variability found over the used reference probes may indicate if the quality of the normalization was adequate For more information about these calculations please see published articles about the methodology behind Cofaflyser NET J Coffa 2011 J Coffa 2008 Results of all samples of the same sample type for each probe are be displayed by a box plot differently from the earlier discussed box plot this one displays the estimated 68 1 1 standard deviation confidence range by the box and the 95 confidence range by the outer whiskers 2 standard deviations The statistics over the reference sample collection are loaded on default by a green box test sample population by a blue box and the positive sample population by a yellow box A found single probe result thus has a higher probability to be different from the reference population if the estimated 95 confidence range of that signal does not overlap with the outer whiskers of the green box or 95 confidence range of the reference sample population Single sample probe results that fall within the 95 confidence range of the reference sample population will be displayed as by black round markers figure 45a if the results fall outside of the 95 confidence range but are still between the set arbitrary borders they will be displayed by purple circular markers figure 45b and in case they also fall outside the arbitrary borders they will be displ
132. rget sequence of the same sample without digestion In case only one of the two copies is methylated the amount of target sequences available in the digested result will be 50 lower as compared to that of the undigested result 3 RNA analysis RNA RNA experiments are quite similar to copy number experiment except that the probe target sequences are directed to RNA sequences Sample DNA is therefore often purified from genomic DNA in order to minimize contamination and required reverse transcriptase In the analysis you may also set reference samples e g zero control or RNA from control tissues in order to make a relative comparison Alternatively users may only evaluate the intra normalized results thereby comparing each probe signal against one or two reference probe signals within the same sample fragment analysis comparative analysis experiment type DNA MLPA default CE device ABI 3130XL schootje channel type channel content analysis method DNA type marker probes PO81 NF1 mix 1 LOT1109 2011 08 02 block defaut iset per sample size marker 65500 250 m show all channels Figure 21 Experiment fragment analysis settings window 6 5 Setting the channel contents After settings the correct analysis method you need to set for each dye channel what the expected contents are The channels are usually set 3
133. ro LAURA MLPA 2027178 U LURAMLPAAZTTS CIDI LAURA MLPA 2027113 C DigestedSample URAMLPA2007171U LAURA MLPA 2027171 C D Mra 2057086 U LAURA MLE ecm LAURA MLPA 2037153 U LAURA MLPA 2037153 C D LAURA MLPA 2037327 U LAURA MLPA 2077060 U LAURA MLPA 2077060 C D LAURA MLPA 2077192 U R 150 LAURA MLPA CF U R LAURA MLPA CF C D hg18 location Figure 53 Sample explorer result chart of digested reference sample Different from the DNA MLPA analysis digested samples are not normalized against the set reference samples The reference sample are however used to create distribution statistics thereby having something to compare to a so called normal situation In figure 54 you may for instance recognize the earlier described reference box plots chapter 8 4 1 which in this case do not fluctuate around 1 This is the result of the normal methylation status of the SNRPN gene A genomic imprint causes the maternal copy of this gene to be always methylated while the paternal copy remains unmethylated The reference sample population box plot of the methylation status for this gene therefore falls around 50 since the signals of the paternal copy are cut away in the digested sample as opposed to the undigested sample blue arrow figure 54 Signals that are higher than this distribution box and are higher than 75 or ratio 0 75 as opposed to the undigested sample are expected
134. rom only 68 of the reference population Results g is ambiguous the ratio of the signal is higher than the set arbitrary border but both the reference probes and reference samples for this sample were variable resulting in a large standard deviation for sample probe result making the result inconclusive 8 4 2 Comparative analysis sample explorer electropherogram viewer 92 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 The second tab allows you to explore the original electropherograms better Confirmation of found results is often not only desirable but also imperative in order to get an indisputable assessment The displayed electropherogram descent directly from the baseline corrected original data stream created by your capillary electrophoresis device figure 46 Even though a line chart may be visible the original data stream consists of separate time points or data points The sample electropherogram tab on default present the data point on the x axis and the relative fluorescent units on the y axis To make data interpretation easier the design probe length are displayed underneath the x axis at the data point level of the detected peak top of that probe A peak that was related to a probe will furthermore have a circular marker at the detected peak top data point to relative fluorescent units sect Bessie Sols P335 A2 lot 0809 BMP ID 91 HYI
