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1. 2 8 Remove the reaction wells from the centrifuge and replace in an upright position on the work bench Discard the paper towel or tissues 2 9 Prepare fresh solution of 80 ethanol with absolute ethanol and sterile water 2 10 Add 60uL of 80 ethanol to each reaction well Reseal the wells and vortex briefly 2 11 Spin at 2000g for 5 minutes 2 12 Repeat steps 2 6 and 2 7 2 13 Remove the reaction wells from the centrifuge and discard the paper towel Reseal the reaction wells and proceed to the denaturation step Otherwise store at 20 C in the dark It is recommended that the extension products are run on the DNA sequencer within 24 hours of setting up the sequencing reactions 3 Denaturation amp Electrophoresis of Sequencing Reaction Products NOTE The procedure for the denaturation of extension products in Hi Di Formamide described here may not be necessary if purification procedures other than ethanol Page 7 of 14 For In Vitro Diagnostic Use precipitation have been used It is strongly recommended that users validate alternative procedures before proceeding 3 1 Add 12uL of Hi Di Formamide to each reaction well Vortex and centrifuge the wells plate briefly 3 2 Incubate the reaction wells at 98 C for 5 minutes Following incubation ensure that the reaction wells are cooled quickly to room temperature e g place on ice or use the thermal cycler to perform the denaturation and cooling steps before being pla2. 50 tests C1 TC98 F 20 C1 AG203 F 20 C1 AT362 F 20 C1 TA368 F 20 C1 CC144 F 20 C1 CA309 R 20 C1 AC362 F 20 C1 CC486 F 20 C1 GG572 R 20 C1 CG134 F 20 C1 CC341 R 20 C1 CT379 R 20 RB 09 F 20 RB TT197 F 20 RB AT258 F 20 RB GT344 R 20 Page 12 of 14 SBT Resolver HLA DRB1 kit 20 and 50 tests C1 TA98 F 20 C1 GT240 F 20 C1 GG362 AR 20 C1 CT423 F 20 C1 AC206 F 20 C1 GAA309 R 20 C1 GC363 F 20 C1 AC559 R 20 C1 GAG601 R 20 C1 CA176 F 20 C1 CA343 F 20 C1 GG539 R 20 RB 15 F 20 RB GT196 F 20 RB GC258 F 20 RB TG344 R 20 C1 CA102 F 20 C1 TT368 F 20 C1 GG362 R 20 C1 AG453 R 20 C1 GA206 F 20 C1 GAT309 R 20 C1 GG363 BF 20 C1 CT559 R 20 C1 AG270 F 20 C1 CG343 F 20 C1 TG539 R 20 RB 52 F 20 RB GA196 F 20 RB CT257 R 20 For In Vitro Diagnostic Use Self certified SBT Resolver typing kits SBT Resolver HLA C kit 20 and 50 tests HH PD3 2 2 20 HH PD3 2 2 50 PQ PD6 2 2 20 PQ PD6 2 2 50 AN PD6 2 3 20 AN PD6 2 3 50 HH PD10 1 20 HH PD10 1 50 KD PD10 2 1 20 KD PD10 2 1 50 SBT Resolver HLA DQBI1 kit 20 and 50 tests SBT Resolver HLA DPB1 kit 20 and 50 tests Self certified HARPS QB TA122 F 20 QB GA316 R 20 PB GC112 F 20 PB GT313 R 20 QB GC134 F 20 QB CG353 R 20 PB TT113 F 20 PB AG341 R 20 QB CT173 F 20 QB GG353 R 20 PB TAC121 F 20 PB GG341 R 20 QB TA173 F 20 QB GG361 R 20 PB GC19
3. area Table top centrifuge with plate adapters and capacity to reach 2500 x g a 2 te oN Vortex 10 Thermal cycler with heated lid These HARPS have been validated using the following thermal cyclers Page 4 of 14 For In Vitro Diagnostic Use 11 12 13 14 15 MJ Research PTC 225 DNA Engine DYAD Applied Biosystems by Life Technologies Veriti Thermal cycler Gene Amp PCR System 9700 and Eppendorf Mastercycler Pro 125mM EDTA pH8 0 Available for purchase from Conexio Genomics product code EDTA 3 0 200 or EDTA 3 0 5000 Absolute and 80 Ethanol Each run requires freshly prepared 80 ethanol consisting of absolute ethanol and sterile water DO NOT USE DENATURED ETHANOL also known as methylated spirits in some countries Hi Di Formamide Applied Biosystems by Life Technologies product code 4311320 Automated DNA Sequencer and accessories e g Applied Biosystems by Life Technologies ABI Prism 3730 including data collection software These HARPS have been tested and validated on the Applied Biosystems by Life Technologies 3100 3730 and 3730x1 capillary sequencers and software HLA Sequencing Analysis Software e g Assign SBT version 3 6 or higher Conexio Genomics Pty Ltd Sample Requirements Locus specific ExoSAP treated amplicons prepared according to the HLA SBT Resolver kit Instructions for Use Mites and Safety Precautions This k
