Home

Chapter II: DNA Based Testing Section: Application - ASHI-U

1. Remove combs from gel tray and submerge it under 0 5X TBE containing 3 ul of ethidium bromide Load 12 5 ul of each PCR product in each well on the gel Run gel at 150V 15 minutes Disconnect from the power supply and take the gel to the electrophoresis documentation apparatus Take a photo and attach to the SBT documentation 8 Sequence Based Typing 4 13 Document positive amplifications and confirm size of PCR products 4 14 Eliminate any failed amplification from the Cycle Sequencing map for the current run 5 PCR product cleaning using ExoSAP IT 5 1 Take the vial of EXoSAP IT from the freezer 5 2 Add 3 ul of ExoSAP IT to each positive PCR product and cap the tubes 5 3 Vortex briefly and spin them down for few seconds 5 4 Place in the thermocycler and run the following profile ExoSAP IT __ thermocycler profile 80 C 15minutes 4 C co 37 C 15minutes 1 cycle 5 5 Remove ExoSAP IT treated PCR products from thermocycler Note For DRB1 amplifications ONLY add 20 ul of molecular biology grade water to each treated PCR product At this step you can freeze the products to continue the sequencing later Cycle Sequencing step 1 Thaw the primer mixes to room temperature vortex and spin down before opening vials 2 Create a sequencing tray panel using a 96 well tray paper template already having the primer identification as a plating guide 3 Pull a 96 well optical tray and label the run
2. Users manual 3 Assign SBT 3 2 7 User Guide 21 Sequence Based Typing PROCEDURE II HLA CLASS I amp II SEQUENCE BASED TYPING USING GROUP SPECIFIC AMPLIFICATION Principle This procedure describes the chemistry protocol for group specific HLA sequence based typing SBT strategy It gives the most reliable and accurate information of the DNA sequence of a gene and it is therefore of particular interest to fully characterize the genetic complexity of the HLA genes in the human Major Histocompatibility Complex The allelic diversity in HLA class and class II makes SBT the method of choice for HLA typing Recent developments have made sequencing equally simple and robust making it attractive for patient related diagnostic bone marrow registry typing and genetic investigation The method described here amplifies the alleles in a group specific fashion providing medium to high resolution results Each HLA loci have the alleles grouped in 8 or 15 reactions tested simultaneously under identical conditions Group Specific SBT strategy Group specific SBT is designed to reach a maximal level of allele specific sequencing and in turn lowering the number of ambiguities This is achieved by applying either 7 or 14 Group Specific Amplifications GSA and 1 Locus Specific PCR Amplification LSA in parallel allowing in most of the cases identification of sequence data for both alleles present separately hemizygous If the GS
3. 7 amp 10 e Dispense 8ul of the Exon 2 R Sequencing Primer Mix to columns 2 5 8 amp 11 e Dispense 8ul of the Codon 86 Sequencing Primer Mix to columns 3 6 9 amp 12 ZOQ TMMOjO D gt N ar N A O N g N A O N p N A O N H N A O Sequence Based Typing 7 Using a multi dispensing pipette add 2 ul of each ExoSAP IT treated PCR product pre diluted with 20 ul of H2O to each combination of 3 columns containing the same sequencing primer mixes Use the sequencing tray template as guide to add the samples in the correct order 8 Cover tray using a micro amp full plate cover Mark the top left corner to prevent switching the position in further steps 9 Spin tray for 30 seconds at 1200 rpm and immediately place it in the thermocycler and start the Cycle Sequencing profile Cycle Sequencing thermocycler profile 96 C 20 ae 25 cycles 60 C 2 minutes 4 C o0 10 Remove tray from thermocycler and proceed with the ethanol precipitation 11 If not performing precipitation immediately centrifuge the tray 1 minute at 1260g wrap tray in foil or plastic and store at 4 C protected from light for few days limit should be set from a Friday to the following Monday Ethanol Sodium Acetate precipitation Ethanol 100 and EDTA NaOAc precipitation will eliminate unincorporated fluorescent dyes from the sequencing reactions
4. Perform Cycle Seq per SOP and analyze data per SOP Fill the QA form indicating result concordance and document any comments Analyze data print the short form of report indicating in the QC form any observation about primers and concordance or not of results obtained Confirm that Allele database utilized is the same Result may present ambiguities if there is an Allele Database update between both tests 52 Sequence Based Typing PART V ASHI STANDARDS APPLIED TO SEQUENCE BASED TYPING The flowing ASHI Standards Version 2005 can be used as guidelines for Sequence Based Typing D 2 2 4 Laboratories performing amplification of nucleic acids must D 2 2 4 1 Use physical and or biochemical barriers to prevent nucleic acid contamination carry over D 2 2 4 2 Perform pre amplification procedures in a work area that excludes amplified nucleic acid that has the potential to serve as a template in any other amplification assays performed in the laboratory e g PCR product plasmids containing HLA genes or relevant STR VNTR sequences Restricted traffic flow is recommended D 2 2 4 3 Use dedicated lab coats gloves and disposable supplies in the pre amplification area D 2 2 4 4 Ensure that for methods that utilize two consecutive steps of amplification addition of the template for the second amplification occurs in an area isolated by physical or chemical barriers from both the pre amplification work area and post amplification wor
5. ase B compl HWY max gs 097 compl description of all columns shown in this dialogue Column Description Gene Examined gene a State State of the gene This can be e incompl e incompl TV e incompl MV e compl If you point with the mouse pointer at a result file the software shows a tip with the following items 40 Sequence Based Typing Item Description File name Name of the result file Direction Sequencing direction fwd forward rev reverse Amp Module Used amp module Amp Lot Used lot number of the amp module Seq Primer Used seq primer Used lot number of the seq primer Seq Lot Run date Run date and time of the result file TV Date and time of the technical validation as well as the name of the user who gigit Result File Positions Resultfiles anns Hl Pos Pr Stas Ras Zya LHT mp Mosa 270 hemi 1 S382 y1 270 het 1 2 53B gvl 276 hem 1 S3B2 v1 276 het 142 S3B8_ 1 276 het 142 S3BE_vI b Join to both haplotype If the assignment of a result file to both haplotype could not be done automatically by the program HLA or you think the automatically assignment is wrong you can join it manually If you join a result file to both haplotype the sequence is checked for mismatching positions with other result file sequences also assigned to both haplotype If there are any mismatches an error message appears whic
6. terminators are the four different dideoxynucleotides ddNTP each labeled with a different fluorescent dye fluorescing at different wavelengths During the cycle sequencing reaction the template DNA is denatured and the primer is annealed As the polymerase is synthesizing a new complementary DNA strand it has a choice of nucleotides for the incorporation If a normal nucleotide dNTP is incorporated the chain will continue to extend If a dye labeled dideoxynucleotides ddNTP is incorporated the chain extension halts In this way the reaction generates a mix of dye labeled oligonucleotides of different lengths that begin from the primer and terminate randomly at the residue of the dye labeled ddNTP The oligonucleotides of different lengths are separated either by a polyacrylamide gel electrophoresis or by a capillary electrophoresis A laser detects the fluorescence of the dye carried on each of the DNA fragments passing by a light detector Data collection and analysis software generate a DNA base calling The nucleotide sequences determined by the base calling are compared to sequences from the IMGT database and identify various alleles Sequence Based Typing There are at least four SBT strategies developed for HLA typing 1 Generic Multiplex amplification and sequencing All alleles of a HLA locus are amplified by one pair of primers or by a mixture of multiple primer pairs in one tube Each exon is sequenced in two tubes o
7. 40 seconds 64 C 60 seconds 15cycles 72 C 2 minutes 96 C 20 seconds 60 C 60 seconds 15cycles 72 C 2 minutes 96 C 20 seconds 56 C 60 ae 10cycles 72 C 2 minutes 4 C oo 4 PCR verification 4 1 Prepare a 2 Agarose gel using 0 5X TBE 4 2 Cool gel and prepare documentation 4 3 Remove combs from gel tray and submerge it under 0 5X TBE buffer 4 4 Add 6 ul of loading dye to a 96 well PCR tray 4 6 Once PCR profile is complete remove 3 0 ul of each PCR product and add to the tray with loading dye 4 7 Re cap strips and place PCR products in the refrigerator 4 8 Run gel at 150V 9 minutes 4 9 Take photo following SOP and attach to SBT documentation 4 10 Fill paperwork for each sample identifying all positive PCR reactions 4 110pen Pipetting Assistant software to start documentation necessary to identify PCR reactions that will proceed to sequence 4 12 Order amplifications in Pipetting Assistant software PPA A Select Sample Input tab B Select Template button and choose format 8 vs 15 and locus from the drop down menu 25 Sequence Based Typing C In Sample ID box enter 1 sample using each sample unique ID D Select Add button E Repeat for subsequent samples F Once 96 well tray is complete or all samples have been entered G Select Complete I Name the plate PPA requires some type of identification l Click Print amp Complete 5 Create the Purification ExoSAP IT tray 5 1
8. HLA are based on elucidation of exon 2 for HLA DRB1 and elucidation of exons 2 3 and 4 for HLA A B and C loci The most common commercial HLA SBT reagents involve one single initial PCR reaction per locus using primers that will amplify both alleles present The DRB1 primers will amplify the fragments of all DRB1 alleles between the first hypervariable region codon 13 and the 3 end of exon 2 codon 90 Its product is in the range size of 250 300 base pairs bp The PCR reaction for HLA class alleles has a larger size product since it amplifies exons 2 3 and 4 of each locus While the PCR product size for HLA class II is 250 300 bp only each PCR product for HLA class genes is in the range of 1000 bp The presence of a PCR product obtained is verified using a 2 agarose gel followed by an enzymatic cleaning step which will eliminate the unincorporated primers The products once treated are diluted and mixed with the sequencing primers for exon 2 Forward exon 2 Reverse exon 3 Forward exon 3 Reverse exon 4 Forward and exon 4 Reverse individually Codon 86 primer for DRB1 is used to distinguish alleles which are heterozygous at codon 86 of exon 2 The primer will only prime alleles that contain GTG sequence motif at codon 86 resulting in a hemizygous sequence which will permit the identification of one allele in the ambiguous combination Purified PCR products serve as templates for cycle sequencing reactions utilizing Big Dye Terminat
