Manual for Demo Data SEQUENCE Pilot
Contents
1. For automatic generation of orders special sample naming conventions have to be used to ensure that the program gets all necessary data This information has to be entered within the sample naming field of the sequencer in the following way name of the used amp module family ID 12 COL1A1 ears 2222 name of the used seq primer Any information can be entered before and after the parentheses The DNA number is obligatory for automatic joining of result files to an order So what can we see in our demo data The COL 1A1 files could automatically be joined to an order due to correct sample naming conventions The order is found in the Lower Table and is ready for analysis All result files joined to this order are listed if you press the in the column Order To see the used sample naming right click on one of the result files and select the item show annotations from the context menu D 928570003 LA ert unjoin edit bases settings OW show protacal Manual for demo data SEQUENCE Pilot module SegPatient 7 The marked line shows the used sample naming conventions E Annotation gt Q NewBResultFiles ABI i lZ C LIAI EUl CIlAIEIF 2222 abl comment 3071 2688 2359 3311 18 L LIAI EUI CIlAIEIF 11 16114 008 Irunstart 15 27 2005 runstop D5 z7 2005 runstart time 17 172. m The files are then moved to the Lower Table Now look at the column Alleles Variations in the Lower Table First press in front of the order line in the Lower Table to expand the order and see all joined result files The column Alleles Variations shows the mutations variations that were detected in each result file C base change insertion D deletion M missing bases 12 Worklist How to get there Category SeqPatient Operation Worklist This operation lists all existing orders and their joined result files The dialogue part Select Orders and the Worklist Table are the same as Select Orders and the Lower Table in Joining chapter 11 To proceed with the analysis select the first order in the Lower Table of Joining or in the Worklist Table and switch to the operation Sequence 13 Sequence How to get there Category SeqPatient Operation Sequence The operation Sequence is the main operation for the analysis Before analysing our demo data important features of this operation are explained below Manual for demo data SEQUENCE Pilot module SegPatient 9 Order statistic Protocol Family all 8 distinct 4 other 4 filter o C allGenes selected Gene Nrvescesosussensenscovesersnneneet Order No 150120001 Patient P 12 COL1A1 ENST00000225964 13 C t gt g bc rs176394 c 850 14 0 4 of 13 intronic v DNA No 12 2 COL1A1 ENST00000225964 E22 z yes gt
3. Note In case SNP databases are installed information from the SNP database for a mutation can be shown in additional columns in the Demo Data no SNP databases are installed for filtering For more information please have a look at our User Manual SeqPatient Menu Help Manuals or contact our support team 13 6 Electropherogram In this dialogue part the electropherogram curves of the result files are shown The reference sequence the result file sequences and the combined sequences of both result files for bases and amino acids are also shown The location in the result file exon intron is also displayed background bar reference sequence combined sequence result files diagram amino acid sequence amino acid sequence amino acid sequence PME result files location overview combined sequence base sequences S Exon 22 B s 1 COL1A1 G A D G V A combined seq G A D D G V A G G G c G G A F G T G T G T G T amp 1 E22 fwd G A D D G v G G G G T G T G T T G e T G G T COL1A1 E21 22 C1A1E21 22F 2 E22 rev COL1A1 E21 22 C1A1E21 22R Tak Y Caramean te combo box count mode 22225 horizontal scroll horizontal vertical function compression compression numbering There are different co
4. 35806 gt p Asp119 5 00000225964 E52 12 C g a het 5224949 4240 1 5 7 of 15 intronic v The table is divided into the tabs a l distinct other and filter The number of mutations present on each tab is shown as well all lists all mutations distinct lists mutations where the base is mutated in both sequencing directions other lists mutations where one sequence forward or reverse shows the reference sequence filter lists mutations that were filtered using the SNP database filter in the Demo Data no SNP databases are installed for filtering For more information please have a look at our User Manual SeqPatient Menu Help gt Manuals or contact our support team If an entry is selected within this table the corresponding position will be shown in the Electropherogram For the analysis it is usually enough to check the here listed entries The Variation Mutation Table is explained in the following table Column Description Print mouse click mouse click The mutation is not printed in the report The mutation is printed in the report table The mutation is printed in the report table and the corresponding electropherogram is also printed mut DB Mark the box if the mutation should be added to the mutation database during the archiving process chapter 15 Index Here the table entries are indexed Lo
5. Login with name and passwora p D t n D h e 2n 4 1 medical systems a 4 categories operations Sequence Pilot i Manual for demo data SEQUENCE Pilot module SegPatient 4 8 Create your own user How to get there Category Administration Operation Users After the first login create a new user After the new user has been created the user 5 is deleted automatically System SeqNext SeqHLA SeqPatient MLPA Help Administration Click on the category Administration on the left side of the screen and then click on the operation Users Fill out the field User max 4 letters Mark active and all options user is authorized to Press Save Select the operation Logout and then login with the new user 9 Configuring of the base caller How to get there Category SeqPatient Operation Thresholds master file Before the files can be loaded configure the base caller to ensure that the same results as described on the following pages are shown Ensure that there are the same settings as in the picture below If not change the values and press Save rectory i dame 7 Base sequencer Type Leading Seq Absolut Count o Percent Count 4o Manual for demo data SEQUENCE Pilot mo
6. 1 1 C1A1E10 11R 12 COL1A1 E10 11 C1A1E10 11R COL1A1 12 05 27 2005 E12 fwd COL1Ai1 12 C1A1E12F 12 COL1A1 E12 1 1 12 2222 COL1A1 12 05 27 2005 E12 rev COL1A1 E12 C1A1E12R 12 COL1A1 E12 C1A1E12R 2222 COL1A1 me re 12 06 08 2005 E13 E14 fwd 1 1 1 C1A1E13 14F 12 COL1A1 E13 14 C1A1E13 14F COL1A1 w 12 06 08 2005 13 14 rev COLIA1EL 1 1 13 148 12 COL1A1 E13 14 C1A1E13 14R X COL1A1 12 05 27 2005 15 16 fwd 1 1 1 1 1 15 16 12 COL1A1 E15 16 C1A1E15 16F COL1A1 w 12 05 27 2005 E15 E16 rev COL1A1 E1 C1A1E15 16R 12 COL1A1 E15 16 C1A1E15 16R COL1A1 12 05 27 2005 E17 E18 fwd 1 1 1 1 1 17 18 12 COL1A1 E17 18 C1A1E17 18F 4X COL1A1 w 12 05 27 2005 E17 E18 1 1 1 C1A1E17 18R 12 COL1A1 E17 18 C1A1E17 18R COL1A1 12 06 08 2005 19 20 fwd 1 1 1 C1A1E19 20F 12 COL1A1 E19 20 C1A1E19 20F COL1A1 12 06 08 2005 E19 E20 rev 1 1 1 C1A1E19 20R 12 COL1A1 E19 20 C1A1E19 20R COL1A1 12 06 20 2005 21 22 fwd COL1A1 E2 1 1 21 227 12 COL1A1 E21 22 C1A1E21 22F COL1A1 w 12 06 08 2005 E21 E22 rev 1 1 2 C1A1E21 22R 12 COL1A1 E21 22 C1A1E21 22R AX COL1A1 12 05 27 2005 E23 fwd COL1A1 E23 C1A1E23F 12 COL1A1 E23 1 1 23 2222 m gt
7. 999 2999 2999 999 AM 20 The statistic also shows there are no archived result files where a or any other base at this base position has been detected before That is the insertion C in the forward sequence isn t one To remove the insertion you can left click or right click on the C as done before chapter 14 1 3 1 Left clicking removes the C automatically base of the reference sequence is set You can right click on the base and select edit None You can right click on the base and select edit bases to open the dialogue Edit Bases To remove the C at base position 19 right click on it within the edited line and select the item None from the context menu After the C is removed press OK 14 1 7 Mutation in E49 position 58 The next entry in the Variation Mutation Table is exon 49 position 58 There is a S C G in forward sequence The EMBL reference sequence and the reverse sequence show a G This is again a not distinct mutation Manual for demo data SEQUENCE Pilot module SegPatient 28 P R L T PIR L T Order Statistic Protocol Family Info C1ATEABF E48 58 7 0 0 SPA 0072 0 063 0 528 0207 0 029 0 032 0 029 0 220 RPA 0 049 0 122 0499 0976 RSD 0793 1 243 0988 3499 vol p g mo The statistic shows the result file peak area for base G in forward sequence is close to statistic peak area calculated over 17 archiv