4. each time a sequence reaction is to be performed The composition and volumes for the mix indicated below are per sample Component Volume HARP 2uL Sterile water 11 5uL BigDye Terminators luL 5x Seq Rxn Buffer 3 5uL 1 2 Mix each sequencing reaction mixture gently by pulse vortexing 1 3 Dispense 18uL of the sequencing reaction mix into each appropriate reaction well NOTE For runs which involve few samples with many different HARPS it is acceptable to dispense the HARP 2uL directly into the individual reaction wells A master mix may then be created composing of sterile water BigDye Terminators and 5x Seq Rxn Buffer of which 16uL is to be dispensed into each reaction well It is strongly recommended that use of this alternative procedure is validated by the user prior to implementation 1 4 Add 2uL of purified PCR product to each appropriate well NOTE Care must be taken to prevent cross contamination of sequencing reactions 1 5 Seal the reaction wells mix gently and centrifuge briefly to ensure that the contents are located at the base of each reaction well 1 6 Place the reaction wells into a thermal cycler and run according to the following profile Number of cycles Temperature and time 25 96 C 10 sec 50 C 5 sec 60 C 2 min Page 6 of 14 For In Vitro Diagnostic Use Number of cycles Temperature and time 1 4 C hold 1 7 Once the program is complete remove the r
5. 4 F 20 QB TA185 F 20 PB AT251 R 20 Assign SBT software Self certified ASSIGNssrse ASSIGNssrva7 ASSIGN 5srv47 CE CE Product code CGX0036 Product code CGX00470 Product code CGX00471 For Research Use Only except Australia SBT RESOLVER SBT Resolver HLA DRB3 kit 20 and 50 tests AN PD11 0 0 20 AN PD11 0 0 50 AN PD12 0 0 20 AN PD12 0 0 50 AN PD13 0 0 20 AN PD13 0 0 50 LC PD2 9 20 LC PD2 9 50 SBT Resolver HLA DRB4 kit 20 and 50 tests SBT Resolver HLA DRBS kit 20 and 50 tests SBT Resolver HLA B57 kit 20 and 50 tests General Purpose Laboratory Reagents MgCl2 1 0 50 MgCl2 1 0 3000 SEQ BUF 2 0 400 SEQ BUF 2 0 5000 2mM MgCl 5x Seq Rxn Buffer Page 13 of 14 For In Vitro Diagnostic Use EDTA 3 0 200 125mM EDTA pH8 0 EDTA 3 0 5000 Please contact your local distributor for further details Support and Contact Details Conexio Genomics Pty Ltd Or your local distributor PO Box 1294 Fremantle 6959 Western Australia AUSTRALIA Tel 61 08 9336 4212 email support conexio genomics com Skype conexiocgx Website www conexio genomics com For ordering details please refer to the Olerup website http www olerup com Conexio SBT Resolver Assign and HARPS are trademarks of Conexio 4 Pty Ltd HARPS is a registered trademark in some territories Page 14 of 14 For In Vitro Diagnostic Use
6. CONExXIO SBT RESOLVERiarrs Instructions for Use Version No 6 0 Issue Date August 2015 Conexio Genomics Pty Ltd 2 49 Buckingham Dr Wangara 6065 Western Australia Australia Page 1 of 14 LEC REP Qarad bvba Cipalstraat 3 B 2440 Geel Belgium For In Vitro Diagnostic Use PRINCIPLE sssscssssrssssrsesscssrsesscssnsessccensesscsensesscsssenssssessesscsssensessenenssssenenssssesensesessevessessevesseseevassesees 3 INTENDED USE sessssssssess ssasescsesssessidesssssessscnesscastestsenesonassevsasassassessssussvetosssosssscesabstesseseass coasetsancsoussaesenene 3 KIT COMPOSITION iiecssssisescscssssnssetsnsececssttvsassssusescsessvessedesacescesavensassascusepsdesssasedsdscssdevsnsadecvesesescnssdbions 4 STORAGE REQUIREMENT sssssssssssssssssssssssesesessssssssssscesssssesssssssssesosessssssssssesesesesesessssssssseseses 4 MATERIALS REAGENTS AND EQUIPMENT NOT SUPPLIED ccscssscssscesssscssecesesssesesees 4 SAMPLE REQUIREMENT sscsssssssssssssssssscssssesssessssssssssssesssssssssssesssesesessssssssssevesssesosesesesesesseseses 5 WARNINGS AND SAFETY PRECAUTIONS ccscssssssssssssesesesessssssssssnsnssssssssesesssesesssssssssseseseses 5 SYMBOLS PREE E E A A OE 6 ILOLO DIDI BI VASEA A E E EEEE E 6 1 HARPS SEQUENCING REACTION ucccccsccsscsssscsssescssecsscsessessscsesssssecescsessussuseesesesaseesseeseuseuseuseneees 6 2 PURIFICATION OF SEQUENCING REACTION PRODUCTS ccceseessese