9. PCR purification steps o Equipment failure e g buffer capillary bubbles poor resolution polymer mobility file etc 6 1 2 Not all sequencing reactions failed o If some but not all of the sequencing reactions of an individual sample failed it is likely due to a pipetting error during Sequence reaction cross contamination when adding reagents splashes when vortexing tray Since at least one reaction was successful you know that the PCR product isn t the problem 6 1 3 Random multiple weak or failed sequencing reactions o If the vortexing step is not performed following the addition of Absolute Ethanol NaOAc EDTA then the precipitation of sequencing reactions will be inconsistent Significant variability in sequence signal strengths will result from that 6 1 4 When the following problems are encountered they do not indicate a failure in the amplification or sequencing reactions Rather they are most likely related to the post sequencing precipitation or data analysis steps 6 1 4 1 Small peaks at the beginning of sequence may be due to e Inefficient Ethanol precipitation Ethanol used for wash step may be diluted incorrectly 6 1 4 2 Dye blobs at the beginning of sequence may be due to e Failure to add or incomplete mixing of NaOAc EDTA to the sequencing reaction prior to ethanol precipitation will cause dye blobs e Weak miscellaneous peak size shape due to incorrect volume of BDTv1 1 e When this
10. amplified by PCR and falsify the test result One particular source of contamination is amplified DNA coming into contact with samples which are still to be amplified To avoid contamination with amplified material SBT follows the standard SOP keeping the reagents equipments and utensils physically separated Pre PCR area All work carried out before PCR preparing and storing sample DNA preparing PCR amplification reactions setting up and storing reagents and solutions for DNA isolation and PCR should be done inside the laminar Flow hoods dedicated for Pre Amplification procedures Post PCR area All work carried out after PCR running thermocycler and DNA analyzers preparing and running Agarose gel electrophoresis preparing and purifying sequencing reactions storing amplified DNA or sequencing reactions PCR trouble shooting No PCR product or weak PCR product 1 1 No ethidium bromide in gel Re stain the gel in 1X TBE with 0 5 ug ml Ethidium bromide 1 2 Incomplete mixing of AmpliTaq Gold and PCR reaction mix Repeat PCR with attention to mixing 1 3 DNA concentration out of range ideal is 25 100 ng Recheck DNA concentration 1 4 Blood sample collected in Heparin Treat DNA sample with Heparinase or recollection 1 5 PCR tube caps not well settle in the thermocycler causing evaporation and false negative reactions 1 6 Incorrect thermocycler profile 33 Sequence Based Typing Check the cycling profile and current variation
11. causing profile to stop before completion SBT Troubleshooting SBT problems may be due to low quality sequences or heterozygous sequences of specific allele combinations that can have C and G rich regions causing peak shift or high background The majority of these anomalies occurs in only one sequencing orientation at a certain base position and can be resolved by reviewing data from the other orientation 3 1 Weak signal strength Inappropriate injection time or injection voltage because of variations between instruments adjustments of the injection time and or the injection voltage may be needed to get a signal range from 100 2000 relative fluorescent units 3 2 Too little sequencing reaction applied Increase injection time injection voltage or concentration of sequencing reaction 3 3 Too strong signal strength Inappropriate Injection time or injection voltage because of variations between instruments adjustments of the injection time and or the injection voltage may be needed to get a signal range from 100 2000 relative fluorescent units 3 4 Noisy baseline Inappropriate PCR product purification or poor quality reaction precipitation 3 5 Inappropriate sequencing reaction purification Re purify the sequencing reaction or purify the correct one 3 6 Broad fluorescent terminator artifacts dye blobs Inappropriate sequencing reaction purification 3 7 High fluorescent artifact peaks Air bubbles in the capilla
12. if you want to select the sequences from the upper list of this dialogue yourself for which a report should be printed or a print preview should be shown select all This option is activated by default All sequences in the upper list of this dialogue are selected and printed in the report or shown in the print preview Print Press this button and a report for the selected gene s is printed Preview Press this button and a print preview for the selected genes is shown Cancel Press this button and the dialogue Print Preview is closed References 1 Protrans medizinische diagnostische Produkte GmbH S3 and S4 User Manual 2 Protrans Sequence Pilot Software User Manual 3 Pipetting Assistant User manual 45 Sequence Based Typing QUALITY ASSURANCE PLAN HLA CLASS amp Il SEQUENCING BASED TYPING Introduction The Sequence Based Typing described in this protocol has been well established in the Clinical Transplantation Immunology Laboratory The method has proven to be accurate precise and reliable We follow the guidelines provided by the kit manufacturer and common sense Molecular Biology precautions to prevent contamination or degradation of reagents and or DNA samples This quality assurance plan QA Plan is developed for HLA class I and class II Sequence Based Typing and it can be used as a guideline or reference for the HLA Sequence Based Typing using other SBT reagents source 1 Guidelines Pre Ampl
13. inside the sampler area 9 19Link tray map to tray ID and click on the green arrow to start the run 9 20The run module for this procedure takes about 30 minutes for each injection on the 3130XL and 25 minutes on the 3730 Analyzer when using a 36 cm long capillary array 9 21Genetic Analyzers module 3130XL 3730 e Oven Temperature 60 C 60 C e Cap Fill Volume 184 steps n a e Pre Run Voltage 15 kV n a e Pre Run Time 180 sec 180 sec e Injection Voltage 1 5 kV 1 2 kV 30 Sequence Based Typing e Injection Time 5 sec 5 sec e Run Voltage 8 5 kV 8 5 kV e Data Delay Time 405 sec 120 sec e Run Time 3600 sec 1500sec 9 22 A 3130 XL Analyzer analysis settings e Mobility file 3130 _POP7_BDTv1 1 mob e Analysis module KB_V1_PCR_Mixed Bases_ 10 20 15 e Basecaller KB bcp e Dye set primer file KB _3130 POP7_BDTv1 1 e Run Module Protrans Result Group B 3730 Analyzer analysis settings e Mobility file KB_3730_POP7_BDTv1 1 mob e Analysis module KB_V1_PCR_Mixed Bases_ 10 20 15 e Basecaller KB bcp e Dye set primer file KB_3730_ POP7_BDTv1 1 e Run Module StdSEq36_POP7_IV1 2_IT5 RT25min 10 Identification of the Alleles The final step in sequence analysis consists of the allele assignment using the JSI SeqPilot Allele Identification Software or other computer softwares developed for sequence based typing The SeqPilot Allele Identification Software is compatible or adaptable to all four dye sequencing instruments available The HL
14. of new primer probe sets that have changed from the previous lot D 4 11 10 14 2 Test each new shipment of kits to demonstrate that the integrity of the kits has not been compromised during shipment This can be accomplished by D 4 11 10 14 2 1 Testing with reference DNA samples and assessing the results or D 4 11 10 14 2 2 Testing with non critical clinical samples and assessing the quality of the reactions and the ability to give a clear interpretation of the results or D 4 11 10 14 2 3 Testing the new lot or shipment in parallel with the old lot D 4 11 11 Laboratories performing nucleotide sequencing must D 4 11 11 1 Establish a scientifically and technically sound method for interpretation acceptance and or rejection of sequences especially regions that are technically difficult e g compression ends D 4 11 11 2 Ensure that sequences contributed by amplification primers are not considered in the assignment of alleles 58
15. the sequence file name convention must be defined The dialogue box allows you to e Create your sequence file name convention 16 Sequence Based Typing underscore Locus library e Exclude primer site sequences where primer site is within the exon e Enter the location and sequence details of primers for the resolution of heterozygous ambiguities e Activate or deactivate automated editing of the consensus sequences 5 Click update after making any changes to ensure that the alterations are recorded 6 Use the sample delimiter pane to enable to define the location of the sample ID within the sequence file name The name for the sequence reaction should be Sample ID_locus_ primer name amp orientation Example 004589 A_2F T Use the following features a Create locus code following the name criteria the laboratory uses to call each HLA locus b Set the primer set trimming appropriately Note HLA A B or C don t require trimming only DRB1 SBT data requires trimming settings c Matching Mode is to be set as heterozygous library mode 8 Automated Editing This function uses information from the typing libraries to refine the base calling in the consensus sequence The criteria must be followed before data can be reviewed by the automated editing function 17 Sequence Based Typing a The Base call score of the consensus sequence at a site must b
16. to appropriate Analyzer computer this does not use the Pipetting Assistant software 8 9 Plate Record is stored in the directory C Programs files PipettingAssistant PlateRecords as a txt file_ 8 10 Right click on the txt file and change the name format from txt to plt 28 Sequence Based Typing 8 11Transfer tray format information to the 3130xI or 3730 computer 8 12Copy the plt file into the Plate Import folder on the sequencer computer 8 13In the sequencer software Import and choose the appropriate plt file 8 14Pipette to your Sequencing tray following the map created in PPA 2ul of Big DT v 1 1 6ul of the appropriate primer F or R Spin tray for 30sec 1000rpm 2ul of the Exo SAP IT treated PCR product 8 15Run Cycle Sequencing profile Set volume for 10 ul 96 C 1 min Hold 96 C 10sec 50 C 5 sec 25 cycles 60 C 4min 4C Following cycle sequencing the sequencing reactions have to be purified to remove non incorporated dye terminators which would otherwise cause sequencing artifacts 9 Ethanol 100 and EDTA Sodium Acetate Precipitation Ethanol 100 and EDTA NaOAc will eliminate unincorporated fluorescent dyes from the sequencing reactions This is an important step and has to be done without interruption and in the exact order described The precipitation step will determine the quality signal strength of your sequence data 9 1 Thaw the EDTA NaOAc buffer and Vortex spin down gently before opening
17. with each sample The picture below indicates the information necessary for you to process the data analysis Scrollbar Mismatch Position f Edit Confirmed Manual Edit J 140 J J i150 4 Relerence Copsensus Consensus Base DM eee Pied Sample Call Scores po Consensus EPG Base Soui Bi x iy E ee flecmence Call Scores Peere MLE LL TOI 19 Sequence Based Typing 10b Assignment Pane Sample or perena ST ES First Allele JORB1 3102 223 14 ol Number of bases in the match Liame TOTEAN ol 4 matched against both DREi 07Q DRBi iizif 237 oj uh reference sequences peron REDE HEEX PL 1 Second Allele of E E in the match prer zooa0z oreraa 4 3 14 Number of bases matched against one aq 2 reference sequence COI EE n ppeironoe pREi iio2 237 A 4 Number of ETEA TEET 237 _c miamarehes B paj ou w a The assignment pane displays information about the best matched pairs of alleles from the current libeary The selected test sample ID is indicated in the top belt hand corner of the assignment pane The results window contains the allele pairs whose combined sequence gives the closest matches to the observer sample sequence columns Allele 1 and Allele 2 Homozygous samples contain the same allele name in each column The number of mismatches between the allele pair combinations and the sample sequence are Indicated in the MM columns 11 An allele assignment cannot be considere