8. G gt D 497 14906 A p Gly497 0 10 15 patho m Date 01 12 2015 14 10 54 3 COL1A1 ENST00000225964 E43 34 3070 C G C het D gt H 1024 c 3070G C 5 102 Sample Src COL1A1 ENST00000225964 E43 37 3073 C gt G gt C 1025 c 3073G gt T p Gly102 Project 5 1 1 ENST00000225964 E43 419 C a cf het c 30904 Procedures SEQ COLIA1 COL1A1 ENST00000225964 E49 19 3541 I C homo STOP AA 3540 3 p Gly118 E 2 COL1A1 ENST00000225964 E49 58 3580 C gt D gt H 1194 C 3580G C p Asp119 aM COL1A1 ENST00000225964 E52 12 C g a het 5224949 c 4240 1 5 7 of 15 intronic v State compl gt Reference E A ANN DR a Genes B COLIA1 1 G combined 1 t t c t c t G lt gt 1 E13 fwd G m Positions Resultfiles 1 c t 9 t t G COL1A1 E13 14 PEROT C1A1E13 14F is x Bus E Pe AN 2 El3rev 5 t c 9 t c t a c g G COL1A1 E13 14 A C1A1E13 14R gt M A xA NV show mn Comments mnu reading check TES b ob 1 statistic peii T V M V edited mism lt 0 0 1 s 171 Tw MV bases
9. 0 10 15 patho m 3 COLL 52 12 C g gt a het rs224949 c 4240 1 5 7 of 15 intronic v That s the result of your COL1A1 analysis 14 1 10 Validation It is possible to technically and medically validate all result files of the order The technical and medical validation shows that all validated variations mutations have been looked at and confirmed Manual for demo data SEQUENCE Pilot module SegPatient 30 For the validation of single exons mark the corresponding exon within the dialogue part Positions Resultfiles and use the buttons T V and M V respectively below this dialogue part To validate all exons at one time use the buttons T V and M V respectively shown in the dialogue part Functions Functions Previous Next print Digi bases Q statistic After the technical validation the field Condition in the dialogues Gene and Position Resultfiles is highlighted 8888 and shows the entry V After the medical validation the field Condition is highlighted and shows the entry S The user who did the validation and the date are shown in the columns TValidation and MValidation Moreover after medical validation all edits are locked no further modifications can be made unless the medical validation is removed by clicking V again 14 1 11 Printing of a report After finishing the analyses the report can be printed Which mutations variations are printed and if the electropherog
10. 023 0 064 RPA 0 622 0 007 0 120 0 220 RSD 1 231 0 847 0 438 1 154 11 2 If you have a look at the statistic you see that we have a similar situation as described in the chapters 14 1 3 and 14 1 4 In 11 archived result files where a homozygous A was called there is always a certain background for base C present Moreover the peak area of base C is much smaller than the peak area of base A So probably base position 19 is not heterozygous We remove the m and set an a as done before chapter 14 1 3 1 Therfore you can left click or right click on the m asl J Left clicking sets a automatically base of the reference sequence is set You can right click on the base and select edit gt A You can right click on the base and select edit bases to open the dialogue Edit Bases Right click on the M within the edited line and select the item A from the context menu Then press OK 14 1 6 Mutation in E49 position 19 The next entry in the Variation Mutation Table is exon 49 At base position 19 there is an insertion of a C in the forward sequence The EMBL reference sequence and the reverse sequence show no base This is again a not distinct mutation Manual for demo data SEQUENCE Pilot module SegPatient 27 Order Statistic Protocol Family info C1A1E49F E49 19 0 0 SPA 0 000 0000 0 000 0 000 SD 0 000 0 000 0 000 0 000 RPA 0 004 0257 0 001 1 354 RSD
11. 198 308 1 14 i 17 814 886 886 815 6365 To get an impression how many original base positions within a result file have been changed by the internal Basecaller of SEQUENCE Pilot use the jumper change within this dialogue Edit Bases Alternatively go to the dialogue part Show and select the item change of the jumper check In the result file above 20 bases were changed 2 SEQUENCE Pilot contains an own Auto Edit function This function enables the program to delete insertions when there are none replace base positions by a base extend compressions W gt A T remove heterozygous base positions based on the already existing statistic data opera tion Statistic master file see chapter 15 The statistic data will only be factored into the auto edit function if at least five result files of the same chemistry exist Manual for demo data SEQUENCE Pilot module SegPatient 33 All base positions changed by the Auto Edit function are shown in the edited line within the dialogue Edit Bases Here are some examples extend compressions exon 8 fwd base position 4 A N called by the sequencer was replaced by an M by the internal base caller The M was extended to by the Auto Edit Seq 1 Seq 2 change statistic Gene COL1Ai Direction fwd File 12 COL1A1 E08 09 C s 1 28 gt By edited calculated original original C complement To get an impression
12. Gene X N Opens the next order X P Opens the previous order X T Technical validation of the location X V Medical validation of the whole order X Ctrl Shift 1 Sequence is ignored from the selected position to the beginning X X Ctrl Shift R Sequence is ignored from the selected position to the end X X Pos1 Cursor jumps to the first base X X End Cursor jumps to the last base X X Page up Cursor jumps one page backward X X Page down Cursor jumps one page forward X For a detailed description have a look at the Manual SEQUENCE Pilot module SeqPatient Manual for demo data SEQUENCE Pilot module SeqgPatient 40
13. T C G Mar E10 MAR E10R Exon Intron gt Comments Show Functions reading statistic Previous lt I 1 1 54 13 T v m v print het mism CED E 21 teas Manual for demo data SEQUENCE Pilot module 37 What can be seen in our demo data There is a statistic warning for the Mareno sample as it is shown by the highlighted dot statistic and the entry B in the dialogue part Positions Resultfiles column Warning So there is at least one position with a dissimilarity score above 40 To move to the corresponding positions within the electropherogram use the dialogue part Show Select statistic in the field check and use the jumper behind the field to move to the position in the electropherogram The jumper statistic goes through the base positions sorted by dissimilarity score highest score first as shown above The base at position 248 in exon 10 has a dissimilarity score of 54 The value is shown in the dialogue part Show below the jumper statistic The position in the electropherogram is therefore highlighted with a feen background which indicates a score above the threshold of 40 This position does not look like a heterozygous position There are well defined peaks of G in the forward and reverse sequence Moreover there is a background of C in both sequences But in the statistics there is a significant deviation from the normal peak areas which lead
14. The EMBL reference sequence displays a G LJ Gl JU g m g Order Statistic Protocol Family Into C1 ATE21 22F E2236 SPA 0012 un 1300 0 006 sb 0 023 0 024 0 008 RPA 0198 0686 0003 RSD 65 114 S142 25 595 0 535 Manual for demo data SEQUENCE Pilot module SegPatient 22 The statistic shows that the result file peak area for base G in the forward sequence is just half of the statistic peak area calculated over 27 archived result files Furthermore the result file peak area for base A is as high as the result file base area for base G The reverse sequence shows the same This is a true heterozygous position R In the electropherogram the mutation has a grey background This shows that there is a mutation reference at that position This can also be seen in the column mut Ref of the Variation Mutation Table For detailed descriptions see chapter 14 1 1 14 1 3 Mutation in E43 position 34 The next entry shows an S C G at base position 34 in the forward sequence The EMBL reference sequence and the reverse sequence display a G at this position This is a not distinct mutation since it occurs in one sequencing direction only Order Statistic Protocol Family Info CT1A1EA2 43F E43 34 0 0 SPA 0 066 0046 0 449 0136 SD 0 019 0 040 0024 0018 RFA 0 054 0 108 0 402 0 170 RSD 0634 1529 1993 1 8988 The statistic of the forward sequence does not look like the statistics