135. rrected ratio 0 574 3 azg B AFA EIE sw S28 BS SSAU a WUU AJOU WUU OUUU UUU U o AJ TU I 82285883 83872338 CSRRISERGIR SoRSBLIR ES ZZRES REL RR BB2IBSE BE BBB es se ess ek ST P2ZARSVNAST SOEREN 5 SARS SRS RR FPP PTE FS FFB Length nt Figure 47 Comparative analysis sample explorer electropherogram double sample view the displayed tool tip box shows a peak signal found to have a reduction of 50 as opposed to the reference population Top part of the chart area displays the electropherogram of a reference sample while the bottom shows that of a tumor sample both tested with P335 MLPA mix 94 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 8 4 3 Comparative analysis sample explorer report viewer The third and final tab can be used for reporting services The displayed grid shows information concerning the target sequence of each probe and also all relevant information of the peak signal that was related to that probe figure 48 While most displayed fields are already explained at 8 3 2 there are two extra columns which were not discussed yet the RSQ reference sample quality column and the RPQ reference probe quality column The RSQ accounts for the part of the standard deviation of each probe that is calculated over the ratios when applying multiple reference samples The RPQ account for the part by the usage of multiple reference probes The final st
136. s and comparison to a known quantity of DNA Alternatively the user could try a dilution series with the same template starting with an amount that is obviously too high and ending with an amount which is much too low This method assumes that the user knows the approximate amount of template added to the reaction this may be from previous work using similar DNA preparation methods Use the preheat treatment for highly super coiled DNA Most commercial DNA preparations yield highly super coiled DNA The preheat treatment will knick the super coiled DNA which yields much more efficient DNA reactions linear molecules sequence better than super coiled molecules Low raw data signal due to bad formamide formamide is used to resuspend the DNA sequencing fragment prior to loading on to the electrophoresis deice The formamide solution must be prepared and stored properly to achieve high quality sequencing data If the formamide is not deionized and stored properly it will decompose into ammonia and formic acid The formic acid then destroys the fluorescent dyes and produces low Raw Data signal Corrective actions Use the special Sample Loading Solution SLS Do not freeze thaw the SLS or formamide solutions Store aliquots at 20 in anon frost free freezer and use the aliquots only once We do not recommend using water to resuspend the DNA sequencing fragments prior to loading Some dyes are not stable inoure water solutions and will yield Raw Da
137. s are calculated Without copy number information these percentages would be very difficult to interpret During a DNA MS MLPA analysis the normal DNA MLPA analysis is initially performed normalizing all samples of the type sample and positive reference against all available samples of the type reference sample After the calculation of all distribution statistics the MS MLPA analysis will follow automatically Here each sample of the type sample is matched against available digested samples by using a Smith amp Waterman algorithm on the sample name To ensure that that this matching is successful is it recommended giving the cut and uncut samples equal names in the capillary sample sheets Samples that are for instance named Sample1 Undig and Sample1 dig will ensure correct matching After each sample is matched the methylation status normalization will follow normalizing the data of the digested samples directly against their undigested counter parts During this normalization only a single reference sample exists for each digested sample the undigested counterpart The reproducibility of each probe in the experiment is therefore derived from the DNA MLPA analysis We thus assume that the reproducibility over the reference samples of each probe as found in the DNA MLPA analysis can be applied to the reproducibility of the probes in both the copy number as the methylation status analysis The reproducibility as d
138. stical overview grid The statistical overview grid displays data in a similar fashion as the previously described heat map grid however instead of displaying the samples and their data this grid display the calculated statistical value over each sample type For each probe the following statistical value are calculated over all sample of the same sample type average median minimum maximum standard deviation and MAD median of absolute deviations By using right mouse click menu region coloring may be changed or the grid may be exported in a similar way as described for the heat map grid The right mouse click context menu contains the same options as earlier described for the ratio overview grid 8 4 About the comparative sample results explorer The comparative sample results explorer may provide a more detailed view to the results of each sample separately as opposed to viewing the reporting option of the experiment explorer To open the experiment explorer right mouse click on the grid showing the quality scores and select from the right click menu Open sample results Alternatively the sample results explorer 89 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 may be opened through the experiment explorer as described in 8 3 2 The comparative analysis sample explorer has three tabs allowing getting a comprehended view of the results of the selected sample