18. 03 2007 E2 ev S3C7 v1 0 CE2 Cw06020101 06 76995_S3C7_C E2R_B05 ab1 76995 12 03 2007 E3 ev S305 v1 0 CE3 Cw 06020101 06 76995_S3C5_C E3R _E05 ab1 aN 76995 12 03 2007 E3 rev S3C7v1 0 CER Cw 06020101 06 76995_ 3C7_C E3R _FO5 ab1 76995 12 03 2007 E4 fwd S3C8 v1 0 CEAF Cw 06020101 06 76995_S3C8_C E4F _A06 ab1 76995 12 03 2007 E4 ev S3C8 v1 0 CEAR Cw06020101 06 76995_S3C8_C 4R _C06 ab1 fad 76995 12 03 2007 E2 fwd 54P12 v1 8 DREF DRB1 0804 1501 76995_S4R12_DR E2F _B07 ab1 New AF Dits 76995 12 03 2007 E2 fwd S4R2vi8 DREF DAB1 0804 1501 76995_S4R2_DR E2F _A07 ab1 master fle 76995 12 03 2007 E2 ev S4R12_1 8 DRE2R DRB1 0804 1501 76995_S4R12_DR E2R _B08 ab1 76995 12 03 2007 E2 ev S4R2v18 DRE2R DRB1 0804 1501 76995_S4R2_DR E2R _A08 abt Workspace Li E SegPilot manual_2 2 Gil SEQ Pilot Microsoft JSI medi DNA number you search for DNA Number Please not that in this case all other search criteria are ignored Orders from a certain patient you search for Patient Name Please not that in this case all other search criteria are ignored Patient ID Orders from a certain patient ID you search for Please not that in this case all other search criteria are ignored Orders with a certain validation state you search for 37 Sequence Based Typing Tab Test Order m Order OrderNo 52380001 Patient P M054818 DNA No MO54818 Date 06 26 2005 11 48 55
19. A reactions do not identify two separate alleles the LSA reaction must be sequenced true only for HLA class This ensures in all cases the recognition of both alleles present Special emphasis was put on the complete coverage of exons 2 3 and 4 to sort out nearly all ambiguities caused by genetic polymorphism in these relevant areas of the HLA molecule and the location of the sequencing primers to ensure complete exon sequences in both orientations For ease of use the group specific primer mixes are pre dispensed in one or two 8 well PCR tube stripes 22 Sequence Based Typing Note HLA DRB71 kits only tests exon 2 of the DRB1 alleles HLA A B and C test exons 2 3 and 4 The 8 PCR reactions can reach a high level of allele specific sequence with a lowest number of ambiguities when used for the locus A and C that are not as polymorphic as HLA B and DRB1 Either approach having 7 group specific PCR amplifications GSA and 1 locus specific PCR amplification LSA or 14 group specific PCR amplifications GSA and 1 locus specific PCR amplification LSA in parallel allow in most of the cases both alleles to be analyzed separately If the GSA reactions do not indicate two separate alleles the LSA reaction must be sequenced This ensures in all cases the recognition of both alleles Amplification procedure 1 Creating a panel for PCR amplification 1 1 Label the PCR tube strip using the sample ID The tube containing mix 1 should be
20. A library is updated with each new Sequence Database release of the HLA Informatics group The software performs allele identification allows manual review or editing sequencing data as well as reporting exporting and archiving of sequences and results The following example provides basic steps of allele identification using SeqPilot software analysis version 1 3 10 1 Sample name convention The Sequence Pilot software automatically recognizes the locus and exon sequence as well as the direction of the sequence In order to allow the software 31 Sequence Based Typing to properly pair join the sequences of the same sample the name of sequences has to follow the naming convention as shown Sample ID_amplification mix_sequence primer _any other information 10 2 Process sequencing files using ABI Sequencing Analysis program e Transfer folders to computer with SeqPilot software e Open SeqPilot and choose from System drop down menu Load Seq Results Files e Choose LIS click on Order list e Right click and choose Jump to order Input for each patient listed in order list e Patient ID box will fill with the Sample ID Last name box will fill automatically with P xxx Sample ID e In First name box type patient s first name or any other ID Click Save order list and for all sample data imported into SeqPilot Software e Click the SeqPilot tab and click on Joining It will display the sequenci
21. Click the Result Input tab 5 2 In the Sample ID box type the 1 sample number ID If the chosen Sample number was amplified for more than 1 locus a list of all the amplification trays on which that sample number appears will pop up 5 3 Choose the appropriate tray 5 4 The entire amplification tray that includes the chosen sample ID will appear in the upper window 5 5 On the tray map click on the first amplification product to be sequenced 5 6 On the right side margin click on all the desired sequencing primers exon and direction 5 7 The lower window will display the amplification products to be purified and their positions on the purification tray 5 8 Repeat for 2 amplification product and for subsequent samples 26 Sequence Based Typing 5 9 When all amplification products from the upper tray have been selected click print result to print a grid graphically showing which amplification wells will be purified 5 10 When all amplification trays have been added to the Purification plate click Complete 5 11 Name Purification Tray Date of Purification Pur Loci i e 20050829 Pur ABDR 5 12Click Print amp Complete The printout is saved as an excel file in the directory C Program Files PipettingAssistant PrintFiles This doc can be opened reformatted and reprinted to let you see all the required information if the default printout is truncated 6 Choosing the positive PCR reaction products to proceed to S
22. ID on the front lower side Note We prefer working with each locus separately to prevent mixing up primers 4 SBT testing for HLA A and B allows for 16 samples locus to be tested in a 96 well tray e Dispense 8ul of the Exon 2 Forward F Sequencing Primer Mix to columns1 amp 7 e Dispense 8ul of the Exon 2 Reverse R Sequencing Primer Mix to columns 2 amp 8 Sequence Based Typing e Dispense 8ul of the Exon 3 forward Sequencing Primer Mix to columns 3 amp Diep nse 8ul of the Exon 3 Reverse Sequencing Primer Mix to columns 4 Saree 8ul of the Exon 4 forward Sequencing Primer Mix to columns 5 penne 8ul of the Exon 4 Reverse Sequencing Primer Mix to columns 6 amp 12 5 SBT testing for HLA C allows for 24 samples to be tested in a 96 well tray Dispense 8ul of the Exon 2 Forward Sequencing Primer Mix to columns 1 6 amp ee 8ul of the Exon 2 Reverse Sequencing Primer Mix to columns 2 6 amp D 8ul of the Exon 3 Forward Sequencing Primer Mix to columns 3 7 D onet 8ul of the Exon 3 Reverse Sequencing Primer Mix to columns 4 8 amp 12 ZO TMMOjO o gt N T N ps wo T w Ps N T N Ps wo T w A N T N A wo T w ye 6 SBT testing for HLA DRB1 allows for 32 samples to be tested in a 96 well tray e Dispense 8ul of the Exon 2 F Sequencing Primer Mix to columns 1 4
23. Pre E ae State any not archived Validation any Export Clear Joining Date Bange Worklist a Worklst ile Proc ion Direct i Alleles Variations Sequence STREETER compl TV 12703 2007 5342 v1 0 A 02010101 0201 76995_5342_A E2R _A01 ab1 76995 12 03 2007 E2 tev S344_v1 0 AE2R A 02010101 0201 76995_S344_A E2R _BO1 ab1 76995 12 03 2007 E3 ev S3210 AER A 02010101 0201 76995_ 342_A 3R EO1 abt Archiving 76995 12 03 2007 E3 rey S3A4 v1 0 AER A D2010101 0201 76995_5344_A E3R _F01 ab1 76995 12 03 2007 E4 fwd S3A2 v1 0 AESF 4020101010201 5 342 A E4F _A02 abt Fe 76995 12 03 2007 E4 fwd S2AB v1 0 AESF A 02010101 0201 76995_S348_A 4F _DO2 ab1 76995 12 03 2007 E4 ev S3AB V1 0 AEAR APD2010101 0201 76995_ 348_AE4R _FO2 ab1 E 76995 12 03 2007 E2 ev 54812 v1 0 BE2R B 070201 440301 76995_54812_B E2R _B03 ab1 76995 12 03 2007 E2 ev S4110 BER B 070201 440301 76995_5481_B E2FA03 ab1 76995 12 03 2007 E3 ev SABI2 v1 0 BER B 070201 440301 76995_ 4B12_B E3R _FO ab1 E 76995 12 03 2007 E3 ev S4110 BER B 070201 440301 76995_ 4B1_B E3R _E03 ab1 Seq Primer 76995 12 03 2007 E4 fwd 54B15_v1 0 BE4F B 070201 440301 76995_54815_B E4F _B04 ab1 master fle 76995 12 03 2007 E4 ev S4B15_v1 0 BER B 070201 440301 76995_S4B15_B E4R _D04 abt 76995 12 03 2007 E2 ev S3C5 v1 0 CER Cw 06020101 06 76995_S3C5_C E2R _A0S ab1 76995 12
24. R It is possible to have the wrong PCR product sequenced or a wrong primer ID was used Review Purification tray and Seq tray before repeating discrepancies Be aware of anomalous heterozygous positions and artificial peaks which may cause discrepancy between two sequencing orientation In these cases a repeat may not be necessary 4 6 Review the data available in SeqPilot manually and independent of the list of possible alleles The SBT report is generated by the technologist that performs the first analysis and electronically does the TECHNICAL VALIDATION TV on each test on a given run 5 SBT limitations Using the Protrans SBT kits there is no missing bases at the beginning or end of the exons sequencing data because all the PCR primers are outside of the exons e DRB1 SBT limitation Novel sequence outside of exon 2 will not be detected e Ambiguous allele assignments can occur when two alleles are present and the composite sequence is identical for more than one combination cis trans ambiguities Note See the Anthony Nolan Research Institute publication from April 2005 Exon Identities and Ambiguous Typing Combinations 6 SBT Kit Troubleshooting 6 1 Sequence Failures 6 1 1 Ifall reactions of an individual sample are negative the most likely explanations are o There was a missing faulty common reagent o Sequencing reactions set up precipitation failure 49 Sequence Based Typing o Poor manipulation during
25. Sample Src Project Procedures HLA C high State compl Join function The Join function allows the user to start checking the sequence data upload in the Seq Pilot software This feature indicates the reactions uploaded and also the ones not uploaded for a reason Un joined result files in the upper table can be joined manually by selecting an order in the Lower table For this please select the order by setting a hook into the box in front of the line select one or more entries in the Upper table and press the button Join The selected result files are joined to the selected order and deleted from the Upper table Please note that if the field DNA in the Lower table for a patient result file is shown with a grey background the order and the patient result file have different DNA numbers This can happen when result files are joined manually In this case please check if the patient result file is joined to the correct order 38 Sequence Based Typing If you click on this operation the following dialogue is opened Auto join function In case of entering orders with valid DNA numbers after loading result files or manual changing of DNA numbers of result files the function auto join can be used to join the orders automatically with their corresponding un joined result files Work list function This operation serves to get an overview about all existing patient orders and their joined result files If you