7. age may need to be increased before reapplying samples on the sequencer Problems during purification of Ensure there is sufficient sequencing products mixing of materials during the purification procedure Use extreme care when discarding the supernatant as it may dislodge the pellet Signal intensity is too high Presence of high fluorescent peaks artefacts Too much PCR product Check sequence quality obtained from the sequencing primers supplied with the SBT Resolver typing kits Consider using a higher dilution factor following PCR purification Too much reaction products applied to sequencer Check instrument parameters Consider reducing the injection time and voltage before reapplying samples on the sequencer Noisy baseline high background Poor PCR purification Check sequence quality obtained from the sequencing primers supplied with the SBT Resolver typing kits Ensure ExoSAP treatment is undertaken according to the SBT Resolver typing kit instructions Consider using ExoSAP following the manufacturers procedure increasing the amount of enzyme or consider an alternative purification technique If an alternative PCR purification procedure was used ensure that the procedure was undertaken according to standard operating procedures Poor sequencing set up Refer to weak signal intensity of electropherograms Contaminated sequencin
8. ced on the sequencer If it is not possible to run the plates immediately store at 4 C until required NOTE Ensure that there are no air bubbles in the reaction wells These can enter and damage the capillary 3 3 Load the reaction wells plate onto the automated sequencer and prepare the data collection file according to the sequencer manufacturer specifications 3 4 The following instrument parameters have been validated by the manufacturer using Big Dye Terminator Sequencing Kit v3 1 and POP 7 These parameters may require user validation for other polymers sequencing chemistries and instruments Please refer to the appropriate instrument user s manual for detailed instructions and guidance e g ensure that the dye set setting is appropriate for the chemistry used for example v1 1 Big Dye Terminator sequencing chemistry will require a different dye set Parameter Setting Dye set Z_BigDyeV3 Mobility file KB_3730_POP7_BDTV3 Basecaller KB bcp Run Module Regular FastSeq50_POP7 Injection time 15 sec Run time 3000 sec 3 5 Use the instrument s data collection software to process the raw collected data and create the sequence files Please refer to the appropriate instrument user s manual for detailed instructions and guidance 4 Editing and analysis of electropherograms The SBT Resolver HARPS product range were developed and validated using the Assign SBT and Assign ATF software developed by Conexio Gen
9. e based on the sequence differences at the HARPS annealing site The higher the score the greater the sequence differences and the highest probability of producing hemizygous sequence For further information including exceptions and cautions regarding specific HARPS please refer to SBT Resolver HARPS Technical Notes downloadable from the Conexio Genomics website http www conexio genomics com Bibliography 1 SBT Resolver Typing Kits IFU Conexio Genomics Pty Ltd 2 Assign SBT v3 6 Operator Manual Conexio Genomics Pty Ltd 3 Assign SBT v4 7 Operator Manual Conexio Genomics Pty Ltd 4 Assign SBT v471 Operator Manual Conexio Genomics Pty Ltd 5 SBT Resolver HARPS Technical Notes Conexio Genomics Pty Ltd 6 Current HLA alleles can be found at http www ebi ac uk imgt hla Troubleshooting Problem Possible cause s Solution Weak signal intensity of Weak PCR product Check sequence quality electropherograms obtained from the sequencing primers supplied with the SBT Resolver typing kits Poor sequencing set up Ensure sequencing reactions are set up according to the manufacturer s instructions Ensure correct addition and mixture of samples and sequencing mixture Page 9 of 14 For In Vitro Diagnostic Use Problem Possible cause s Solution Insufficient reaction products applied to sequencer Check sequencer parameters Injection time and volt