139. ta signals similar to that of BadFormamide Low Raw Data Signal Due to Insufficient Sample Injection poor injection of DNA fragments onto the CEQ capillaries will lead to low Raw Data signal Since the CEQ uses electrokinetic injection it is highly sensitive to excess salts in the loading solution The excess salts compete with the DNA sequencing fragments during injection and result in lower loading of the fluorescently labeled DNA molecules The sources of the excess salts are improperly purified sequencing reactions and decomposed formamide Corrective actions Follow a desalting procedure such as ethanol precipitation If using spin column purification methods make sure that the column materials do not contain salts check with the spin column manufacturer for details for using their products with capillary sequencers DNA Polymerase inhibitor if the correct amount of template was added and the preheat treatment does not yield a substantial increase in Raw Data signal increase the number of cycles inthe thermal cycling program from 30 to 40 or 50 If the correct amount of template was added and the Preheat Treatment and or increasing cycle number does not yield a substantial increase in Raw Data signal a DNA Polymerase inhibitor may be present do not resuspend DNA in DEPC treated water In this situation further purification of the DNA template may be required In some cases a simple ethanol precipitation of the plasmid will remove the i
140. tain fragments to properly recognize the size marker peak Problems are indicated when not all marker peaks that were expected were found but size calling was performed because the correlation of the remaining size marker peaks length with their data points still had a good correlation Even though and incomplete marker with a good correlation does not necessarily have to cause problems it does require good manual examination of the data Runs with an incomplete marker therefore get a subtraction of 60 points of the FRSS total Recommendation with problems rerun with alternative injection settings Run also a lane with only marker since contaminant introduced from the sample DNA may also have an effect 7 3 2 FMRS FMRS fragment MLPA reaction score displays the quality of the performed MLPA reaction To get to a final score seven different criteria are evaluated from the probe mix channel Start score of the FMRS is 100 points 7 3 2 1 FMRS check 1 signal Intensity of the probe fragments 51 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Background too little template leads to poor signal strength which in turn leads to poor base calling and increased variation and thus unreliable results Too much DNA results in a greater number of short extension fragments during labeling which are preferentially loaded into the capillary during sequencing This will result in
141. template in the reaction It is recommended that 50 100 fmoles of product DNA be used in the cycle sequencing reaction This provides enough template to generate an adequate amount of fluorescently labeled DNA sequencing fragments yet not so much as to cause current problems Half this amount of DNA template 25 50 fmoles should be used for single stranded DNA templates such as M13 phage DNA and even less DNA is needed for small PCR products 10 50 fmoles for PCR products less than 3KB in length In many cases the amount of template added to the reaction is not determined and therefore insufficient template is present In other cases an incorrect approximation of the DNA concentration is made Spectrophotometer estimation of DNA samples is only valid if the DNA is pure as in the case of commercial DNA template purification methods Crude preparation of DNA templates which have substantial amounts of protein and or RNA will over estimate the concentration of the template and cause the user to add too little DNA to the MLPA reaction as in the case of crude alkaline lysis minipreps Corrective actions Add the correct amount of the template DNA to the reaction This will require quantification of the template DNA by spectrophotometer in the 52 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 case of commercial DNA preparations or by estimation using agarose gel electrophoresi