26. Sequence Based Typing Chapter II DNA Based Testing Section Application Module MODULE HLA SEQUENCE BASED TYPING Authors Dong Feng Chen Ph D D ABHI Angelica DeOliveira MS CHS Clinical Transplantation Immunology Laboratory Duke University Medical Center Durham NC REVISED 05 26 2009 Sequence Based Typing INTRODUCTION OF HLA SEQUENCE BASED TYPING Sequence Based Typing SBT is a technology used to determine the exact nucleotide sequence of a gene or a DNA fragment of interest Therefore itis a powerful tool to characterize the genetic complexity and allelic diversity of the HLA genes Recent technological developments have made sequencing sufficiently rapid cheap simple and robust thus making it feasible to perform high resolution HLA typing by sequencing in clinical HLA laboratories Sequencing is considered the gold standard for HLA allele identification and high resolution typing The Sanger chain termination based sequencing is a method of DNA synthesis It requires a single stranded DNA template obtained either by PCR amplification or by DNA cloning a primer that specifically anneals to the template a DNA polymerase like Taq DNA polymerase or Sequenase nucleotides deoxynucleotide dNTP dideoxynucleotides ddNTP and a fluorescent dye to identify the newly synthesized DNA strand Currently the fluorescent dye terminator technique is the most popular SBT strategy used for HLA typing The four color dye
27. Sequence Based Typing The following format is required by the Sequencer and also by SeqPilot software name of the used seq primer 0306781_BGM7_B E3F DNA number name of the used amplification module The information related to the sequence is enclosed in parentheses Within the parentheses the three sectors are separated by underscores this information is required by SeqPilot Any text outside the parentheses will not be regarded by the SeqPilot software HLA database To install the HLA database go to the download section of our home page www jsi medisys de and execute steps listed Genes and exons The following entries in the section HLA Genes of the lis ini file located in the bin directory of your installation are needed E2 E3 E4 E2 E3 E4 E2 E3 E4 E2 Please note that the program HLA checks these default entries for genes and exons Genes and exons which are not listed here can t be analyzed Load Sequencing Result files When choosing this command the following dialogue is opened The sequencer ABI is pre chosen to open the dialogue Load Result File If you don t want to continue the file import press the Destination ABI button Cancel and the dialogue Load Result files is closed Load Sequencing Resultfiles Cancel 36 Sequence Based Typing Order No DnaNo 76995 Sample G v Date Pat Name Project z Patient ID
28. This is a very important step and has to be done without interruption and in the exact order described The precipitation step will determine the quality signal strength of your sequencing data 1 Prepare an 80 ethanol solution mixing 16 ml of 100 ethanol to 4 ml of molecular biology grade water Mix well 2 Thaw the EDTA NaOAc solution vortex spin down gently before opening vial 3 Add 2 ul of the NaOAc EDTA solution to each sequencing reaction Note The static can prevent the drop to be dispensed from the pipette i Make sure the volume gets added to each well 4 Spin tray for 30 seconds at 2160g 5 Add 25 ul of 100 ethanol to each well 11 Sequence Based Typing 6 Place the full plate cover onto the tray and vortex the tray thoroughly Note Incomplete mixing will result in poor quality data 7 Centrifuge tray at 2000g for 30 minutes Note This centrifugation is important to ensure complete removal of unincorporated dyes 8 Immediately remove supernatant inverting tray on a paper towel stack 9 Place the inverted tray and paper towels in the centrifuge and spin at 500g for 30 seconds to remove the supernatant 10 Add 50 ul of 80 ethanol to the wells 11 Centrifuge at 2000g for 5 minutes 12 Immediately invert the tray on a paper towel stack 13 Place the inverted tray and paper towels in the centrifuge and spin at 500g for 30 seconds to remove the supernatant 14 If not performing capillary sequencing
29. Tools choose change polymer wizard and remove bubbles from the entire path manually Place buffer cup under lower block and close the equipment doors 19 Perform Spatial calibration for the capillaries checking the box Fill Capillaries C Run sequence analyzer 20 Open collection software and click on the NEW button under the Plate View page 21 Select Sequencing and a spread sheet will be displayed for the information required from each sample tested 22 Once the plate editor opens name the plate following the run ID criteria Example A zxzx 002 05 26 Each sample will have 3 entries Spreadsheet is designed as the columns from the plate 27 Names are identified per the following criteria Name locus primer ID P3456_ locus _2F ID locus primer 28 Each DRB1 sample will have 3 entries name locus different primer 29 Repeat for all the samples making sure the positions are correct Note The names given here will be in all the analysis reports 30 Fill the column indicating Dye Set Mobility File Run Module Analysis File Dye Set set by ABI when installing your genetic analyzer e Mobility File DT3100POP6_36cm mob e Run Module 36cm_ 5sec_POP6 module Run temperature 55 C Leak threshold 25 steps Current tolerance 100uAmp Run Current 100uAmp Voltage tolerance 0 6 kvolts Pre Run Voltage 15kvolts 14 Sequence Based Typing Pre Run time 180 seconds Injection Voltage 1kvolts Injection Time 5 seconds Run Vol
30. assay or document the justification for acceptance of an assay when acceptance criteria are not met D 4 11 10 8 If presence of an amplified product is used as the end result include controls to detect amplification failure in every amplification mixture D 4 11 10 9 Utilize oligonucleotide probes under empirically determined conditions that achieve the defined specificity D 4 11 10 10 Perform quality control testing to confirm specificity for each lot and shipment of primers and probes D 4 11 10 11 Use reference material for quality control of new lots 57 Sequence Based Typing D 4 11 10 12 Use reference material for each shipment when it contains a labile reagent D 4 11 10 13 For each new lot or shipment of commercial kits perform parallel tests with reference DNA specimens or spiking positive panel to assess that all components of the typing kits are working properly D 4 11 10 14 Have available a sufficient number of DNA samples of known class I or class II alleles and representative of the ethnic population served by the laboratory D 4 11 10 14 1 For each new lot of kits perform parallel testing using the number of reference samples determined by the Director or designee for the size of the kit and frequency of use D 4 11 10 14 1 1 When possible include testing of alleles known to have demonstrated weak false negative amplification with previous lots of the same kit D 4 11 10 14 1 2 When possible include testing
31. calls in hemizygous sequence data is greater than 40 per single sequence orientation 48 Sequence Based Typing Note Some exceptions may apply when the number of edits exceeds 40 but a confirmation of the present alleles is supported by another method that obtains the same result and has reliable data 4 2 If only one DRBI allele is assigned possible homozygous the result must be confirmed by another method SSP Luminex etc or using different SBT reagents Note HLA Class I reagents have an All Alleles PCR product that once sequenced can identify most alleles missed by the different allele group mixes 4 3 Repeat the entire SBT when rare alleles are identified Use at least two additional separate PCR products to be sequenced and confirm typing using different reagents When a potential novel allele is confirmed sequence data should be submitted to GeneBank and to the WHO HLA Nomenclature Committee for name designation 4 4 Weak PCR amplification usually causes weak signal strengths as a result non reliable assignments can be made The average signal strengths across all four bases should be equal greater than 40 T A C G 4 gt 30 If after data being forced into the software to be analyzed it fails to meet this requirement and requires more than 20 editing exon PCR should be repeated because data may not be reliable 4 5 Any discrepancy between sequencing orientations must have the data reviewed before repeating PC
32. ch between the primers and target DNA that may present a novel allele differing at the binding sites 6 2 3 Technical failure e Allele primer may be not present in the primer strip causing failure of PCR reaction e Lack of AmpliTaq Gold in the PCR reaction may cause failure of PCR reaction e Problem during dilution of PCR reaction to perform electrophoresis can cause failure 6 3 Anomalous Heterozygote Positions With dye terminator sequencing the peak incorporation patterns are not completely uniform For homozygous positions this is not a problem but for certain heterozygote positions one peak may be present at a much lower level than the other Consequently the allele calling software may not correctly identify these positions as heterozygous sequencing in both orientations By sequencing in both orientations the number of these anomalous positions can be minimized DRB1 09 and 10 on S4 DRB1 kits 51 Sequence Based Typing Furthermore the incorporation patterns are very reproducible and most anomalies have been identified by analyzing a relevant panel of DNA samples 7 SBT QC General Guidelines 7 1 PA 7 3 7 4 Ta 7 6 dads 7 8 7 9 Select appropriate number of samples to either QC a new lot 2 3 or a new batch shipment 1 of reagents Find from database the SBT run file they were tested in write down information on the QC form and make a copy of the short form of the SEQ Pilot a
33. click on this operation the following dialogue is opened Worklist 210 2005 js ge oes E fad SAR DREF ORBI 13010 fad S4A2V1 8 DREF DRET tev S3A2 v 0 EIA md SAAD VLD AEF fnd S3A8 4 0 AEF ew vl AE2R SA V1 0 AER 5348 v1 0 Afar id S342V1 0 ACSF ew vi fnd Bca toed tev 5345v 0__ AERA 42601 2524 fud S345 v1 0 AEI A2601 2524 39 Sequence Based Typing SeqPilot Sequence Analysis This operation is the main part of the program HLA If you click on this operation the following dialogue is opened Sequence m Dicer OrderNo 52380001 Patient P M054818 DNS No M05418 Dale 06 26 2005 11 48 5 Sange Sic Picject Piocedses HLA Dw hich mfl a Cute lt 1 cwm Siate cornpl combined seq p Eommets 1 E2fd Haplotypes 1 2 Show adra frame check height T 3 z eigh z E Z pe J r y i of ga dua oe helerozyoous resull s based on haplolypes Zhet 1 2 Co 04 04010102204040 7040 402 0408 0403N 7041304 4 RI E2rev Heplotppe 2 F compl revers HS E2rey Haplotypes 1 2 N comal revere x r ExonAniron z a 4 270 het 142 2 E2 ew te 270 hemi 2 305 2 Cw04010101 3 E2 re Me 270 het 42 S3C8 3 Cw04010101 Cw 0510 4 Cw 040102 Cw D602 060 5 Cwndo103 Ow 0602 060 2 E raenank N EN nen This dialogue part shows all examined genes of an order
34. d correct unless the number of mismatches indicated is 0 unless a novel allele is present 12 Several allele pairs can have 0 mismatches This is because different heterozygous allele pairs may have identical sequence 13 These are heterozygous ambiguities Some alleles are identical in the sequenced region Sample Name Base Selection Layer Selection Confirm Base Call Current Search Position Filters Information Editing is performed on the consensus base that is highlighted by the Editor Bar using the base selection keys 20 Sequence Based Typing 14 The Navigator tool can be launched by selecting Launch Navigator from the menu 15 Checking the BCS and mismatch boxes enables fast verification of bases with low Base Call Scores and consensus sequence which are mismatched with allele pairs within the assignment pane Button Function anc Moves the Editor to the first and last base in the analysed sequence respectively and Moves the Editor to positions that have low BCS or are mismatched with the allele pairs in the assignment pane see 4 1 2 and Moves the Editor by a single base in either direction Sample Selection Moves to the next sample Moves to the previous sample 16 Once each sequence data is analyzed save your changes before closing the software 17 Create a report for each locus analyzed References 1 ASHI laboratory manual 4th Edition 2 Allele SEQR HLA SBT