15. of the heterozyous positions we have seen before chapter 14 1 1 and 14 1 2 Here the result file peak area for base G is close to the statistic peak area calculated over 20 archived result files Moreover the peak area for base C is much smaller than the peak area for base G and also close to the statistic peak area This means that the archived result files where a homozygous G was called have a similar background for base C then our present result file That is we have no heterozygous base position S in the forward sequence and have to change the S into a G 14 1 3 1 Edit Bases There are several options to edit the base Left click on the S the forward sequence Left clicking base sets the base of the reference sequence A G is set automatically Right click the S and open the context menu shown below Manual for demo data SEQUENCE Pilot module SeqgPatient 23 edit bases edit split sequence print T T M A C R A reference 5 K G T V C H A T D A G T B C G T N C G T Y None D ZEE z ignore to left ignore to right Here you can use the items edit 1 or edit bases 2 to do your modifications 1 edit Select the base that should be set in our case edit G 2 edit bases Opens the dialogue Edit Bases Seq 1 seq 2 paie Gene COL1A1 Direction Fwd File 12
16. other dates remove the mark in Date Select Orders and press Search All result files that have the same DNA number are joined to one order Result files that could not be joined to an order are shown in the Upper Table whereas result files that could be joined to orders and are ready for analysis are shown in the Lower Table Manual for demo data SEQUENCE Pilot module SegPatient 6 Upper Table Select Orders Lower Table No DNANp Gene Location Dir Position AmpModule 5 _ RunDate Filename Hint 1 Mareno E10 fwd Mar E 10 MAR E10F 06 04 2005 C A07 mar E10F 159 a4b1 No 2 Mareno E10 rev Mar E10 MAR E10R 06 04 2005 C B06 mar E10R 159 abi no DNA No 3 Mareno E10 rev Mar E 10 MAR E 108 06 01 2005 C 806 mar E10R 160 1 no DNA No 4 Mareno E10 fwd Mar E 10 10 06 01 2005 B07 mar E10F 160 abi no DNA No r Select Orders Join Date Dna No Sample 5 Date Range 7 06 2011 Order No Project x Clear State any not archived y act inact all Validation any v Patient ID Update Order Name State DNANo ProfiefProc Date Location Direc AmpModule amp uD 111780001 P 12 compl 12 SEQ COL1A1 06 27 2011 COL1A1 m 12 05 27 2005 E1 fwd COL1A1 01 C1A1E1F 12 COL1A1 EO1 C1A1E1F 222
17. 01 runstop time 18 13 33 FWO GATC Save as The four Mareno files are listed in the Upper Table They could not be joined to an order automatically because the DNA No is missing Right click on one of the result files and select the item show annotations from the context menu to open the following dialogue The marked line shows the entry that is not compliant with the desired sample naming conventions W Annotation runstart 0620422005 runstop 06 04 2005 runstart time 00 50 09 runstop time 03 15 24 FWO GATC Save Files in the Upper Table have to be joined to an order manually before analysing To join this result files to an order we have to enter a DNA number for each file Therefore right click on all Mareno files in the Upper Table that show the number 159 in the file name and select the item edit DNA Manual for demo data SEQUENCE Pilot module SegPatient 8 delete autosearch Enter the DNA number 159 and then press OK For the other two Mareno files please do the same but enter DNA number 160 To join the files to an order click on Autojoin in the dialogue part Select Orders Select Orders Join Date jvi 27 06 2011 Dna No Sample 5 v Gene Search Ri 211 Project DN Pee act inact all State ps not archived Pat Name Validation any v Patient ID
18. 2 1 foe COL1A1 12 05 27 2005 1 1 1 01 C1A1E1R 12 1 1 01 C1A1E1R 2222 ab1 COL1A1 12 06 07 2005 E2 fwd 1 1 02 C1A1E F 12 COL1A1 E02 C1A1E2F 2222 1 COL1A1 12 06 07 2005 2 rev COL1A1 E02 C1A1E2R 12 1 1 02 C1A1E2R 2222 ab1 AX COL1A1 12 07 08 2005 E3 fwd 1 1 03 1 1 12 COL1A1 E03 C1A1ES3F 2222 1 COL1A1 12 06 08 2005 E3 rev COL1A1 03 1 1 12 COL1A1 E03 C1A1E3R 2222 abi COL1A1 12 05 27 2005 E4 fwd COL1A1 04 1 4 12 COL1A1 E04 C1A1E4F 2222 1 4X COL1A1 12 05 27 2005 E4 rev COL1A1 04 Ci1A1E4R 12 COL1A1 E04 C1A1E4R 2222 1 COL1A1 12 06 07 2005 E5 fad 1 1 _ 0 5 1 1 5 12 COL1A1 E05 C1A1E5F 2222 1 COL1A1 12 06 07 2005 E5 rev COL1A1 E05 1 1 5 12 COL1A1 E05 C1A1E5R 2222 ab1 COL1A1 12 05 27 2005 E6 E7 fwd 1 1 0 1 1 6 7 12 COL1A1 E06 07 C1A1E6 7F 22 COL1A1 w 12 06 20 2005 E6 E7 rev 1 1 0 C1A1E6 7R 12 COL1A1 E06 07 C1A1E6 7R 22 _ COL1A1 w 12 05 27 2005 8 9 fad COL1A1 0 C1A1E8 9F 12_COL1A1 08 09_C1A1E8 9F_22 A COL1A1 12 05 27 2005 8 9 COL1A1 E0 C1A1E8 9R 12 COL1A1 E08 09 C1A1E8 9R 22 COL1A1 12 07 01 2005 E10 E11 fwd 1 1 1 C1A1E10 11F 12 COL1A1 E10 11 C1A1E10 11F COL1A1 12 05 27 2005 10 11 rev 1
19. 41 I C homo STOP AA 3540 3 p Gly118 D 7 ENSTO E49 58 3580 C gt D gt H 1194 c 3580G C 119 8 COL1 ENSTO 52 12 C g gt a het 5224949 4240 1 0 0 of 15 intronic v lt Four mutations are listed on tab sense do not show the wildtype Manual for demo data SEQUENCE Pilot module SegPatient distinct for those position both sequences forward and reverse 18 O M alg distinct 4 other 4 filter 0 all Genes selected Gene Transcript Location T Nuc Change Hint web Ref D mutEffect TValidation MValidati 1 ENSTO E13 14 C t het rs176394 c 850 14 0 0 of 13 intronic v 2 COLL ENSTO 22 38 1490 C gt het G D 497 c 1490G gt A p Gly497 0 0 0815 patho m COL1 ENSTO E43 419 C a c het c 3090 4 COL1 ENSTO E52 12 C g gt a het rs224949 Cc 4240 1 0 0 of 15 intronic v gt The other four mutations are listed on tab other For these mutations only one sequence displays a mutation whereas the other sequence shows the reference sequence Mutations listed on tab other are therefore not as likely to be real mutations as distinct mutations alls distinct 4 other4 fiter o all Genes selected Gene m 4 In Gene Transcript Location Pos T Nuc
20. 99 0 531 i a 1 0 0 Order statistic Protacal Family T Into C1 S1E51 52F 52 12 SFA 0 005 0 000 778 0 046 SD 0 002 0 000 0 020 0 03 0 370 0 000 0 406 0 004 RED 2888 0 000 18 921 1 118 I z 1 0 b 0 homozygous g was detected in 6 archived result files lower statistic To see statistic click on the G in the tab Statistic Here the result file peak area for base g in the forward sequence is just half of the statistic peak area Furthermore the result file peak area for base a is as high as the result file base area for base g The reverse sequence shows the same heterozygous base position This is a true heterozygous base position r There is also a web Ref and a mut Ref entered which shows that it is a known mutation that was detected before For detailed descriptions see 14 1 1 14 1 9 Result of the COL 1A1 analysis The analysis is finished and there are three remaining entries in the Variation Mutation Table tab all All of them listed on tab distinct initially The mutations listed on tab other were no real mutations all 3 distinct 3 other 0 filter 0 C allGenes selected Gene NucChange Change c HGVS p HGVS mut Effect_ TValidation MValidation 1 COLT ES 14 t gt g het rs176394 c 850 14 0 4 of 13 intronic v 2 COLL JEZ 38 1490 gt G gt D 497 14906 gt p Gly497
21. COL1A1 E42 43 C1A1E Bll io Js be LM edited HEN Eus 1c HRS gt edited calculated original sel T2 a0 a 38 z View reverse 5 a original E pu HHHH Exon Intron Extras gt 2 i EH 280 255 C complement 104 m 113 E To set a G at base position 34 right click on it within the edited line and select G from the context menu Then press OK Manual for demo data SEQUENCE Pilot module SegPatient 24 The Mutation in E43 at position 34 disappears in the Variation Mutation Table 14 1 3 2 Undo Function There is a function available to undo manual edits in the electropherogram Therefore press the button Extras in the dialogue part Functions and select undo ET comment Next FeO print Protocol order state J Extras gt The following dialogue opens Undo Redo lt change base E43 278 5 gt The latest edit in the electropherogram is listed on top If you select it and press OK the edit will be removed In our case the listed base change is all right Therefore we do not use the undo function 14 1 4 Mutation in E43 position 37 The next entry in the Variation Mutation Table shows a K G T in exon 43 at base position 37 in the forward sequence Manual for demo data SEQUENCE Pilot module
22. Change AA Change S Hint web Ref c HGVS p HGVS mut Entr mutEffect TValidation MValidati 1 1 ENSTO E43 34 3070 G gt C het D gt H 1024 c 3070G gt C p Asp102 2 COLi ENSTO E43 37 3073 C G T ht G gt C 1025 3073G gt T p Gly102 Bu COLi ENSTO E49 19 3541 IC homo STOP R 3540 3 p Gly118 Bo 4 COL1 ENSTO E49 58 3580 C G gt Cthet 0 gt 1194 c 3580G C p Asp119 lt gt 14 1 1 Mutation in E13 position 14 Please click the first entry exon 13 of the Variation Mutation Table tab all The electropherogram view changes to this position There is a k g t in the forward and reverse sequence at base position 14 The EMBL reference sequence 9888 line shows a t X Now look at the statistics Select the tab Statistic on the upper right part of the screen Manual for demo data SEQUENCE Pilot module SegPatient 19 Order Protocol Famil Infa 61A1E13 14F E13 14 70 0 SFA 0058 1164 0008 0004 sb 0 038 0 120 0006 0 002 RFA O052 0 4659 0 418 0001 RSD 0150 5 642 64 760 1 255 Eos 0 18 0 0 i For each base position of a result file this tab shows the corresponding statistic values The statistic values are calculated over all existing result files of the same chemistry which are already archived Archiving and Statistics is explained in chapter 15 The number below the bases shows t