10. eaction wells from the thermal cycler and either proceed directly to purification of the reaction products or store in the dark at 4 C until required It is recommended that samples are purified and run on the DNA sequencer within 24 hours 2 Purification of Sequencing Reaction Products NOTE Purification of the reaction products may be carried out by procedures other than the ethanol precipitation method described here It is strongly recommended that users validate these procedures before proceeding 2 1 Briefly centrifuge the reaction wells plates before proceeding If reusable lids caps have been used during thermal cycling label the lids caps to avoid cross contamination 2 2 Carefully remove the seals 2 3 To each reaction well add SuL of 125mM EDTA pH8 0 Ensure that the EDTA reaches the base of the reaction well 2 4 Add 60uL of 100 ethanol to each reaction well Seal the wells plate and vortex briefly but thoroughly to ensure thorough mixing 2 5 Pellet the extension products by centrifuging at 2000g for 45 minutes IMMEDIATELY PROCEED TO THE NEXT STEP If this is not possible re centrifuge for an additional 10 minutes before proceeding 2 6 Remove the seals to the reaction wells and discard the supernatant by inverting the reaction wells onto paper towel or tissues 2 7 Place the inverted reaction wells and paper towel or tissue into the centrifuge Centrifuge at 350g for 1 minute to remove any residual supernatant
11. ed until the indicated expiry date and can tolerate up to 25 freeze thaw cycles Accelerated stability testing for the HARPS indicated a shelf life of five years when stored at 20 C While confirmatory real time testing is underway it is strongly recommended that these HARPS are NOT to be used beyond their expiry date To maintain optimal kit performance the HARP should be removed from the 20 C storage location and thawed rapidly at room temperature before use The HARP should then be gently vortexed to ensure that the components of each tube are appropriately mixed after thawing After use the kits components should be returned immediately to 20 C Materials Reagents and Equipment Not Supplied NOTE The use of materials reagents equipment or procedures other than those detailed within this Instructions For Use requires validation by the user prior to use 1 Sterile water 2 BigDye Terminator Cycle Sequencing Kit v3 1 or v1 1 Applied Biosystems by Life Technologies 3 5x Sequencing Reaction Buffer Conexio Genomics product code SEQ BUF 2 0 400 or SEQ BUF 2 0 5000 or BigDye Terminator v3 1 or vl 1 5X Sequencing Buffer Applied Biosystems by Life Technologies 4 Electronic or mechanical pipettes and aerosol resistant tips Ji 0 2mL thin walled thermal cycling reaction tubes 8 well strips or 96 well plates Use those recommended for use with your thermal cycler Sterile 1 5mL tubes Sterile work
