1. HLA Genotyping Data Generated by 454 Sequencing Cherie Holcomb, Ph.D. Roche Molecular Systems picture placeholder NGS Data Consortium October 8, 2012

2. Medium, High, and Very High Resolution HLA Genotyping Systems Targeted Amplicon sequencing – Primers target up to 9 loci (format as fusion primers or “4 primer system” w/use of Fluidigm Access Array™) NOTE: After amplification of gDNA, amplicons contain all adapters & MIDs etc. for NGS sequencing – Workflow: Amplicons processed either individually or pooled (fusion primers) or pooled (“4 primer system”) – 454 Life Sciences GS FLX or GS Junior for NGS – Conexio Assign ATF 454 software (commercially available will perform MR and HR; for VHR limited early access to Conexio Assign MPS v1.0)

3. Amplicon Sequencing using 454 GS FLX* *GS Junior can also be used, ~8x fewer samples per run ∆ Abstract #1025-LB ǂ Abstract #ORO1-02 NOTE: All current products are for Research Use Only

4. Primer Set Comparison GS GType MR, HR with added VHR * amplicons MR Primers HR Primers VHR CLASS I HLA-A: 2, 3 2, 3, 4 1, 2, 3, 4-5 HLA-B: 2, 3 2, 3, 4 1, 2, 3, 4, 5 HLA-C: 2, 3 2, 3, 4 1, 2, 3, 4, 5, 6-7 CLASS II DPA1 exon: N/A N/A 2 DPB1 exon: N/A 2 2 DQA1 exon: N/A 2 2 DQB1 exons: 2 2, 3 2, 3 DRB1 exon: 2 2 2, 3 DRB 3, 4, 5 exon: 2 2 2, 3 *Numbers shown are exons, “ - ” indicates intron 454 GS GType HLA Primer Sets

5. Primer Plate Layout for Very High Resolution Sequencing 9 loci, 22 primer pairs, 11 MIDs, 10 samples per set ( 3 plates) Commercially avail from 454

6. 454 Amplicon Sequencing File Generation 454 Pyrosequencing Image Acquisition Image Processing Image Processed CWF Signal Processing PNG Examine seq Signal Processed CWF Signal Processing SFF FNA (FASTA) Consolidation (454 AVA software) Consolidated FNA (FASTA) Genotype Report Genotyping Sequence Export + (Conexio Assign)

7. Conexio Assign ATF 454 Interface Genotypes automatically assigned, sequences visible

8. Conexio Assign ATF 454 Interface Genotype Report allele name format and output format can be chosen

9. Conexio Assign MPS v1.0 Genotyping Report All fields, MS Excel format 454 GS GType HLA +VHR primers—only part of report shown; assay includes DQA1, DQB1, DRB1 and DRB3/4/5 A CWD filter OR “highlighting” of CWD alleles in report (preferred) has been requested

10. GS GType HLA HR primers, Conexio Assign MPSv1.0 References for gDNA; Noncoding sequence can be considered HLA-A genotype: Ambiguity String includes A*03:01:01:02N NC seq not activated Null

11. GS GType HLA VHR primers, Conexio MPSv1.0 Noncoding sequence is activated, ambiguity string greatly reduced HLA-A genotype: A*02:01:01:01/02L, A*03:01:01:01 NC seq activated; Null resolved CHALLENGE : How could/should these sequences be reported?

12. Reporting of Sequence Information Currently Can report out (combined) consensus exon sequence that has given rise to list of possible genotypes for a given sample/locus. Can do this easily for all samples. (Q: If community decides sequence is necessary for publications, is this sufficient?) – Cannot report component (consensus) sequences (with exons matched) to give individual allele(s) – Doesn’t include intronic sequence (but can report consensus of each intron individually—too laborious to be practical) – FASTA format in notepad (Q: Sufficient for publication?)

13. Reporting of Sequence Information Preferred Option to report component sequences (with exons and introns matched) to give allele calls—imp for reporting new alleles For (combined or individual alleles) consensus sequence – Option to report Coding only OR Coding plus Noncoding – Format options including XML (accepted by IMGT)—imp for reporting new alleles Works in Progress

14. GS GType HLA VHR primers, Conexio MPSv1.0 New allele can be identified Genotype has 1 mismatch w/IMGT database; can determine in which allele

15. GS GType HLA VHR primers, Conexio MPSv1.0 DRB1*12 allele is a perfect match with IMGT database

16. GS GType HLA VHR primers, Conexio MPSv1.0 New allele is identifiable DRB1*07:01 allele has 1 mismatch w/IMGT database (A at b259 instead of G) Proposed acceptance criteria: Sequenced multiple times (2 different runs); Minimum read depth of 25 for each direction, each allele for (all) amplicons of prospective new allele; Mutation(s) defining new allele observed in both F and R direction Confirmed by Sanger sequencing

17. Reporting Sequences for New Allele Using info from Res Layers, manually harvest sequences and assemble 1 allele

18. “Copy Sequence” Output Simple Text file: Copy into Word Pad, Excel, Bioedit Assume XML is most appropriate for submission to IMGT database Not in FASTA or XML format (currently no way to convert to latter) In discussion with Conexio

19. Gaps in IMGT database create ambiguity in typing Gaps indicated by “orange bar” in user interface but not in Genotyping Report etc. Issue: Would be good if alleles lacking sequence were flagged in Genotyping Report In discussion with Conexio

20. Additional info & Summary Using HR or VHR 454 sequencing HLA genotyping system including Conexio Assign ATF 454 or MPS v1.0 software, respectively: – Ambiguity string lengths are reduced to a practically reportable size – Genotype/ allele ambiguity strings (in various formats using combinations of delineation in columns, “+”, “or”, “,”) can be reported in Excel, text and XML(??) format at 1, 2, or all field level. – NMDP codes supported – Most recent IMGT nomenclature and references supported (updated with periodicity, 6 months); version of references used is reported – Export of consensus sequence used to make genotype calls for all loci/all samples is easily accomplished in FASTA format—currently doesn’t include NC sequence. – New alleles readily identifiable, however, reporting of amplicon sequences currently only possible by manual “harvesting” into text file.

21. Acknowledgements Roche Molecular Systems – Henry Erlich Conexio Genomics – Damian Goodridge

22. We Innovate Healthcare 22

23. 23 Back-up slides

24. Ambiguity C*03:03/ 03:20N GS GType HLA HR primers

25. Ambiguity Resolution C*03:03/ 03:20N VHR primers