23. E SES E 9 AAE AE Am T Ts 10 T an TEA 11 ERIE i EE AE A ordi EA AE E A E AEE EEA T EESE ATTAT EEEE 11 laa Foston esrin ESE EA EA E EAE EE E 12 13S Varato Mutation TaNiEc nn ETR sees 12 g N E MN E OIT 14 ETES AAEE EE EREE EEEE AE EEA E 16 OAN E A E AA AEEA A AA AA ER 17 S pu O me damno NE 18 jpeg I TTE E 18 BOS 19 EZA POSON Ret a ERE ena dn bip A ERT exo iat EM Mo 22 15 1 5 M tation in E43 POSION OA accede scares aa ind PRECIO HEEL DE HR eI 23 ONSE SET RR ENTE 23 IS once aR Ee Ren fe en near tener een ee DNE 25 14 14 Mutation in E43 DOSIDUD ORRHDUKE DEM E UP FR EM ERU M ME 25 151 9 Mutation in E43 poslan P TEL eese oci Xni Edd ua eina 26 Te LO NUEU in POSON Teipin omi EE ERTES 27 TLF WM N E49 org RIA Bou ERR Mua bed an aed 20 TE L S DAUERN IE ERE a oauiq ibtebeH Had vbt bd UFU ER PERIOD
24. Ht ECCE REF 29 14 1 9 Result of the COL1A1 30 TEL odERMPEUMTEFUDUE EIN NOD ote csi ba kan seen MANU SK RR AMI ale 30 PELLIT PIENE STA ee eo aieo sss piers sarees DUM MUR EID EL ND INIM EB RP MIRO EIDEM M Ci ME 31 HDD DN Daria DPI Ub cH FO 33 14 1 13 How to analyse the order ETT mE 35 E AA 36 14 2 1 Sample with DNA number 99 36 14 2 2 Sample with DNA number 160 Statistic 36 Discuss BT THO pad ERES EPI ERE ENIM ARTE HERR MM GU GLA 38 15 Archiving Statistics and usasareeemtapEl9yAA YE FHEEYA E YRAEAFERIdIRES RE REREFETMKKERI S RP PME REIS 39 Perte RTT T me 40 Manual for demo data SEQUENCE Pilot module SegPatient 1 1 Introduction to SEQUENCE Pilot SeqPatient The module SegPatient of SEQUENCE Pilot is used for bioinformatical analysis of Sanger sequencing data It is a simple easy to use desktop application including a var
25. Manual for Demo Data SEQUENCE Pilot module SeqPatient Pi lo medical systems GmbH HE E n Hr sis C git ssi P eU EU a ae RZGBBEFZZAHEEC USES ENS SEQUENCE 24 015 a nals developed by JSI medical systems GmbH JSI medical systems Corp 77975 Ettenheim One Boston Place Suite 2600 Boston MA 02108 USA phone 49 7822 440150 0 phone 1 61 7 933 7241 fax 49 7822 440150 20 fax 1 617 933 7246 email mail jsi medisys com email mail us jsi medisys com web www jsi medisys com for research use only Table of Contents 1 Introduction to SEQUENCE Pilot EOP BUG ath n abend dodi Park d biu Pa ER Een ds 2 SEQUENCE PI eere un Ed he EP ERAN A 2 CECI rg ec ee ee 2 4 Installation of demo 3 2211 1 to RE 3 sE ilc DER PEE 4 TEUER E PI Miu TM INI INIERUNT HMM bx RENE 4 SRI ic diuo WR PER TE E Tn 5 or ho Dase Caller TE TID UIT E I I MM 5 10 Load seguencng result TES NR 6 iE iom EIT PEET A AT EET TAT 6 EA gt il EE ETE S E ONA EERO ee T EEEE E A E A
26. SegPatient 25 gt Order statistic Protocol Family Info CIATE42 435F E43 37 SPA 0059 01798 0595 0 032 sp 0032 0072 O022 0 014 RFA 0036 0 262 0532 0046 RSD 0 740 1450 2 795 1 134 guid pm diim The EMBL reference sequence and the reverse sequence have a G As the mutation we just edited chapter 14 1 3 this is also a not distinct mutation The statistic shows that we have the same situation here as described in chapter 14 1 3 This is not a real heterzogous position the K in the forward sequence needs to be replaced by G Therfore you can left click or right click on the K as done before chapter 14 1 3 1 all aJ Left clicking sets G automatically base of the reference sequence is set You can right click on the base and select edit G You can right click on the base and select edit bases to open the dialogue Edit Bases Right click on the K within the edited line and select G from the context menu Then press OK 14 1 5 Mutation in E43 position 19 The next entry in the Variation Mutation Table shows a m in exon 43 at base position 19 in the forward sequence The EMBL reference sequence has an a at this position This is a distinct mutation because no reverse sequence is present Manual for demo data SEQUENCE Pilot module SegPatient 26 Order Statistic Protocol Family Info 42 43 43 19 0 0 SPA 0650 0015 0 130 0122 SD 0023 0 008 0
27. cation Location e g exon of the detected variation mutation Pos Nucleotide position of the detected variation mutation The first number shows the position in exon intron count mode the number in parenthesis shows the position in CDNA count mode The later is only shown if the mutation is localized within an exon In case of insertions or deletions only the forward sequence position is shown Type Variation Mutation Type C change of a nucleotide SNP D deletion insertion M missing bases Nuc Change The changed bases AA Change The resulting changes in the amino acid sequence State State of the loaded result file V technical validated S medical validated Hint The entry is RF changed if the reading frame is changed by the variation mutation Reading frame has to be available web Ref Reference index of the detected variation if it is described in the downloaded gene file HGVS nomenclature Here the HGVS nomenclature for each table entry is listed mut Entry In case the mutation is present in the internal mutation database the column mut Entry shows the frequency homo hetero of the mutation mut Effect If the mutation is in the mutation database and a information is entered it will be shown in this column Manual for demo data SEQUENCE Pilot module 13 T Validation User and date of technical validation MValidation User and date of medical validation
28. d files not loaded yet So what is shown in this case The complete COL1A1 gene has been sequenced in both directions We have two result files for all exons one forward and one reverse direction 13 4 Positions Resultfiles This dialogue part shows all loaded result files belonging to the selected gene in the dialogue part Genes To display the result file base sequences of an exon within the electropherogram click on it once 13 5 Variation Mutation Table This table shows all detected variations mutations Manual for demo data SEQUENCE Pilot module SegPatient 12 al8 distinct 4 other 4 filter 0 P m Index s D 4 5 COL1A1 7 OM lt alGenes Transcript Location Pos T Nuc Chanae AA Change cT Hint web Ref HGVS HGVS mut Entry mut Effect ENSTO0000225964 E13 14 C t gt het 5176394 c 850 14 0 4 of 13 intronic v 5 00000225964 E22 38 1490 C G A het G gt D 497 14906 gt p Gly497 0 10 15 patho ENSTOO000225964 E43 34 3070 C G gt C het D gt H 1024 c 3070G gt C p Asp102 ENST00000225964 E43 37 3073 C G gt T het G gt C 1025 3073 gt p Gly102 ENSTOO000225964 E43 19 C a gt c het c 3090 ENSTO00000225964 E49 19 3541 I C homo STOP R 3540 3 p Gly118 ENST00000225964 E49 58 3580 C G gt C het D gt H 1194
29. d select the context menu item delete order Now you can load the order again as described in chapter 10 Administration p gt Lis Select Orders Order Mo Date Date Range 1 15 02 2011 State lany active Orderlist Jump to Order Input D2 export order show protocol 14 2 Mareno 14 2 1 Sample with DNA number 159 Now go to the second order first Mareno sample To do this click on Next in the dialogue part Functions The buttons Next and Prev will jump to the next or previous entry of the Lower Table of Joining or the Worklist respectively Alternatively you can switch to the operation Joining or Worklist select the second entry DNA No 159 and return to the operation Sequence There is only 1 mismatch at position 288 which has to be checked But this position really is a R and is not changed So there is one remaining entry in the Variation Mutation Table all 1 distinct 1 other 0 In Location Pos Type Nuc Change AA Change HGVS nome mut Entry mut Effect Tvalidation MValidation 1 10 288 2080 gt het M gt V 694 c 2080A G That s the result of this Mareno analysis Technically and medically validate all result files of this order and print the report like described in chapters 14 1 8 and 14 1 9 14 2 2 Sample with DNA number 160 Statistic Warnings Now go to the last order second Mareno sample To do this click on Next in the d
30. dule SegPatient 5 10 Load sequencing result files How to get there Menu SeqPatient Item Load Sequencing Resultfiles SORE Pi zZ m If this item is selected the following dialogue is opened Load Sequencing Resultfiles Destination ABI cons Within the field Destination select the sequencer being worked with By default the sequencer type ABI is already preselected Please do not change this entry because the demo files are made with Loading of the sequencing result files for COL 1A f Press Files Open the directory SeqPilot Demo COL1Al1 Select all result files with ctr A Press Open to load the selected result files Load the Mareno files in the same way They are located in the folder Mareno of the Demo directory Follow the process of loading by looking at the status bar shown at the lower right of the SEQUENCE Pilot screen The dot will be 88 if the loaded files are still being imported The number of remaining files to import is shown after the dot The dot will be green if all loaded files are imported The number after the dot is then Depending on the hardware used the loading of all demo result files needs approximately two to three minutes 11 Joining How to get there Category SeqPatient Operation Joining By default this operation lists all result files or orders loaded at the current date To show orders from