35. e less than 70 b There must be signal present at the edited position for each base in the new call c The algorithm is weighted towards including extra bases rather than changing heterozygous to homozygous It minimizes the possibility of incorrect calls d All the automatically edited positions are stored in the Edit list and highlighted in red above the consensus sequence e Clicking the Undo button can change all the automatic or manual editing 9 The Assign SBT test sample analysis pane is composed of 3 panes A Sample Pane B Sequence Pane C Assignment Pane e The first line contains the library name and date e The active sample is highlighted dark blue with white text e Either the mouse or the scroll bar can let you select the sample to be analyzed e The consensus sequence the electropherogram display and the allele assignment are updated automatically as you move between samples e Samples highlighted in orange have warnings associated with them Show the warnings by a right click on the sample 18 Sequence Based Typing beiieie i jaiicie 2 BaiilPartisn 3 foetal mh Cones BS fa See E a a NEIUT 10 The Sample Pane Samples highlighted in orange may have warnings associated with them Click on the sample and you can read the warning 10a The Sequence Pane displays information about the reference sequence the consensus sequence and the electropherogram associated
36. e vortex well and spin down Place buffer and Taq Gold on an ice box rack Note From this point work inside the laminar flow hood Add the appropriate DNA volume 2 ul for DRB1 or 4 ul for each HLA class to each pre labeled PCR tube Prepare the master PCR mix for the locus tested using the chilled thawed PCR buffer and Taq Gold Sequence Based Typing 2 7 Vortex master mix well spin down briefly and add 10 or 16 ul to each PCR tube containing the different DNA samples 2 8 Cap the tube strips and take it to pre warmed thermocycler 3 Amplification Profile 3 1 Place the tube strips in the thermocycler and run the following profile Locus Specific SBT PCR profile 95 C 10 minutes 1 cycle 96 C 20 seconds 60 C 30 seconds 32cycles 72 C 2 minutes 4 C oo 4 PCR verification 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 4 10 4 11 4 12 While PCR profile is running prepare a 2 agarose gel using 0 5X TBE Use 2 grams of agarose in 100 ml buffer and 3 ul of ethidium bromide Cool gel and once profile is finished remove tubes from thermocycler Spin strips containing PCR products before opening them Remove 2 5 ul of each PCR product and dispense on a 96 well tray Close strips back using a new set of tube caps and place in the refrigerator if continuing with the procedure immediately Otherwise freeze at 20 C Add 10 ul of loading dye to each 2 5 ul of PCR product
37. e caps ABI 801 0535 Lint free tissue Kimwipes MicroAmp Optical 96well reaction plate ABI N801 0560 MicroAmp Support Base ABI N801 0531 Genetic Analyzer Plate Septa 96 wells ABI 4315933 Genetic Analyzer Plate Retainer ABI 4317241 1 2 3 4 5 6 MicroAmp Full Plate Cover ABI N801 0550 7 8 9 1 0 Capillary Array 16x 36cm ABI 4315931 Sequence Based Typing Equipment OONBANA WN Vertical laminar flow hood Thermocycler with heated lid and 96 well format Pipettes volumes 1 20ul 20 200uI and 100 1000uI Agarose Gel apparatus Gel documentation camera Electrophoresis power supply Variable speed vortex Table top centrifuge 96 well trays holder ABI 3130XL Analyzer 16 capillary 10 Computer 11 Printer Procedure l Creating a panel for PCR amplification dl 1 2 1 3 Create a SBT PCR list indicating order of samples to be tested for each locus Arrange DNA samples in an order that follows the SBT PCR list to prevent pipetting error Label the PCR tubes using the numerical order from the SBT PCR list Preparing the PCR reaction These steps are done in the Pre amplification area 2 1 2 2 2 3 2 4 2 5 2 6 Remove the DNA samples concentration already adjusted to 20 30 ug ml from the refrigerator and vortex spin down each one before opening them Remove PCR buffer and Taq Gold from freezer Let buffer thaw at room temperatur
38. equencing 6 1Choose a minimum of 2 positive PCR products or in case of homozygosity 6 2Select the appropriate LSA for the loci being tested 6 3ldentify in PPA the chosen positive reactions these will proceed to Cycle Sequencing 7 ExoSAP IT PCR product treatment The PCR products have to be purified before being used as sequencing templates because residual PCR primers and nucleotide triphosphates dNTPs can interfere with the SBT chemistry resulting in lower data quality An easy way to perform PCR purification is by using Exonuclease and Shrimp Alkaline Phosphatase enzyme mix ExoSAP IT treatment of PCR products that will remove unincorporated primers and dNTP s 7 1 Take the vial of EXoSAP IT from the freezer 7 2 Add 4 ul of ExoSAP IT to the required wells of the Pur tray following the Purification tray map generated previously in PPA 7 3 Transfer 10ul of each positive amplification selected to be sequenced into the appropriate wells of the Purification tray created following the PPA location for each positive reaction chosen to be sequenced 7 4 Seal the purification tray with micro amp full plate covers or caps 7 5 Spin tray down at 1000g for 20 30 seconds 7 6 Place in the thermal cycler and run the following profile 27 Sequence Based Typing ExoSAP IT Thermocycler_ Profile 37 C 15minute 80 C 15minutes 1 cycle 4 C o0 7 7 Remove treated PCR from Thermal Cycler Note To each DRB1 product
39. facing the user to the left side 1 2 Cross check as you go to ensure that the ID in the tube strip and the ID from the DNA tube are the same 8 PCR reactions format Mix 1 is marked in black 4 OQOO0O0OO0OO0OO0G 15 PCR reactions format Mix 1 at the cut corner 4 YOOOO Q QVYVVDVDVWVO Sequence Based Typing 2 Preparing the PCR reaction These steps are done in the Pre Amplification Area inside the laminar flow hood 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 Remove PCR mix PSD and Amplitaq Gold from freezer Let the PCR mix thaw to room temperature RT Let PSD sol thaw vortex well and spin down Place PSD on ice box rack Note From this point work inside the Laminar Flow Hood Prepare the master mix for each sample tested using the chilled thawed PSD solution and Taq Gold Add 15 ul of the PCR Amplitaq mix to the negative well before adding the DNA sample to the PDS mix Add the appropriate DNA volume to each PCR mix tube vortex master mix well spin down briefly Dispense carefully to the wall of each tube Follow SOP procedure used in the lab regarding pre amp and post amp precautions to maintain pipettes gloves lab coat racks separated between pre and post PCR areas 2 10 Runthe SBT PCR amplification profile using a thermocycler 3 Amplification Program Group Specific SBT PCR volume 15ul 95 C 2 minutes 1 cycle 24 Sequence Based Typing 96 C
40. h states the mismatching positions In this case the sequence is not joined Note hemizygous result file sequences only can be joined to haplotype 1 or 2 Heterozygous result file sequences always belong to both haplotype 41 Sequence Based Typing Hide sequence Show sequence The item hide sequence is shown if the selected result file is not hidden yet If you select this item the result file is hidden within the Electropherogram and in the column State of this dialogue part an H is shown The item show sequence is shown if the selected result file already is hidden If you select this item the result file is not hidden any longer and shown again in the Electropherogram Result Within this dialogue part the total results calculated for the selected gene in the Dialogue part Genes are shown The first line indicates whether it is a heterozygous or a hemizygous result and on which haplotype the result calculation is based on If the Tab Haplotype 1 or the Tab Haplotype 2 of the Matching table is selected this dialogue part shows additionally the hemizygous results for the selected haplotype tab in the first lines If you select this item of the corresponding context menu within the Upper table or Lower table of the Operation Joining or within the Electropherogram the following dialogue Is opened W Edit Bascs 7 i i f 1 xj 5091 642 Seq3 Seq Seq5 3 edited het pos mism Gone Cw Directicn Fwd Fie HLAseque
41. he selected gene order in the Dialogue part GenesTab Order of the Dialogue par Order Statistic is not TV the state is set to _ TV If the state already is TV the state is removed to the state it had before it you press this button and the state of the selected gene order in the Dialogue part Genes Tab Order of the Dialoque part Order Statistic is not MV the state is set to MV If the state already is MV the state is removed to the state it had before Print If you press this button the dialogue Print Preview Print Reports opens Comment f you press this button the dialogue Comment is opened In the appearing comment box you can enter new comments or edit comments Option Print If you select this item the following dialogue is opened Sequence Print Preview Si HLAsequencing M054818_53C8_C E2F 008 ab1 Seq HL4sequencing_ M954618 5901_C E2R 001 sbi Seq3 HLAsequencing_ M054019_S3C5_CE2A _002 0b1 Seqd enmi POC 8 S3CE_C E2R _ 004 561 H j g 53C9_CE2RL0CE ab Sereen I soroerparl oriy total approx bases per line a ssbctal Emn 44 Sequence Based Typing Printing Reports After the analysis phase you have the possibility to print reports The following dialogue is opened if you press the button print of the Print function i x HLA Patient Report long HLA Sequenci HLA Patient Report short HLA Sequene Printing Options Activate this option
42. ification Area 1 1 PRE PCR 1 1 1 All DNA preparations and PCR setup are handled in a Laminar Flow Hood contamination control chamber in a Pre Amplification room 1 1 2 All Pre PCR reagents are thawed to room temperature vortex spun and kept cold until ready to be used 1 1 3 Wear a dedicated pre amplification lab coat and fresh gloves when preparing samples or reagents for PCR amplification 1 1 4 Open and close all sample tubes carefully to avoid reagent or sample splashes Always vortex spin down vials of DNA and or reagents before opening with intention to use it 1 1 5 Use positive displacement or air displacement pipettes with filter plugged tips Change tips after each use 1 1 6 Keep all the racks and lab coats used in pre amplification area Amplified material should never re enter the Pre PCR room 1 1 7 A new lot of reagents must be QC d and approved before use in clinical typing e New Lot QC d with 2 3 DNA samples previously tested e New batch shipment QC d with 1 DNA sample previously tested e Unusual shipment delays will be QC d as new lots 1 1 8 A new lot is identified by a new version ID indicated in the heading of documentation associated with the Lot Amplification and Sequencing Unit number Sequencing Primers for the different loci exons tested are listed for QC purposes in the QC documentation only 1 1 9 New lots of AmpliTaq Gold BDTv1 1 Big Dye Terminator and ExoSAP IT tested along