142. tes Next you will see the download form click on Start Update to download the latest MLPA sheet In case you are using restricted products from MRC Holland please email to Coffalyser mlpa com to receive the download code This code can be entered in the box which appears by clicking on the Add Code button Adding codes to the database will enable certain restricted lots to be used which are normally not downloaded figure 5 Directly after you will see an update window showing the progress of the current update figure 6 When the update is finished you will receive a message declaring that the download was successful or unsuccessful If the download was unsuccessful please check if your internet connection is active or if you firewall is blocking internet access of the program 14 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 click Start Update to download the latest sheets from MRAC Holland Figure 5 Coffalyser NET start update screen downloading a sheet update package from MRAC Holland Figure 6 Coffalyser NET start screen and location to download sheet updates 3 3 About products lots and active sheets To circumvent problems with designated products and lots and to make sure you will be able to find the correct product and lot you are using the sheet manager has viewing and editing capabilities to compare your product
143. th is important to be properly visible because without sufficient signal it is very unlikely that accurate base calls can be made Optimal size marker signals 49 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 fall between the 1 10 of the detectable maximum and should be at least 3x the signal of the baseline Problems are indicated when in case the measured median peak signal intensity of the recognized size marker peaks is below 1 of the machine detectable maximum or above 70 of the absolute maximum a warning will be given and 20 points will be subtracted from the FRSS Notifications will be given in case the median peak signal intensity is between the 1 and 1 25 or between the 60 and 70 of the machines absolute maximum For an ABI 3130xI for example the minimum intensity for the marker median peak signal intensity is 100 units for warning and 125 units for a notification the maximum median peak intensity is set at 5600 for a warning and 4800 for a notification It should be noted that the minimum demands are often set by the maximum minimum intensity for proper peak quantification and not so much as the percentage of the detectable maximum Next to the median peak signal intensity we also check the maximum peak signal intensity of all detected marker peaks In case the measured peak maximum intensity of the recognized size marker peaks is above 87 5 of the absolut
144. than the maximum set threshold These values are called the minimum and maximum peak amplitude to total fluorescence 3 Model based criterion The application of this criterion can consists of 3 4 steps 26 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 e Locate the start point for each peak a candidate peak is recognized as soon as the signal increases above zero fluorescence e Check if the candidate peak meets minimal requirements the peak signal intensity is first expected to increase if the top of the peak is reached and the candidate peak meets the set thresholds for peak intensity and peak area ratio percentage then the peak is recognized as a true peak e Discarding peak candidates if the median signal of the previous 20 data points is smaller then the current peak intensity or if the current peak intensity returns to zero This value is called detect fake peaks reset peak start datapoints e Detect the peak end the signal is usually expected to drop back to zero designating the peak end In some cases the signal does not return to zero a peak end will therefore also be designated if the signal drops at least below half the intensity of the peak top and if the median signal of the 14 last data points is lower than the current signal This value is called Minimal peak stutter distance datapoints 4 Median signal peak filter The media
145. that fall outside of the arbitrary borders are depicted with a bold font ratios of probes that were found to be statistically different from the reference sample population are depicted with a single apteryx in case they fall outside of 68 of the population and two apteryxes in case they fall outside of 95 of the population Finally the last page of the report contains the statistical measurements over each sub population of samples We measure the average median minimum maximum standard deviation and the median of absolute deviations MAD over the reference samples test samples and positive reference sample for both copy number and methylation status COFFALYSER Sampie OC list Project p335 Experiment p335 Performed by Administrator Machine AB Report date 1 10 2071 Sample name Analysis status Sample type Gender TPF E RPF E DNA DD Primer P335 A2 tot 0809 BMP ID 05 HY ComparativeAnalysed Sampie Female 41 41 10 1 OK OK 11 P335 A2 iot 0809 BMP ID 27 HY ComparativeAnalysed k Female 41 P335 A2 lot 0809 BMP ID 28 HY1 m sed Female 41 Figure 41 Part of the sample quality control list of the experiment pdf report 87 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 Project p335 Experiment p335 Performed by Administrator Machine ABI Report of 2011 D nt Gene Exon Chr band Hg16 loc Probe Nr 454 EBF1 16 05q33 3 05 15805