31. ed result files that showed a G The archived result files also show a certain background for base C The reverse sequence doesn t show a heterozygous base position That is the heterozygous base position S in the forward sequence isn t one To remove the insertion you can left click or right click on the S as done before chapter 14 1 3 1 Left clicking sets a automatically base of the reference sequence is set You can right click on the base and select edit gt You can right click on the base and select edit bases to open the dialogue Edit Bases To set the right click on the S within the edited line and select the item None from the context menu After the G is set press OK 14 1 8 Mutation in E52 position 12 The last entry in the Variation Mutation Table exon 52 shows ar atg at base position 12 in the forward and the reverse sequence The EMBL reference sequence shows g The statistic shows that there are 10 archived result files where a was detected homozygously upper statistic Compared to the statistic values the result file peak area for base a is only half of the statistic peak area Moreover there is a peak for g Manual for demo data SEQUENCE Pilot module SegPatient 29 Order Statistic Protocol Family Info 1 51 52 t E52 12 70 0 c c SFA 0 809 0 001 0 000 0 008 sb 000r 0 003 000 0 005 0 370 0 000 0 408 0 004 RSD 56 799 0 500 29
32. er folder in case you already have an existing SEQUENCE Pilot installation on C SeqPilot Otherwise your existing installation will be overwritten A warning appears that updates are only free for customers with maintenance contract This does not apply to trial licenses Ignore this warning by pressing OK Select the components which should be installed By default all SEQUENCE Pilot modules are selected Then press Next Select the start menu folder where the setup should place the program s shortcuts By default this is c SeqPilot Then press Next Check the box Create a desktop icon if a SEQUENCE Pilot icon should be placed on your desktop Then press Next Press Install Press Finish when the SEQUENCE Pilot installation is completed 3 Get your license get your license please do the following a otart SEQUENCE Pilot by clicking on the desktop icon The program stops with the message Manual for demo data SEQUENCE Pilot module SegPatient 2 PC not licensed Please send file JSl cfg to mail si medisys de Path C XDemodatenNSeqNext HLAXbin Press OK to close the dialogue Please send the JSI cfg file as an email attachment to mai si medisys de It is located in the bin directory of the SEQUENCE Pilot installation by default SeqPilot bin Moreover please let us know which module s SeqPatient SeqHLA SeqNext SeqC or MLPA you want to test We will answer as soon a
33. erage Normal fluctuations of the peak areas and backgrounds result in dissimilarity scores between 0 and 10 By default we have defined two limits 20 and 40 for the dissimilarity score If the dissimilarity score passes the first limit 20 the program gives a warning by showing an s within the corresponding field of the column Warning of the Positions Resultfiles table and colours it orange The corresponding position in the electropherogram is not highlighted If the dissimilarity score passes the second limit 40 the program gives a warning by showing an S within the corresponding field of the column Warning and colours it fed The corresponding position the electropherogram is highlighted green Order Statistic Protocol Family all 1 distinct 1 other o nfo warcior ero2ee s41 156 Location Pos Type nuc change AA change HGVS nom mut Effect Tvaidaton Mvaidaton SPA 0 001 0 019 1 133 0 000 490 2282 C G gt A homo R gt H 761 c 2282G gt A SD 0 002 0 033 0 047 0 000 RPA 0 000 0 001 0 901 0 122 RSD 0740 0544 4890 gt 999 e _ E IEs EE _ Genes Mareno combined seq State Co TValidatic L 5 Mareno compl A G T G T 1 E10 fwd L 5 lt gt T G T c G Mar E10 Positions Resultfiles MAR E10F genomic cDNA toc dir w j j 1 P E 2 E10 rev L S A C T G
34. exon 10 of the Mareno sample DNA number 159 edit the heterozygous position 288 in the forward reverse or both sequences to this simulates a not recognized heterozygous position and check the dissimilarity scores Try the same with the found heterozygous base position 12 in exon 52 of the COL1A1 sample Set a g here Statistic warnings appear with dissimilarity scores of 325 and 170 respectively AN Mareno DNA No 159 E10 postion 288 Col1A1 E52 postion 12 Now it can be seen why result files do not have to be checked when no variations are found and no statistic warnings exist SEQUENCE Pilot checked the files for us already by calculating dissimilarity scores 15 Archiving Statistics and Mutation database How to getthere category SeqPatient operation Archiving operation Statistic master file and operation Mutation master file respectively Medically validated orders can be archived with the operation Archiving The electropherogram data is moved out of the database during archiving All other information is still stored in the database The advantage to this is that the database is much smaller and faster During archiving the statistics is created automatically which can help to detect mutations variations Moreover detected mutations variations are added to the internal Mutation database The operation Archiving is divided into two parts With Select Orders it is possible to search for orders to archive using di
35. fferent criteria Only processed orders with the state compl MV complete medical validation are automatically listed Then select the orders to be archived in the list below and press archive The present statistics for all genes can be viewed in the operation Statistic master file One statistic is created out of all archived orders of the same chemistry Same chemistry means same gene exon sequencer sequencing direction amp module and seq primer Here also the reference sequences are listed Manual for demo data SEQUENCE Pilot module SeqgPatient 39 The Mutation database can be viewed in the operation Mutation master file Here all detected mutations are stored For stored mutations information will also be obtained in further analyses There will be an entry in the column mut ref in the Variation Mutation Table chapter 13 5 if the mutation was detected before and added to the mutation database If detected mutations are added to the Mutation database during archiving will depend on the settings in the Mutations Variations Table in the column mut db chapter 13 5 16 Shortcuts There are shortcuts available to make the program more convenient Some of the short cuts can be used in the operation Sequence others in Edit Bases and some in both Short cut Description Operation Edit Sequence bases Ctrl arrow key r
36. ground is present there is a blue bar above the line for forward bases and a purple bar below the line for reverse bases typical heterozygous position bars present for forward and reverse bases a middle line COL1A1 B combined a t t t c t g 1 E13 fwd COL1A1 E13 14 C1A1E13 14F mm E FIN peak height ratio peak area highest not reference bases peak area highest not reference base peak area reference base 2 E13 rew COL1A1 E13 14 C1A1E13 14K 12 Exon Intron p HEUS lt Calculation of the bars e g reference base is T highest not reference base is G peak height ratio peak area G peak area G peak area T Manual for demo data SEQUENCE Pilot module SegPatient 15 The peak height ratio is shown as a bar in the graph for each base position It is possible to show the electropherogram data in a separate window Therefore right click one of the result file sequences and select result file view from the context menu e E E EE edit bases show family edit split sequence print reference X m mm The result file view gives a better overview in case many sequences are present for one location 1 E13 fwd COL1A1 E13 14 C1A1E13 14F 2 13 rev WWM G Gi T COL1A1 E13 14 C1A1E13 14R Al Pf s