43. immediately seal the tray with the full plate cover and plastic wrap and store tray at 20 C 15 If running electrophoresis immediately add 15 ul of Hi Di formamide to each well and place tray for 2 minutes at 95 C and place tray on ice for at least 2 minutes 16 Spin tray for 30 seconds at 1260g and place it on the ABI 3100 Support Base observing that the position A1 is at the top left corner 17 Place a Full Plate Septa over the tray and follow with the 96 well Plate retainer Note The position of the retainer over the Septa should be perfectly set to prevent damage to the capillary when collecting samples 18 Place Support base Tray Septa retainer onto the ABI 3100 Analyzer and get the run started 3100 Analyzer preparation for a run A Calibration of 3100 Analyzer Spect36_POP6 default module Run temperature 55 C Leak threshold 25 steps Current tolerance 100uAmp Run Current 100uAmp Sequence Based Typing Voltage tolerance 0 6 kvolts Pre Run Voltage 15kvolts Pre Run time 180 seconds Injection Voltage 1kvolts Injection Time 22 seconds Run Voltage 15kvolts 1 Select the spectral calibration parameters SeqStd any dye Set par 2 Link the plate just created from the Pending Plate Record to the corresponding graph of the loaded tray A or B then click to start the run 3 Once the run is complete accept the result by clicking OK The software will then assign calibration values to passed failed capilla
44. ing algorithm The Assign software was developed by scientists with extensive experience in DNA sequencing based HLA typing in a clinical HLA laboratory It was specifically designated for HLA SBT but applicable to any re sequencing application including SNP scoring Note The allele database utilized by Assign SBT 3 2 7 will have updates performed twice yearly following the updates provided by the IMGT HLA Sequence Database The following analysis procedure was established based on the version Assign SBT 3 2 7 Sequence Based Typing Equipment 1 Computer using Windows XP 2 External Zip Disk Drive 3 HP color printer Procedure 1 Once data is analyzed by Sequencing Analysis close out of software and open data from the Extracted files folder and save to a zip disk naming the copied folder with the ID given to that specific locus SBT run 2 Operator Login can be done by clicking the Assign exe icon The operator login dialogue prompts the user to the operator ID admin and the password 3 Click submit to login Operator Err Current Password DOO O Update New Password DO O O O Retype Password Po Edit Operator o Remove User Default settings ReH x Add User Encoding jur x Quit e The Analysis window will appears once you logged in rer No samples loaded nesar mwr msan aS o D TH a a Weta o sor ee E e ee SEB assem 4 In order to perform sequence analysis using Assign SBT
45. k areas D 4 1 Laboratories performing nucleic acid testing must have written criteria or protocols for D 4 1 1 Accepting the validity of each molecular assay D 4 1 2 Preventing DNA contamination using physical and or biochemical barriers for assays involving amplification of templates D 4 2 Laboratories performing HLA typing must have written criteria or protocols for D 4 2 1 Preparation of cells or cellular component isolations for example soluble antigens and nucleic acids as applicable to the HLA typing technique s performed D 4 2 2 Selecting typing reagents whether prepared in house or commercially D 4 2 3 Ensuring that reagents used for typing are adequate to define HLA specificities or alleles that are appropriate to the clinical application D 4 2 4 The assignment of HLA antigens and alleles D 4 2 5 Determining when antigen or allele redefinition and retyping are required D 4 2 6 Documentation of antigens and or alleles that are defined by each test system used in the laboratory D 4 6 11 Laboratories performing nucleic acid testing must D 4 6 11 1 When applicable interpret data using the IMGT HLA nucleotide sequence database or equivalent The database that is used must be updated at least every six months D 4 6 11 2 Ensure that the laboratory has criteria for accepting each lot and shipment of primers or probes 53 Sequence Based Typing D 4 6 11 3 Have acceptable limits of signal intensity for posi
46. nalysis Using database prepare new SBT work list using the letters QC behind each DNA sample to use as a guide for the SBT procedure remainder steps Find the DNA samples from their appropriate location Make the dilutions of the DNA and have the appropriate typing strips PSD buffer AmpliTaq Gold Set up the SBT PCR per SOP When setting up the sequencing map in PPA use the letters QC behind the DNA number for each sample This will allow each sample to be analyzed in SeqPilot independently from previous tests Confirm that a new lot of ExoSAP IT is or not available and also the BDTv 1 write down the lot numbers of these reagents in the SBT run report form filled for the QC run Confirm that the Sequencing Primers identification for each locus exon direction matches the information provided When a new lot is received for QC purposes run the primers that react for each positive PCR mix in BOTH Directions The expected primer reactivity for each PCR mix can be obtained from the Appendix available for each locus SBT appendix The primer boxes that don t have anything written on them indicate that the mix primer mixes are listed in rows and exons directions are listed down in columns don t react with that specific exon direction Mark the positive reactions observed in the photo documentation per SOP Use the Appendix as the guide for the Primer mixes tested along with the PPA forms filled in the computer
47. nd calibration verification procedures D 4 9 2 For thermal cycling instruments the appropriate target temperatures must be achieved Accuracy of these temperatures must be verified and documented at least every six months D 4 10 4 Laboratories performing nucleic acid testing must D 4 10 4 1 Use a method to prepare DNA that provides sufficient quality e g purity concentration and quantity to ensure reliable test results Written guidelines will specify the minimal acceptable sample in terms of volume or numbers of nucleated cells D 4 10 4 2 Handle and store specimens under conditions that maintain sufficient integrity of nucleic acids to ensure reliable test results 56 Sequence Based Typing D 4 10 4 3 If nucleic acids are not used immediately after purification ensure that samples are stored under conditions that preserve the integrity of the nucleic acids that will be tested D 4 10 4 4 Ensure that samples containing nucleic acids that will be amplified e g blood DNA isolates are stored under conditions that do not result in artifacts inhibition of the amplification reaction or exposure to sources of carry over contamination D 4 10 4 5 Ensure that samples containing nucleic acids that will be used in a primary amplification are not exposed to post amplification work areas D 4 10 4 6 Have sequencing templates with sufficient purity specificity e g locus or allele specificity quantity and quality to provide inte
48. ne in the forward direction and the other in the reverse direction This method has a great potential for high throughput typing but yields a high percentage of ambiguities mainly caused by the incapability to determine the cis trans linkage of the polymorphic motifs Other HLA typing methods may be needed to resolve the ambiguities Group specific amplification and sequencing A set of group specific primer pairs are used to separate alleles in different tubes wells and each allele is then sequenced separately This approach dramatically reduces the ambiguity rate to a minimum and additional ambiguity resolving tests are rarely needed Generic multiplex amplification and group specific sequencing Upon the generic amplification of all alleles in one tube with one pair or a mixture of multiple primers a set of group specific primers is used to sequence the PCR products Individualized allele specific amplification and sequencing Based on the availability of the low resolution typing results allele or group specific primers are selected for individual allele amplification and sequencing For example for a DR4 and DR15 positive sample DR4 and DR15 16 specific primers can be chosen for the amplification and sequencing The following factors for selection of SBT method and reagent may be considered e Robust and reliable e Typing resolution and ambiguity rate e Turn around time Sequence Based Typing e Labor bench work incl
49. ng data to be analyzed e Choose a sample to analyze and click on Sequence Choose first gene and the first exon to analyze most of the time it will be exon 2 e Scroll through entire sequence data watching for low peaks indicating background noise irregular migration compression areas peak shoulders heterozygous positions or presence of dye blobs e Compare sequences of the same exon on opposite orientation to make sure they are complementary to each other Edit data as necessary e Check all the exons for the gene being analyzed to verify that there are no mismatches to rule out the possibility of recombination e After analyzing all the exons if there are still mismatches or if the software can t agree upon an allele or pair of alleles reanalyze sequencing data and look for missed ambiguities or basecall and check the initial PCR gel documentation e When a result can t be reconciled repeat sequence reaction using the same PCR product and include other PCR positive and or other primers in both directions When allele combination or single allele with no mismatch is clearly achieved click the TV Technical Validation box to validate the test result print out the HLA and the HLA short reports 11 Troubleshooting Guide 32 Sequence Based Typing PCR is an extremely sensitive method which can efficiently amplify the very small amount of DNA Therefore even trace of contaminating DNA in a sample can be
50. obes D 4 6 16 3 4 Define the HLA locus and allele designation s for each template primer and or probe combination D 4 7 7 Nucleic acid testing D 4 7 7 2 Laboratories performing sequencing must D 4 7 7 2 1 Ensure that the methods employed for preparation of sequencing templates do not alter the accuracy of the final sequence e g mutations created during cloning preferential amplification D 4 7 7 2 2 Ensure that the conditions for primer extension in cycle sequencing reactions e g polymerase type polymerase concentration primer concentration concentration of nucleotide triphosphates concentration of terminators are appropriate for the template e g length of sequence GC content D 4 7 7 2 3 Establish criteria for acceptance and interpretation of primary data e g correct assignments for non polymorphic positions definition of sequencing region criteria for peak intensity baseline fluctuation signal to noise ratio and peak shapes Document established sequence specific artifacts and utilize the information in routine interpretation of data D 4 7 7 2 4 For heterozygous templates if only one strand is sequenced ensure that sequencing of only one strand consistently yields accurate sequence assignments Sequencing of sense and anti sense strands is strongly recommended If assignments are routinely based upon data from one strand of DNA periodic confirmation of complementary strands is recommended D 4 9 Calibration a
51. only add 20 ul molecular biology grade H20 to each PCR product treated with ExoSAP IT 7 8 At this step you can freeze the products to continue the sequencing reactions later 8 To Create the Sequencing Tray using PPA software Note Choose on the upper right corner box drop down menu 3130xl format or 3730 format and keep track of this choice all the way because the equipments 3130XI 16 capillaries and 3730 48 capillaries use a different approach during sample injection 8 1 Click on the Sequencing Plates tab 8 2 In Purification Plate ID type the purification tray name EXACTLY case sensitive and click Search or you can leave the tray name blank click search and scroll down the list to find the correct Purification Tray 8 3 Click on the desired locus exon direction tab then click Join 8 4 Type a name for the sequencing tray ABI analyzers only accepts underscore as a connecting character when creating sequence tray identification 8 5 Continue to Join locus exon primer orientation tabs until all positive reactions are added to the sequencing tray 8 6 Once finished Joining click Plate Record this will prompt you for a name the default tray name is the one assigned above Just click OK to accept it 8 7 Click Print This will also prompt you for a tray name just click OK This will also tell you that the tray already exists and asks if you want to continue Click Yes 8 8 Transfer Sequencing Plate template