146. the procedure makes use of every MLPA probe signal set as a reference probe to produce an independent ratio when comparing an unknown sample against a reference sample The median of all produced ratios is then taken as the final ratio This allows for the presence of aberrant reference probe signals without profoundly changing the outcome This process will then be repeated for each probe of each sample to each available reference sample producing as many ratio results as there are reference samples The final ratio will then estimated by calculating the average over these ratios In case no reference samples are set each sample will be used as reference and the median over the ratios be calculated During the normalization the software also calculates the average median and the standard deviation reproducibility of each probe over sample results that have the same sample type in the performed experiment Reference samples are assumed to be genetically equal so the effects of sample to sample variation on the inidividual probe ratios can then be estimated by the reproducibility of these results This data can later also be used for sample profile comparison of unknown samples to the results over a group of samples e g a set of positive or negative reference samples In order to get an estimation of the reproducibility of each independent unknown sample probe ratio result the algorithm combines the variation found over the set reference samp
147. the situation and apply the most robust normalization method based on the biology and quality of the data Most information required for the analysis is extracted directly from the MRC Holland database producer of the MLPA technology 8 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 needing only little user input about the experimental design to define an optimal analysis strategy In the next chapters we explain how we can use this software to analyze a MLPA experiment create experiment overview reports sample reports and chart and how we can make sense of the found results Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 2 Logging in 2 1 Organizations and user accounts Our software uses a SQL client server database model to store all project experiment related data The client server model has one main application server that deals with one or several slave applications clients Clients may communicate to a server over the network allowing data sharing within and even beyond their institutions Even though this system may provide great convenience e g for people who are working on a single project but are working on different locations both client and server may also reside in the same system Having both client and server on the same system has some advances over running both separately
148. then clean the capillaries 7 3 1 1 FRSS check 3 signal Intensity of the marker peaks Background while peak detection and peak size calling are very important processes for sequencing applications peak quantification is not so important Due to the relatively nature of the MLPA data peak quantification is particularly important and has a large influence on the final results Most capillary electrophoresis devices use electro kinetic injection procedures to introduce the sample into the flowing mobile phase which differ from LC in two ways the injection volume is not as well defined and the injection is performed with the electric field turned off Both of these features can contribute to quantitative errors of analysis Because the entire internal volume of a 50cmx50 um inner diameter capillary is only 981 nL the injection volume must be kept quite small Larger volumes will have more band broadening and band broadening may further be effected caused by diffusion Lower strength ionic solution as the sample diluents may allow sample stacking and permits larger volumes for injection Since we are not interested in quantification of the size marker peaks but only use the size marker for comparing the relative migration of the size marker fragments with those of the probe fragment in the same lane the amount of the size marker should be as minimal as possible allowing more injection and better quantification of the MLPA fragments Signal streng
149. tion is only useful when doing an RNA analysis It is then possible to view the Intra normalized ratio get see how each signal relates proportionally to the set reference probes Alternatively DNA can be selected which will display the ratio as they are normalized against the reference samples When a copy number methylation status analysis is performed either DNA results MS results or both may be viewed at the same time On default both copy number and methylation status are loaded and placed directly next to each other Conditional format changed the conditional method of the grid result cells Other than the earlier described default color coding cells may also be colored by 1 Arbitrary borders only comparing the magnitude against the set arbitrary borders Cells that have a probe ratio result higher than the upper border will be red cells that have a result under 85 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 9 than the lower border will be blue 2 Gradient comparing probe ratios against an array of arbitrary borders with steps of 0 1 where each value that is higher than 1 becomes more blue while each value lower than 1 becomes red 3 Hierarchical heat map gives a cell a color based on the rank of that result in either the results in that same sample the results of all sample of the same type or the results of all samples 4 Probability scores cells w