37. h appeared in Variations Mutations Table were checked Three mutations variation in the COL1A1 sample and one mutation in the first Mareno sample DNA number 159 were found All of them were distinct mutations The second Mareno sample DNA number 160 could not really be analysed because there was a problem with the PCR There were many exons in our COL1A1 sample that were not checked because there was no mismatch found As described for the Mareno sample with DNA number 160 all positions are checked against the average statistic peak areas and there was no warning for any COL1A1 or Mareno DNA number 159 result file Check the dissimilarity scores in the Mareno sample DNA number 159 or the COL1A1 sample to see if there are any statistic aberrations Therefore press Prev in the dialogue part Functions and to switch to the Mareno sample DNA number 159 or the COL1A1 sample Select any result file in the Manual for demo data SEQUENCE Pilot module SegPatient 38 Positions Resultfiles table Then check the dissimilarity scores with the jumper statistic In most cases there will be no entry this means there is no peak with a dissimilarity score above 1 For some exons there will be dissimilarity scores in the range of 1 to 3 The highest dissimilarity score is in exon 4 position 15 of the COL1A1 sample with a value of 9 05 Another test for the dissimilarity scores remove the medical validation before you do your edits Move to
38. he number of archived orders where the base was called homozygous Those are used to calculate the statistic peak area SPA shown in blue the standard deviation SD and the relative standard deviation RSD The statistic peak area SPA is a measure for the average peak area The result file peak area RPA of the result file being analysed at the moment is shown in 8881 The relative standard deviation RSD shows to which extent the result file peak area exceeds the statistic peak area For heterozygous positions peak areas are smaller than for homozygous positions That is heterozygous positions can be detected not only by the existence of a second peak but also by comparison of the peak area with the statistic peak area The statistic shows that the result file peak area for base t at base position 14 in the forward sequence is just half of the statistic peak area calculated over 18 archived result files Furthermore the result file peak area for base g is as high as the result file peak area for base t This is typical for heterozygous postions The reverse sequence shows the same The peak height ratio diagram below the location overview also shows a typical heterozygous position there are background bars present for forward and reverse bases In the electropherogram the selected mutation has a grey background This means it is a known mutations that is present in the internal Mutation database see chapter 15 It therefore al
39. how many base positions within a result file have been edited by the Auto Edit function of SEQUENCE Pilot use the jumper edited within the dialogue Edit Bases In the first example below 2 bases were edited in the second example 3 bases were edited Alternatively the jumper edited of the dialogue part Show shows all edited positions remove heterozygous base positions based on the already existing statistic data exon 43 fwd base position 2 Seq1 seq2 change check statistic Gene Direction fwd File 12 COL1A1 E42 43 C ver gt statistic Ell l s edited het pos mism far IQ 1 OO E j af ajj f original C complement Manual for demo data SEQUENCE Pilot module SeqgPatient 34 By default the auto edit is activated If desired it can be deactivated 14 1 13 How to analyse the order again You have two possibilities to remove modifications if you want to repeat your analysis First remove the validation by pressing T V and M V in the dialogue part Functions Recalculate Function Recalculates already loaded files Additionally to removal of manual edits thresholds can be modified and changed settings such as an updated software version updated gene files reference sequence or a modified 1is ini file are automatically used to recalculate the result Edits done in the Variation Mutation table column Pri
40. ialogue part Functions There is only 1 mismatch at position 490 which has to be checked But this position is really an A and is not changed So there is one remaining entry in the Variation Mutation Table Manual for demo data SEQUENCE Pilot module SegPatient 36 all 1 distinct 1 other 0 1 Location Type NucChange AA Change HGVS nome mut Effect TValidaton 10 490 2282 C G A homo R gt H 761 c 2282G gt A No we are not finished There is a statistic warning for this file Below the dialogue part Positions Resultfiles there are the two warnings bases and statistic ig bases statistic 1 bases There is no warning if the dot is highlighted grey and a warning if the dot is highlighted 88 The warning shows that there are missing bases Missing bases result if there are amp modules and or seq primers defined and some of the expected bases are missing in the result file 2 statistic There is no warning if the dot is highlighted f en and a warning if the dot is highlighted or 88 What do statistic warnings mean SEQUENCE Pilot calculates dissimilarity score for each base position of the result files The score value depends on the deviation of the peak areas and background areas from the average statistic values and is a measure of the difference between the peak areas and the background to the statistic av
41. iety of functionalities to fit the program to specific needs The information is clearly displayed in one screen SeqPatient possesses an unique learning base caller which helps to detect and analyse base changes deletion and insertions The used statistics help to find mutations Moreover SEQUENCE Pilot has convenient features like an internal mutation database and individual adaptable reports to print 2 Installation of SEQUENCE Pilot For the installation of SEQUENCE Pilot please do the following E bL E ELE EL E amp Go to our website http www jsi medisys com free trial license to obtain a free trial license Please use the link you will receive via e mail to download our latest version of SeqPilot Execute the downloaded file Press Execute in case of an security warning Press Next in the dialogue Setup Sequence Pilot Press Next after reading the Sequence Pilot module information Check the box accept the agreement after reading the Software License Agreement and press Next Press Next after reading the nstallation Instructions Select a destination folder where SEQUENCE Pilot should be installed By default this is NSeqPilot Then press Next Note Please make sure that SeqgPilot is not installed under Nprogram files C program files x86 or in another directory with limited rights for executing and using SeqPilot full reading and writing rights are required Warning Use anoth
42. ight Cursor jumps one base to the right X Ctrl arrow key left Cursor jumps one base to the left X Ctrl arrow key right Cursor jumps one curve point to the right X Ctrl arrow key left Cursor jumps within one curve point to the left X Ctrl Shift arrow key right Cursor jumps one base to the right X Ctrl Shift arrow key left Cursor jumps one base to the left X Ctrl arrow key up Cursor jumps one sequence up X X Ctrl arrow key down Cursor jumps one sequence down X X E Cursor jumps to the next edited position X X Shift E Cursor jumps to the previous edited position X X M Cursor jumps to the next mismatching position X X Shift M Cursor jumps to the previous mismatching position X X H Cursor jumps to the next heterozygous position X X Shift H Cursor jumps to the previous heterozygous position X X C Depending on the selected entry in the jumper check dialogue X part Show the cursor jumps to the next position Shift C Depending on the selected entry in the jumper check the cursor X jumps to the previous position Ctrl Shift C Changes the entry in the jumper check dialogue part Show Ctrl GT M R W S Yor K Replaces the selected base in the electropherogram by A C G T X M R W S Y or Ctrl Space Replaces the selected base in the electropherogram to the one in X X the reference sequence Ctrl Del Deletes the selected base in the electropherogram X X G Opens the dialogue Gene map dialogue part
43. ined genes of an order To open a gene map which displays the analysed parts of the gene and the existing result files right click on the gene entry COL1A7 and select the context menu item show gene map Genes combined seq 1 E13 fwd E ignore toggle COL1A1 E13 14 C1AIE13 14F statistics k editing FI P n export protocol requirement k gene version y Ee J Manual for demo data SEQUENCE Pilot module SegPatient 11 The dialogue Gene Map opens Sas Order 110390001 Gene COL1IA1 TOS 5325 5D DD DTD STD SE FE Ee Ea Ez ILe C C4 CE 4 CX C4 CIE 0 a The shown gene parts are highlighted with different colours Green highlighted exons show that these exons are sequenced at least one result file exists Exons that are not sequenced are highlighted orange Light green parts at the beginning and or end of a sequenced exon are analysed intron parts Introns parts that are not sequenced are highlighted pink highlighted parts within an exon or intron mark detected mutations variations The arrows above and below the gene parts show the existing result files in forward and reverse direction respectively The arrows can be coloured differently aJ T s Result file exists a gt 1 136 14 gt Result file is technical validated chapter 14 1 7 1 138 14 Result file is require