52. or v1 1 from ABI Cycle sequencing products are precipitated using ethanol and sodium acetate EDTA to eliminate the unincorporated nucleotides The sequencing products are re hydrated in formamide and injected in the capillary electrophoresis genetic analyzer utilizing appropriate module and mobility files 5 Sequence Based Typing The data is pre analyzed using ABI sequencing analysis software and ABI file formats are imported to the HLA analysis software that will compare the sequence obtained to the IMGT HLA sequence library Innumerous factors are relevant to the data analysis Specimen 2 4 ul genomic DNA locus in molecular biology grade water The integrity of the genomic DNA used is very important Reagents A Provided with the kit AUN o Locus Specific PCR Pre Mix AmpliTaq Gold 5U w ExoSAP IT Three DRB1 Sequencing primer mixes Exon 2 F 2R and Codon 86 Six HLA A HLA B and HLA C Sequencing Primer mixes Exon 2 F 2R Exon 3F 3R Exon 4F 4R per locus NaOAc EDTA Buffer B Additional Reagents needed but not provided with the kit 1 2 4 5 6 Supplies Molecular biology grade water Ethanol 99 anhydrous molecular biology grade Sequencing buffer 10X with EDTA POP 6 Performance Optimized Polymer from ABI Hi Di Formamide from ABI Pipette tips for the following volumes 1 20ul 21 200ul 100 1000ul PCR tubes 0 2 thin walled ABI N801 0838 PCR tub
53. rcing 054918 Gli Gv TA 2 1 Ltt W Phill IT CELLET TI consensus IT tt Ri edted PA calculated original 3 i 1 sel i 42 43 Alo if ep poate eee es 7 Count mode p ik reverse Edies T 442 56d 6y 663 G12 S01 f 353 ep orairal G lg 12 p Sa E fares jase 5 complement 5 A ro fo a Cancel Within this dialogue you can see and edit the original electropherogram s of the selected result file 42 Sequence Based Typing Functions IN mvj pit comment Matching Table Total Result Haplotype 1 Haplotype 2 Haplotypes 1 2 0 2 Cw 04010101 Cw 0607 1 E2ft 3 Cw 04010101 Cw 0610 1 E2f1 4 Cw040102 Cw 0602 060 1 E2 1 5 Cw 040103 Cw 0602 060 1 E2 1 xi E CaeAAne CaetRN ZARA 1 Foni This table shows all possible hemizygous results allele combinations sorted by their probability table consists of up to four tabs e Total Results e Haplotype 1 e Haplotype 2 e Haplotypes 1 2 43 Sequence Based Typing Button Previous Next TV Technical Validation MV Medical validation Function Press this button and depending on previously created list in the Operation Joining or Operation Worklist the previous order is shown Press this button and depending on previously created list in the Operation Joining or Operation Worklist next order is shown If you press this button and the state of t
54. ries as well 4 Under File click on Override Spectral Calibration to allow the examination of the data for each capillary B Refilling capillary array syringes and buffer water reservoirs 5 Remove both syringes from equipment and dispose the leftover POP 6 6 Rinse them thoroughly using warm tap water followed by dH20O followed by molecular grade water 7 Prime them both using a small volume of POP 6 and reserve 8 Disconnect the capillary array from the upper block making sure to protect the light path window 9 Flush them both with warm tap water followed by dH2O and then molecular grade water 10 Using the vacuum line dry both syringes very well 11 Remove all the buffer water cups and rinse them thoroughly using molecular biology grade water 12 Prepare 50 ml of 1X ABI running buffer using 5ml from the 10X vial mixed with 45 ml of molecular grade water 13 Fill the analyzer buffer cup using about 15 ml of 1X ABI buffer 14 Fill the reservoirs for waste and washing with molecular grade water approx 15 ml each and fill the front left reservoir with 15 ml of 1X ABI buffer 15 Place the septa in each one and place them back on the Autosampler 13 Sequence Based Typing 16 Place the blocks back and reinsert the capillary array making sure is tightly set in the proper position Do not twist the capillaries 17 Fill the large syringe with 2 3 ml of POP 6 and fill the small syringe with 300ul 18 Under
55. rpretable primary sequencing data D 4 11 Control procedures D 4 11 4 4 For each electrophoretic procedure include concurrent with patient specimens at least one control material containing the substances being identified or measured e g molecular weight markers D 4 11 10 Laboratories performing nucleic acid testing must D 4 11 10 1 Routinely monitor for contamination of the most common amplification products that are produced in the laboratory D 4 11 10 2 Routinely monitor pre amplification work areas with wipe tests D 4 11 10 3 Monitor potential contamination using a method that is at least as sensitive as routine test methods and that uses the routine testing primers At least one negative no nucleic acid and one positive control must be included in each amplification assay D 4 11 10 4 If amplified product is detected clean the area to eliminate the contamination and document re testing as well as the measures taken to prevent future contamination D 4 11 10 5 To minimize the detection of minor contaminants and the occurrence of stochastic fluctuation during thermal cycling set the number of cycles at a level sufficient to detect the target nucleic acid but insufficient to detect small amounts of contaminating template D 4 11 10 6 Monitor the quantity of specific amplification products e g gel electrophoresis hybridization D 4 11 10 7 Adhere to the established criteria for accepting or rejecting an amplification
56. ry Clean blocks and refill the capillaries with fresh polymer 34 Sequence Based Typing Incorrect 10X Buffer dilution Consider the option of change the capillary if problem persists after replacing buffer and flushing polymer block with water before refilling with fresh polymer Data Analysis using Sequence Pilot Software After the sequencing run is complete results can be displayed analyzed in the Sequencer computer and produce ABI data files sequence trace file that can be imported into the HLA SBT software of choice Sequence Pilot features require that the files to be imported following the guidelines for sample naming dye set analysis format and data quality All this information is available to the user in Sequencing Analysis 5 2 SeqPilot features include a base caller for accurate base calling of most of heterozygous data an algorithm for determination of the consensus sequence and a sequence alignment algorithm SeqgPilot was developed for a high throughput sequencing system to typing hundreds of samples daily It is developed for general sequence data analysis with a special component for HLA SBT Sample naming conventions of SeqPilot For automatic joining of patient orders and their result files the program HLA needs special information for each result file DNA number unique number for each examined DNA in your laboratory Name of the used amplification module Name of the used sequence primer 35
57. sion e g polymerase type polymerase concentration primer concentration concentration of nucleotide triphosphates are appropriate for the template e g length of sequence GC content D 4 6 11 13 9 Ensure that for each set of primers conditions that influence the specificity or quantity of amplified product have been demonstrated to be satisfactory for the range of samples routinely tested D 4 6 11 13 10 Ensure that template quantity and quality are sufficient to provide interpretable data for a locus or loci or allele s D 4 6 11 13 11 Ensure that the amount of amplification template in each amplification reaction is in an acceptable range D 4 6 11 13 12 Define and document the specificity and sequence of primer targets The genetic designation e g locus of the target amplified by each set of primers must be defined and documented For each locus analyzed the laboratory must have documentation that includes the chromosome location the approximate number of alleles and the distinguishing characteristics e g sizes sequences of the alleles that are amplified D 4 6 11 16 Laboratories performing sequencing methods must D 4 6 11 16 1 Ensure that the method for preparing sequencing templates reliably generates appropriate length sequencing templates that are free of inhibitors of subsequent reactions e g residual primer extension and free of contaminants that cause sequencing artifacts D 4 6 11 16 2 Ensure the use of a scien
58. step is performed correctly minimal dye blobs will be present in the sequencing data 6 1 4 3 Noisy sequences may be due to e Failure to add correct volume of ExoSAP IT to PCR products prior to setting up the sequencing reactions e Inactive ExoSAP IT added to the PCR products e Incorrect Module 50 Sequence Based Typing e Weak miscellaneous peak size shape due to incorrect volume of BDTv1 1 6 1 4 4 Incorrect Ethanol precipitation may result in excess salt remaining in the sequence reaction e These salt ions will preferentially inject onto the capillary over the sequence DNA molecules resulting in weak sequence Be sure to perform the ethanol precipitation correctly and do a well measured correct concentration wash step afterwards 6 2 Allele drop out 6 2 1 Rarely a particular allele may not be amplified e This may be caused by a denaturation failure which may be related to proximity to regions of very high GC content which serve as clamps during denaturation e Ifthis occurs the allele will usually amplify after heating the DNA for 5 10 min at 65 C and placing the sample immediately on ice just before re setting the PCR 6 2 2 Allele drop out may also occur when a DNA sample with poor quality is used e If this is the reason the typing will be repeated using a re purified DNA isolate with optimal quality Check DNA on a 0 8 Agarose gel to check quality e Allele drop out can also be caused by mismat
59. t quantity and quality of DNA for testing including specimens containing a low number of cells D 4 6 11 13 Laboratories performing amplification based methods must D 4 6 11 13 1 Ensure that pre amplification procedures are performed in an area that excludes amplified DNA which has the potential to serve as a template for amplification for any of the gene targets that are amplified in the laboratory D 4 6 11 13 2 Ensure that equipment used for post amplification products with the potential to cause contamination is not used for pre amplification procedures D 4 6 11 13 3 Ensure that each work area i e pre amplification secondary amplification and post amplification has dedicated equipment Positive displacement pipettes or filter barrier tips are recommended for pre amplification and secondary amplification work areas D 4 6 11 13 4 Ensure that thermal cycling instruments achieve the appropriate target temperatures during cycling D 4 6 11 13 5 Ensure that all batches of aliquoted reagents solutions containing one or multiple components utilized in the amplification assay are demonstrated to be free of contamination 54 Sequence Based Typing D 4 6 11 13 6 Ensure that reagents used for primary amplification are not exposed to post amplification work areas D 4 6 11 13 7 Ensure that reagents used for secondary amplification are stored in a contamination free area D 4 6 11 13 8 Verify that the conditions for primer exten