150. to a log scale before creating the regression line Major outlier filter high low this first outlier filter is used to ignore signals based on the pre normalized ratios By setting a very rude filter you can ignore signals that are very aberrant this will help with better fitting of regression lines Ignore major outlier filter for dynamic detection determines whether or not the probes that were detected as major outliers should be left out the regression line dynamic detection method see outlier detection method Outlier detection method determines the way how outlier signals probes should be determined before plotting a regression line through the signals Note Outlier detection methods should only be applied on regression lines of the type Least squares or polynomial The local linear method and least squares local median are methods that already ignore outlier by their methodology and extra outlier detection may make these methods less robust 72 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sloping correction auto f gt 10 default gt metric peak length default Y metric probe signal default use log transformation signals v major outlier ratio filter low high 10 2 i 25 ignor major outlier filter for dynamic detection outlier dection method dynamic default
151. to originate from 2 uncut copies or two methylated copies in case this sample was found to be diploid for target sequence In case of a Prader Willi syndrome this would most likely be caused by a uniparental disomy where the 2 copies of this gene are received from the maternal DNA and thus both being methylated figure 53 amp 54 In case we found by the DNA MLPA analysis that this sample only has a single copy for this target sequence then this copy will also be methylated still resulting in two non functional copies figure 55 amp 56 From this we thus may deduct that it is always necessary to evaluate the methylation status in combination with the copy number status When analyzing tumor samples situation may be even 102 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 more complex samples that have target sequences that are triploid may for instance have a methylation percentage of 0 33 66 or 100 coming from respectively 3 methylated copies 2 methylated copies 1 methylated copy or no methylated copies at all LAURA MLPA CF U Reference Ratio hg18 location Figure 54 Sample explorer result chart of undigested reference sample matched undigested sample to the result of figure 53 103 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 LAURA MLPA 2037153 C Digested
152. us Release candidate Classification Confidential Versie 0 1 Comparative Analysis Settings romain sp carci wai normalization metric calculate best signal to noise default normalization factor intra auto default normalization factor inter auto default arbitrary ratio border low high 07 2 13 Figure 33 Basic comparative analysis normalization settings Slope correction settings The second tab contains the analysis details concerning the slope correction of the data Slope correction of data may be necessary in case there is too much difference in the signal to size drop between reference samples and test samples A difference in signal to size sloping may cause the ratios of the shorter probes seem to be gained and the ratios of the longer probes may seem to be losses while this is actually caused by a difference in fidelity of the polymerase between the reference sample PCR reaction and the unknown sample PCR reaction In case the difference in signal to size drop is minimal no slope correction is necessary and we also recommend it in such cases since regression analysis is much more sensitive as compared to regular normalizations This signal to size drop is caused by a decreasing efficiency of amplification of the larger MLPA probes and may be intensified by sample contaminants or evaporation during the hybridization reaction Signal to size drop may further be influenced by injection bias of the c
153. utat 6 428 33 23 Wilkinson Leland APA Task Force on Statistical Inference 1999 Statistical methods in psychology journals Guidelines and explanations American Psychologist 54 594 604 doi 10 1037 0003 066X 54 8 594 24 Yau SC Bobrow M Mathew CG Abbs SJ 1996 Accurate diagnosis of carriers of deletions and duplications in Duchenne Becker muscular 107 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 dystrophy by fluorescent dosage analysis J Med Genet 33 7 550 558 doi 10 1136 jmg 33 7 550 25 Zar J H 1984 Biostatistical Analysis Prentice Hall International New Jersey pp 43 45 108 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 12 Appendixes Criteria for each machine for quality control checks 109 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1