44. lect the context menu item web Ref gt show show 1517639446 local Info web Ref um mut Ret show missing bases r a add mut to DB intronic var i setting 4 i export k If the context menu item mut Ref show is selected the dialogue Mutation is opened This dialogue gives information about mutations that were detected in earlier orders and are stored in the Mutation database see chapter 15 Manual for demo data SEQUENCE Pilot module SegPatient 21 Mutation Position zs Type Ir Muc Change t 2g AA Change Name c 850 14T 2 AA Mame Frequency 0 4 of 13 Reset Info Intern Show exist Info Extern Show ig exist Er Disease No Show Mut Effect intronic variation p mut Effect undefined 3 Web Ref Ethnicity po p mut Causality undefined E Print MEM Show Color M W show Default O Changed Date 02 15 2007 mae je ee eee http www nchi nlm nih gav SMP snp ref cgi type rs amp rs rs17639 NCBI entry Add Remove Location r Orders Organ Phenotype Order No 2ygosity No Nationality 704650003 70460009 70460011 20450015 Add Remove Delete Cancel 14 1 2 Mutation in E22 position 38 The next entry in the Variation Mutation Table exon 22 shows a R A G at base position 38 in the forward and reverse sequence
45. module SegPatient 17 14 Analysis of the demo data 14 1 COL1A1 Now look at the first order with gene COL1A1 DNA No 12 Look at the result files in the dialogue part Positions Resultfiles column Var Positions Resultfiles genomic cDNA T o o L gt T Lm He m c m ma m m There no variations mutations found in exons 1 to 12 14 to 21 23 to 40 42 44 to 48 and 50 to 51 no entry in the column Var These sequences do not need to be checked The result files of exons 13 22 43 49 and 52 show at least one mismatch entry in the column Var There are eight variations mutations detected which are listed in the Variation Mutation Table tab all These positions need to be checked all 8 distinct 4 other 4 filter 0 C allGenes selected Gene P m In Gene Transcript Location OLL 4 mutEffect TValidation MValidati T Riven COLL ENSTO 13 E C 5176394 850 14 intronic v 2 COLi ENSTO E22 38 1490 C 6 gt 6 gt 0 497 c 1490G A p Gly497 0 00 15 patho m auus ENSTO E43 34 3070 C G C he 0 gt 1024 c 3070G C p Asp102 0 4 1 ENSTO E43 37 3073 C G gt Tthet G gt C 1025 c 3073G T p Gly102 1 ENSTO E43 19 C a gt c het 3090 O 1 ENSTO E49 19 35
46. nt Mut DB and ignores to left or to right are not deleted To recalculate all result files for a gene right click on the gene in the Genes table and select editing gt recalculate Single result files can be recalculated with the same context menu items in the dialogue part Positions Resulttiles penes Gene state TWalidatic SO 12 25 ignore toggle statistics k combined editing d reanalyse ls 1 UM recalculate InDel gaps export frameshift analysis d requirement j gene version J y S i The following dialogue opens the Threshold for the internal Basecaller and Auto Edit can be modified Basecaller Auto Edit or single Auto Edit functions can be deactivated by removing the mark Press OK to recalculate the file Threshold Ex Basecaller gt Absolut Count Chromosome set Diploid Percent Count 40 Dis Recalculate heterozygous bases Slope 1 20 Slope 2 10 Auto Edit M Bases Statistic iM Compression Mosaic Score E RSD v Decompression lw Statistic Min statistics 5 N s Manual for demo data SEQUENCE Pilot module SegPatient 35 Delete the order and load the files again With this option all edits and settings are deleted To delete the order select the category L S and the operation Orderlist Then right click on the first order DNA No 12 an
47. o ANN o Exon Intron m Ses original Y lt edited het pos mism gi oo gt gi gt grim o OL 1 13 7 Show This dialogue part offers different functions which enable to jump quickly to the corresponding positions within the electropherogram Manual for demo data SEQUENCE Pilot module SegPatient 16 Show reading check statistic statistic 1 2 edited hel pas mis ef oc gt f t gt reading the selected reading frame is shown in the electropherogram check there are different items that can be checked The jumper aside serves to move forward or backward to the corresponding positions within the e lectropherogram edited jump to edited base positions auto edit function or user het pos jump to heterozygous positions mism jump to mismatches 13 8 Functions This dialogue part comprises different functions to edit the orders Functions Mext T V MV print Extras gt Protocol Previous and Next jump to the previous or next file in the Worklist or Lower table of Joining respectively T V and M V to technically and medically validate the order print to print a report L Extras gt undo redo function enter a comment or change the order state LE b E LE Protocol to open the protocol which shows all recent modifications of the order Manual for demo data SEQUENCE Pilot
48. on 4 2 0 Pam 1 0 Print 28 122034 With a little usage you will see that complete analysis of COL1A1 gene can be done in less than 5 minutes Manual for demo data SEQUENCE Pilot module SegPatient 32 14 1 12 Basecaller and Auto Edit Even though there are many exons in our COL1A1 sample only seven entries were found in our Variation Mutation Table There are two reasons for this 1 SEQUENCE Pilot contains its own basecaller This means it does not take the bases set by the sequencers Basecaller During the import of a result file it calculates the bases by curve values The rules for calculation can be defined in the thresholds operation Thresholds master file There is one exception heterozygous base positions set by the sequencers base caller are never replaced In the dialogue Edit Bases the bases calculated by the internal basecaller are shown in the calculated line whereas the bases set by the sequencers basecaller are shown in the original line To see the following example select E3 in the Position Resultfile table select base position 4 in the electropherogram Then right click on the C in the forward sequence and open Edit Bases calculated line original line Seg 1 Sea 2 change statistic Gene COLIA1 Direction fwd File 12 COL1A1 E03 1 20 gt 14 statisti Ell a show edited het pos all KJ gt x ovo gt gi ua 0 0 gt Ic 1 sel
49. osited within the Demo directory of your installation The COL1A1 txt and Mareno txt gene files previously downloaded from www ensembl org are copied to the GeneFiles directory of the installation View them with any text editor or check the contents with SEQUENCE Pilot menu item SeqPatient Gene Admin For each gene information Manual for demo data SEQUENCE Pilot module SegPatient 3 such as gene name loaded files information for each sequence version coding sequence CDS mRNA translation cDNA and known variations are listed Several master files for Amp Modules Sequencing Primer and Thresholds are added to the system They are shown within the corresponding operations in the category SeqPatient on the left side of the screen Please refer to the Manual SEQUENCE Pilot module SeqPatient for a detailed description Moreover reference sequences statistic values and found mutations from previously analysed samples for COL1A1 and Mareno are loaded 6 The first login Once SEQUENCE Pilot is installed and licensed login using the username jsi leave the password field empty 7 SEQUENCE Pilot screen In SEQUENCE Pilot a menu is available which consists of the items System SeqPatient and Help Furthermore several categories and operations are shown on the left side of the screen Open a category or an operation by clicking on the corresponding icon Then different screens with different functionalities are opened
50. ram data is printed depends on the settings in the Variation Mutation Table in the column Print see chapter 13 5 To print a report click on print in the dialogue part Functions options C selection only select all C select all not printed Manual for demo data SEQUENCE Pilot module SegPatient 31 Here you can choose what to print Check genes to print The options selection only select all and select all not printed helps checking the items n the field Variation you can choose which tab of the Variation Mutation Table to print all distinct or other By default the tab all is selected n field report two report forms can be selected by default The report Seq Variation short contains all information of the report Seq Variation except the resultfile information and the gene map Open print preview using Preview and use Print to print your results JSI JSI medical systems GmbH Tullas tr 18 D 77855 Ettenheim medical systems www imedis ys de Urder Ho 14253200205 DHA Mo 12 Propet Patient 17 FateantiD 12 Fami 30 2222 12292014 Gene fENSTOUOOODTTZ 5864 MV as 1229 2014 15713236 dqEzCXCqAILET 014 22752 0851 ETS KT wer d am 1918 H Iz ED peur Amm GS Day OA ES ee ATES ER Gipecigr peers m 64 Versi