12. eeeececsessaesecececseseeaseeeeeeseeseaaees 7 3 DENATURATION amp ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS cccssccccceseeseeeees 7 4 EDITING AND ANALYSIS OF ELECTROPHEROGRAMSG sssseseseceseseseseseseseseececeeeeeeeeeececeeeeeeaeaeateteeeeees 8 PERFORMANCE CHARACTERISTICS ccccsssssssssssssssssssssssesssssssssssnsnssssssssssesesesesssesssssssssseseses 9 LIMITATIONS AND CAUTIONG csssssssssssssssssssssssssssssssssssssesssssssssnsnsnssssssssesesesssssesesssssssseseseses 9 BIBLIOGRAPHY visessccssaicasesssendesvosicieassetesensnebabcasessscscteebeieasesteccosentusansecsicensonisberiietecscsesdssoieest ecdesdutunies 9 TROUBLESHOOTING cscssssssscscssscsssssssssssssesesesssesecscecesessssssesesesesesesesesesesssssseseseseseresecesesescsceseses 9 RELATED PRODUCTS vsssssisessssssssescsesesssnesaceasestssaeseesssessasessasseasssascteressccnesdsodsasteesosbesaseasdeaseaedsusssonene 12 SUPPORT AND CONTACT DETAILS ccccsssssssssssssssssscssesesssessssssssnsnsnssssssesessseresesessssssssssesesees 14 Page 2 of 14 For In Vitro Diagnostic Use Principle The SBT Resolver HARPS Heterozygous Ambiguity Resolving Primers are sequencing primers developed by Conexio Genomics that are used to resolve heterozygous ambiguities by producing hemizygous sequence that enable the phase linking of HLA polymorphisms in locus specific sequencing based HLA typing SBT Following SBT with SBT Resolver kits the data is analy
13. g reactions Ensure that all steps are taken to prevent Cross contamination Change pipette tips wherever possible Add liquids at the top of the reaction wells Prevent aerosols Page 10 of 14 For In Vitro Diagnostic Use Problem Possible cause s Solution Contaminated sequencing reagent s Check sequence quality obtained from the sequencing primers supplied with the SBT Resolver typing kits Check sequence quality of other samples using the same reagent batches aliquots Repeat sequencing using fresh reagents Poor purification of sequencing products Refer to weak signal intensity of electropherograms Poor or incorrect matrix Check spectral calibration and matrix Repeat application of sequencing products Presence of Dye blobs Poor purification of sequencing products Refer to weak signal intensity of electropherograms Ensure products are washed sufficiently with 80 ethanol Ensure all traces of ethanol are removed Heterozygous sequence obtained Incorrect HARP selected from HARPS report Check HARPS score is above recommended threshold Check the SBT Resolver HARPS Technical Notes for any comments or exceptions that may apply to the HARP Incorrect HARP used Ensure that the correct HARP is used No sequence obtained Random sequencing failure Check sequence data obtained from SBT Resolver ty
14. it must be used by trained and authorized laboratory personnel All samples equipment and reagents must be handled in accordance with good laboratory practice In particular all patient material should be considered as potentially infectious The use of gloves and laboratory coats is strongly recommended Handle and dispose of all sample material according to local and national regulatory guidelines There are NO dangerous substances contained in any of the SBT Resolver HARPS products Please refer to the MSDS that is available on the Conexio Genomics website http www conexio genomics com Do NOT use reagents beyond their expiration date Use of reagents or equipment not listed under Materials Reagents and Equipment Not Supplied in this Kit is NOT recommended Such use may affect the performance of the assay Care should be taken to prevent cross contamination of specimens Change tips between samples wherever possible The use of aerosol resistant tips is highly recommended Pre and Post PCR activities must be strictly physically separated Use specifically designated equipment reagents and laboratory coats Page 5 of 14 For In Vitro Diagnostic Use Symbols The following non standard symbols have been used Symbol Description q Date of manufacture required for non EU markets Procedure 1 HARPS Sequencing Reaction 1 1 Prepare a fresh solution of sequencing primer mix for each HARP on ice