154. when you add a Coffalyser work sheet of any existing product you basically make a copy of the original worksheets of MRC Holland This copy can then be adjusted to your wishes the originals however never change 16 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 To view the content of a product lot version right click on a sheet and select open This will open the details properties related to that version figure 14 You can find who created this MLPA mix who modified it to which organization this version is related what type of control fragments were added to this mix what the default analysis method is and at which date this version was created in the Coffalyser NET database By clicking on the tab called probes you can view what probes are present in this MLPA mix and obtain information about these probes and their related target sequences figure 8 amp 9 created by product remarks by MAC Holland Ve modified by he product code P146 Colon Gain lot code version LOT1010 an control fragments yellow QDX analysis method block default remarks lot remarks by MRC Holland pa Figure 8 Coffalyser NET sheet library product lot version details window lal ge pra MV location os MVstat probe order
155. xplorer window This can also be done by double clicking on the samples row on one of the QC icons This fragments analysis explorer will allow you to examine each of the separate analysis steps of the 59 Title Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 fragment analysis and also allows you to pinpoint more accurately where possible problems related to the fragment separation may have occurred The fragment results explorer consists of 8 different tabs The first tab contains the results of the separate factors that were used to calculate the FRSS and the FMRS figure 29 E P335 A2 lot 0809 BMP ID 43 HYI DNA Position B2 e igen emmcsmm a Correlation R2 0 9999 Baseline size marker Median signal height Size Marker Maximum signal height Size marker signal sloping Size marker signal widening Size marker complete E Baseline probes Baseline probes percentage cut Primer Probe Signal Check Median probe signal Maximum probe signal Probe signal sloping Probe signal widening a Found reference probes Found mutation probes Found Probes Found Y probes Found total probes Percentage noise peaks a Q fragments median signal D fragment 92 nt signal D fragment 88 nt signal D fragment 96 nt signal X fragment 100 nt signal Y fragment 105 nt signal fi dgn median sianal ratio wl 69 598 683 7 28 56 86 True 24 2 14 28
156. xtended to all probes that pass the criteratia at point 2 If this option is off the criteria will only be applied at the reference probes that are set in the first round of normalization this selection is dependent on the used analysis method If the analysis method was set to block then all selected reference probes in the active sheet were used if the analysis method was set to population then all probes were used as reference probes Only use equal called reference probes this option limits the use of the earlier selected reference probes to those that were earlier found to be equal to the reference samples collection What the criteria are for a probe to be equal to the reference sample collection is explained further down in this chapter In short probes that are equal to the reference 75 Coffalyser NET analysis manual beta version Status Release candidate Classification Confidential Versie 0 1 sample collection are fall within the 95 confidence range of this population and do not cross the arbitrary set borders default 0 7 1 3 5 Probe minimal reference samples this settings defines the minimal number of reference samples that should be left in the end of the analysis per probe In most cases the set reference samples will be equal for each sample however when using the options only use equal called reference probes and reference sample filter lt median Z score the used reference sample and probes may be differe
157. xtent of this change meets certain criteria The results are finally translated into easy to understand bar charts figure 2 and sample reports allowing users to make a reliable and astute interpretation of the results Data signal to size sloping An effect that is commonly seen with MLPA data is a drop of signal intensity that is proportional with the length of the MLPA product fragments This signal to size drop is caused by a decreasing efficiency of amplification of the larger MLPA probes and may be intensified by sample contaminants or evaporation during the hybridization reaction Signal to size drop may further be influenced by injection bias of the capillary system and diffusion of the MLPA products within the capillaries In case the drop in signal is equal between each unknown sample and reference sample no problem exist because this effect will be normalized out of the equation However when a difference in this extent exists results may be biased In order to measure and if needed correct for this Coffalyser NET follows several steps 1 Normalization of all data in population mode Each sample will be applied as a reference sample and each probe will be applied as a reference probe 1 Determination of significance of the found results by automatic evaluation using effect size statistics and comparison of samples to the available sample type populations 67 Title Coffalyser NET analysis manual beta version Status Release c

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