51. s possible 4 Installation of demo data The demo data package SeqPatient DemoData exe includes the complete configuration and example files for the genes COL1A1 and Mareno and shows step by step how to load and analyse the data Please note that this package shouldn t be installed to systems already including data for genes COL1A1 or Mareno This might overwrite your local configuration and statistic values In this case please contact our support associates support si medisys de for a separated installation For the installation of the demo data do the following Stop SEQUENCE Pilot The program should not run during installation Go to our website http www jsi medisys de DownLoadFiles DemoData SeqPatient_DemoData exe Press Execute in the dialogue file download security warning Press Execute again in case of another security warning Press Next in the dialogue Setup SeqPatient Demo Data Check the box accept the agreement after reading the Software License Agreement and press Next Press Next after reading the nfo and Installation Instructions for Sequence Pilot demo data Select the destination folder where the SEQUENCE Pilot demo data should be installed By default this is c SeqPilot Then press Next Press Install Press Finish when the SEQUENCE Pilot installation is completed 5 What is configured All sequencing result files and configuration data are extracted and dep
52. s to the statistic warning Moreover in the background bar diagram below the location overview you can see that there is a forward and reverse bar present which usually is the case for heterozygous positions forward and reverse bar 0 In the bar diagram you can also see that there are multiple base positions showing background You can also use the jumper statistic in the dialogue part Show to find base positions with a dissimilarity score above 20 This is very unusual What happened to this sample There was an allelic drop out this means a mismatch at a PCR Primer position for one allele This allele was not amplified with the PCR as expected and is only present with a small copy number Therefore several peaks do not match to the normal relative peak areas and we get high dissimilarity Scores At position 248 the G peak is too small and we have a C background Knowing the allelic drop out we see that the C is no background but a real peak and the small peak area is a result of the small copy number SEQUENCE Pilot can not define positions like 248 to be heterozygous but it will give a warning if something differs from the statistic average Knowing this position 490 can also be defined to be heterozygous which was previously assumed to be homozygous So the warning means do not trust the results and think about what could have happened 14 3 Discussion of the analysis All variations mutations whic
53. so has an entry in the Variations Mutations Table in the column mut Entry alla distinct 4 other 4 P In Location Pos NucChange AAChage s Hint mut Entry 1 E13 14 C t gt g het rs17639446 c 850 14T gt G 8 0 4 of 13 ntronic var 2 E22 38 1490 C G gt A het G gt D 497 c 1490G A 0 1 of 15 patho mut 3 E43 19 a gt c het c 3090 19 52 12 g gt a het rs2249492 c 4240 12G 5 7 0 15 intronic var MValidation 1 61 6 E E El El A Moreover there is an entry in the column Web Ref of the Variations Mutations Table This means that the mutation is also described in the downloaded gene file from EMBL or Genbank in our case COL1A1 from EMBL Manual for demo data SEQUENCE Pilot module SeqgPatient 20 Move the mouse to a base within the EMBL reference sequence 81888 line A tool tip window shows information about this position This information is only shown if there is a web Ref and or a mut Ref at this position 1 icem Heplace T DB dbSHP IDi2re1 7639446 950 147 gt 05 Changest gt g AS Change Effect intronic variation Frequency 0 4 of 13 If the computer is connected to the internet the web reference can be looked at by right clicking on base a within the EMBL reference sequence or on the corresponding entry in the Variation Mutation Table and se
54. statistic E 95 5 gt 21 Extras gt Protocol gt y lt 13 1 Order Statistic Protocol Family Order This tab shows all important information about the order like order number patient DNA number date etc a Statistic SD standard deviation Order Statistic Frotacal Family Info 42 43 SPA 0059 0 175 SD O052 0072 RPS 0036 10 282 0 740 E43 37 0 000 0 593 0 0532 0022 0014 0532 0 048 1 134 SPA RPA statistic peak area result file peak area Sequences of the same chemistry mostly show the same electropherogram characteristics that is background peak compression curve height etc Same chemistry means same gene exon sequencer sequencing direction amp module and seq primer Therefore they can be Manual for demo data SEQUENCE Pilot module SegPatient 10 used for statistic evaluation For each selected base position of a result file this tab shows the corresponding statistic values statistic value of the peak area statistic value of the background standard deviation These were calculated over all existing result files of the same chemistry already archived see chapter 15 where the selected base was detected homozygously Heterozygous positions are not used to calculate the statistic The present statistics are listed in the operation Statistic master file see also chapter 15 The advantage is tha
55. t all analyses can be compared with previous analyses Therefore the statistic helps to decide whether a postion is heterozygous Protocol This tab shows a list of all base changes as well as ignored bases to left and to right of the currently selected gene within the dialogue part Genes for each result file By selecting an entry the cursor jumps to the corresponding base position within the E lectropherogram Family This tab shows a list of all orders belonging to the same family ID The current order is always shown in the first line Click on one of the other orders to open it 13 2 Reference A reference sequence is shown that corresponds to the sequence of the result file in the electropherogram A reference sequence is a sequence of the same chemistry gene exon sequencer direction amp module and seq primer which was analysed previously and defined as a reference Only one reference sequence can be saved for each chemistry To save a result file sequence as a reference right click on a base of the result file sequence see 13 6 Electropherogram for result file sequence and select the item reference save as from the context menu If a reference sequence with the same chemistry has been saved already there is the option to overwrite it All reference sequences are listed in the lower table of the operation Statistic master file see chapter 15 for Statistic master file 13 3 Genes This dialogue part shows all exam
56. unt modes possible They can be changed with the combo box count mode Exon lintron the first position of every exon intron is 1 CDNA refers to the absolute position of the base in the gene excluding introns AA every amino acid is counted File first base of the file is 1 Gene abs absolute position in the gene Genome Position genomic position Manual for demo data SEQUENCE Pilot module SeqgPatient 14 Above the electropherogram the location overview is shown giving an overview of the selected location in the dialogue part Position Resultfiles 1 Exon 49 em je Introns are marked yellow whereas exons are marked blue Entries of the mutation database see chapter 15 Mutation master file are indicated as bars below the overview here in grey color is user defined Web Refs and Local Infos are indicated as bars above the location overview here in light blue and blue respectively Web Refs are known variations described in the downloaded gene file in our case COL1A1 from Ensembl Local Infos is information deposited at a base position by the user color is user defined You can jump to a location within the electropherogram by clicking on the overview The peak height ratio diagram is shown graphically for each position below the location overview The bars show positions with a high background In case there are no or only small bars above below the middle line there is no high background In case a back
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