15. omics Pty Ltd Users are recommended to use Assign SBT v3 6 and higher as these versions of the software utilise setting and reference files specifically designed for the SBT Resolver typing kits and HARPS For more details in relation to the operation of these software please refer to the applicable user manuals available for download on the Conexio Genomics website http www conexio genomics com For further information regarding the Assign reference files to be used for analysis please refer to the SBT Resolver kit Instructions For Use Page 8 of 14 For In Vitro Diagnostic Use Performance Characteristics Well characterised samples that contained unresolved heterozygous ambiguities were sequenced using the recommended HARP reported from Assign SBT v3 6 and higher Each HARP produced sufficient hemizygous sequence for the resolution of heterozygous ambiguities Limitations and Cautions These products are for professional use only It is strongly recommended that these products are validated by the user prior to implementation in the laboratory using samples whose HLA type has been determined by other molecular based procedures In particular any deviations from this procedure e g the use of alternative DNA sequencing purification procedures must be validated by the user prior to implementation Assign SBT v3 6 and higher calculates the HARP S required to resolve an ambiguity and includes a scor
16. ping kit Check sequence data obtained for other samples that were sequenced with the same HARP Repeat sequencing reaction and ensure that all reagents and template have been applied Poor purification of sequencing Ensure procedure is products undertaken according to the manufacturer s instructions If an alternative purification procedure was used ensure that it was undertaken according to standard operating procedures Incorrect HARP selected or used Refer to Heterozygous sequence obtained Page 11 of 14 For In Vitro Diagnostic Use Related Products CE marked IVDs SBT RESOLVER XH PD1 1 2 20 XH PD1 1 2 50 BS PD2 1 2 20 BS PD2 1 2 50 HH PD5 2 5 20 HH PDS5 2 5 50 LG PDS5 2 7 20 LG PDS5 2 7 50 SBT RESOLVERharrs Product codes C1 TT98 F 20 C1 CT102 F 20 C1 GG307 R 20 C1 GG363 AF 20 C1 AC497 F 20 C1 BTA F 20 C1 GC209 F 20 C1 CG319 F 20 C1 CC387 F 20 C1 GA559 R 20 C1 CT97 F 20 C1 AC302 R 20 C1 CC355 F 20 C1 AG595 R 20 RB 01 F 20 RB GG125 F 20 RB TA164 F 20 RB AT257 R 20 C1 AC98 F 20 C1 CC102 F 20 C1 GT355 R 20 C1 TA363 F 20 C1 CG570 R 20 C1 BCG F 20 C1 CG285 F 20 C1 AG360 F 20 C1 TA420 F 20 C1 CG572 R 20 C1 CT112 F 20 C1 GC302 R 20 C1 GA361 F 20 C1 AA601 R 20 RB 04 F 20 RB AA197 F 20 RB TT227 F 20 RB TT321 R 20 SBT Resolver HLA A kit 20 and 50 tests SBT Resolver HLA B kit 20 and
17. sed with Assign SBT sequence analysis software Once the software produces a HARPS report the PCR product is re sequenced with the reported HARP S and the resulting sequence data is analysed with the original data to resolve the heterozygous ambiguity Intended Use Conexio Genomics SBT Resolver HARPS kits are used to assist in the resolution of heterozygous ambiguities arising from HLA sequencing based typing obtained from the use of the SBT Resolver typing kits Selection of the appropriate HARP is based on the analysis of the resultant DNA sequencing data from the typing kits by Conexio Genomics Assign SBT sequence analysis software Page 3 of 14 For In Vitro Diagnostic Use Kit Composition Each SBT Resolver HARPS product is supplied as a single vial containing a single HARP sufficient for 20 tests 44uL The SBT Resolver HARPS product names are assigned according to the following nomenclature system Locus terminal nucleotides terminal nucleotide position HARP direction Class I HARPS contain the C1 prefix e g C1 TT98 F while those for Class II contain either the RB HLA DRB1 QB HLA DQB1 or PB HLA DPB1 prefixes For the complete list of available HARPS please refer Related Products section located at the end of this document Storage Requirements When stored at 20 C temperature range of 15 C to 25 C is acceptable the kit components can be us
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