60. tage Skvolts e Analysis Module BC 3100RRv2_SeqOFFFToff saz 30 Save information Click close New tray ID will be displayed on pending plate record screen Link plate clicking on its ID and on the now yellow graph A or B that represents where the plate was placed in the Autosampler 31 The Tray ID will be displayed on Linked plate Record and the green triangle button from the heading of the screen can be selected to start the run 32 It will take about 10 20 minutes until the samples start to be collected In order to expedite the heating of the over set the manual control to pre heat the oven at 55 C 33 All the data obtained will be available on the extracted files folder 34 Analysis proceeds using the sequencing analysis software that will allow looking at the raw data and adjusting the beginning and end of data collection for each sample Also the signal intensity of each individual reaction will be taken in consideration HLA SBT DATA ANALISYS USING ASSIGN SOFTWARE After a sequencing run is completed raw data can be visualized in the computer screen Assign SBT is designed for use with ABI format sequence files after they have been analyzed by the version of sequence analysis available in the Genetic Analyzer computer Assign SBT features include a base caller for accurate base calling of heterozygous sequence an algorithm for determination of the consensus sequence a sequence alignment algorithm and a sequence match
61. tifically and technically sound method for interpretation acceptance and or rejection of sequences especially in regions that are technically difficult e g compression ends D 4 6 11 16 3 Determine the sequences of both sense and anti sense strands if a sequence suggests a novel allele or a rare combination of alleles D 4 6 16 HLA typing D 4 6 16 1 Laboratories performing HLA typing must D 4 6 16 1 1 Conform to all pertinent Standards for the methods used D 4 6 16 1 2 Ensure that the level of resolution of HLA typing is appropriate for the clinical application and based on established criteria D 4 6 16 1 3 Define the criteria used for the assignment of HLA types D 4 6 16 1 4 Use HLA antigen terminology that conforms to the latest report of the World Health Organization W H O Nomenclature Committee for factors of the HLA System Potential new 55 Sequence Based Typing antigens not yet approved by this committee must have a designation that cannot be confused with W H O terminology D 4 6 16 3 Laboratories performing HLA typing by nucleic acid analysis must D 4 6 16 3 1 Recognize and document ambiguous combination s of alleles for each template primer or template probe combination D 4 6 16 3 2 Define the specificity and sequence of each primer and or probe for each HLA type D 4 6 16 3 3 Define and document the genetic designation e g locus of the target amplified by each set of primers or hybridized with pr
62. tive and negative results If these are not achieved acceptance of the results must be justified and documented D 4 6 11 4 Have an independent review of the data and its interpretation D 4 6 11 5 Include in each electrophoretic process controls that verify that the specific targets can be detected D 4 6 11 6 If the size of a nucleic acid is a critical factor in the analysis of the data D 4 6 11 6 1 In each gel include size markers that produce discrete electrophoretic bands spanning and flanking the entire range of expected fragment sizes D 4 6 11 6 2 The amount of DNA loaded in each lane must be within a range that ensures equivalent migration of DNA in all samples including size markers D 4 6 11 7 Ensure that each lot and shipment of primers or probes is monitored to confirm stability and performance of the primers or probes D 4 6 11 8 Ensure that oligonucleotide probes and primers are stored under conditions that maintain specificity and sensitivity D 4 6 11 9 Define the specificity and critical polymorphic sequence of each primer and probe D 4 6 11 10 Document and validate the methods used to purify nucleic acids If tests are performed without prior purification of nucleic acids the method must be documented and validated in the laboratory D 4 6 11 11 Ensure and document acceptable electrophoretic conditions used for each gel electrophoresis D 4 6 11 12 Ensure that the DNA isolation method used provides sufficien
63. uding initial SBT and additional ambiguity resolving test data analysis and review e Final cost per typing including all reagents supplies and labor In this chapter we introduced two procedures One represents the generic multiplex amplification and sequencing strategy using Abbott SBT kits and the other represents the group specific amplification References 1 Sanger F Nicklen S Coulson AR DNA sequencing with chain terminating inhibitors Proc Natl Acad Sci USA 1977 74 5463 5467 2 Petersdorf EW Hansen JA A comprehensive approach for typing the alleles of the HLA B locus by automated sequencing Tissue Antigens 1995 46 73 85 3 Ross J Sequencing Based Typing Histocompatibility Testing Editor Bidwell and Navarrete 2000 Imperial College Press 4 Wu J Bassinger S Griffith BB Williams TM Analysis of HLA class Alleles via Direct Sequencing of PCR Products ASHI Laboratory Manual Forth Edition 2000 5 Blasczyk R HLA Diagnostic Sequencing Conception Application and Automation J Lab Med 2003 27 9 10 359 368 6 Hurley CK Ellis JM DNA methods for HLA typing a workbook for beginners Version 6 2004 Sequence Based Typing PROCEDURE I HLA SEQUENCE BASED TYPING SINGLE LOCUS AMPLIFICATION Principle The Human Leukocyte Antigen HLA encoded within the Major Histocompatibility Complex MHC is one of the most polymorphic gene complex in the human genome Sequence based typing SBT methods for
64. ument Profile interruption and use caution to judge photo documentation from the problem PCR b Run the instrument performance test per manufacturer s recommendations and repeat amplification 2 Guidelines Pre Analytical Area A PCR run is considered to be Invalid and needs to be repeated if 47 Sequence Based Typing 2 1 No positive PCR products are visualized in the photo 2 2 PCR products with a band of wrong size 2 3 The sequencing product generates an unreadable data e g low signal strength noisy or artifact artificial peaks unexpectedly truncated large spacing between peaks for the Forward and or Reverse sequence reaction e Due to Genetic Analyzer malfunction e Due to operator error e Due to faulty reagents 2 4 If a given sample fails to amplify repeat the test on that sample in the next run If again no sequence is seen confirm with other technologists how the performance of that sample with other tests reconfirm OD readings and DNA dilution made Check anticoagulant used to collect blood sample Heparin will cause fail in SBT amplification 2 5 If a given sample fails to generate good quality data repeat the Cycle Sequencing reaction using the same ExoSAP IT treated PCR product available Note 1 ExoSAP IT treated PCR products can stand a small dilution sometimes necessary to repeat a given Cycle Seq reaction usually 6 to 8ul of Molecular grade water Note 2 Make sure to add the water co
65. ver the tray vortex briefly and spin for 1 minute before using it for a new Cycle Sequencing reaction 3 Guidelines Analytical Area 3 1 All nucleotide positions must be as expected unless there is a confirmed novel substitution 3 2 The data is reviewed by at least two qualified individuals a technologist plus a designee or director 3 3 Homozygous typing rare types and types that are unexpected for any reason e g linkage disequilibrium prior typing family analysis race ethnicity should be confirmed using a different typing method or reagents 3 4 Reliability of test results should be monitored by periodic use of positive controls of known HLA types Sequencing of both directions of each sequencing reaction product is recommended when 2 distinct groups of alleles can t be distinguished by initial PCR 4 CRITERIA FOR ACCEPTANCE amp REVIEW OF SBT RESULTS To ensure typing accuracy sequencing of at least one forward or reverse direction on the same exon is mandatory When using Protrans strategy the alleles get separated in different PCR products amplicon that are sequenced separately and it is not necessary to perform sequencing in both directions if the alleles are distinct in the PCR step Note When the PCR products can t clearly separate both alleles each exon must be sequenced in both directions 4 1 Repeat the PCR amplification when the number of constant positions errors E g heterozygous base
66. vial 9 2 Add 3 ul of the NaOAc EDTA buffer to each sequencing reaction 9 3 Cover tray using full plate cover and spin tray for 30 seconds at 1000 rpm 9 4 Add 25 ul of the 100 Ethanol solution to each well 29 Sequence Based Typing 9 5 Place the Plate Septa onto the tray and vortex the tray thoroughly using an Eppendorf Mix mate for 30 seconds 9 6 Centrifuge tray at 3160 rpm for 30 minutes 9 7 Prepare an 80 Ethanol solution mix well and keep it closed 9 9 Immediately remove supernatant invert tray on paper towel stack 9 10Place the inverted tray and paper towels in the centrifuge carrier and spin at 1000 rpm for 20 seconds to remove the supernatant 9 11Add 50 ul of 80 Ethanol to the wells spin down at 3160 g for 5 min 9 12Immediately invert the tray onto paper towel stack Place the inverted tray and paper towel stack in the centrifuge and spin at 1000 rpm for 20 seconds to remove the supernatant 9 13Dry reactions for 15 minutes protected from light 9 14Add 15 ul of Hi Di Formamide cover with full plate cover and spin tray for 30 seconds at 1000 rpm 9 15Incubate for 2 minutes at 95 C and cool on ice for 2 minutes 9 16Spin tray for 30 seconds at 1000 rpm and place it on the Support Base Specific for each Analyzer 9 17Place a Full Plate Septa over the tray and follow with the 96 Well Plate Retainer 9 18Place Support base Tray Septa retainer onto the ABI Analyzer Wait the tray to go back
67. with the QC performed for the new lot of SBT reagents received 46 Sequence Based Typing 1 2 PCR Amplification Failures Amplification failures can be identified by Agarose gel electrophoresis or by the failure of all the sequencing reactions from a single sample Failures at the PCR step may be due to the following 1 2 1 AmpliTaq Gold not added to PCR Taq Mix In this case all samples are failed to amplify 1 2 2 PCR Taq Mix not well mixed e In this case samples will show variable intensities or absence of a PCR product in the Agarose gel photo e SBT PCR Amplification that fail to identify a specific allele that have been picked up by the All Alleles Mix only HLA Class I should be used to re amplify for QC documentation Result can be confirmed by other method in case result is needed to state in the patient record Consult patient information to make decision e Unacceptable DNA quality or quantity a check the 260 280 ratio of DNA sample b In case of low ratio below 1 5 ethanol precipitate DNA sample to remove protein contamination Examine the integrity of genomic DNA in a 0 8 Agarose gel electrophoresis Re isolate DNA if the amount of DNA is insufficient 1 2 3 DNA not added e Repeat PCR set up 1 2 4 Thermocycler problems e Incorrect thermocycler profile a Confirm the profile used fix if appropriate and repeat amplification e Thermal cycler failure or interruption during the PCR run a Doc

Chapter II: DNA Based Testing Section: Application - ASHI-U

Contents

Download Pdf Manuals

Related Search

Related Contents