HLA Genotyping Data Generated by 454 Sequencing

Cherie Holcomb, Ph.D.Roche Molecular Systems

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NGS Data Consortium October 8, 2012

Medium, High, and Very High Resolution HLA Genotyping SystemsTargeted 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)

Amplicon Sequencing using 454 GS FLX*

*GS Junior can also be used, ~8x fewer samples per run∆Abstract #1025-LB ǂAbstract #ORO1-02

NOTE: All current products are for Research Use Only

ResolutionPossible/Future

Applications# Primer

Pairs LociMethod of Amplicon

Generation# Samples per GS FLX

run achieved Advantages Disadvantages454 fusion primer plates

(simplification of workflow by pooling amplicons possible)

88 (limited by MID/PTP region combination if use commercial

product) Commercially available

Storing primer plates at 4oC; Limited to 11 MIDs-limits # of samples per run

Fluidigm Access Array™∆ 192

More MIDs→higher throughput; Less

sample consumption & PCR reagent consumption

Need to purchase Fluidigm instrumentation &

disposables

454 fusion primer plates (workflow as above)

88 (limitation as above)

Fluidigm Access Array™∆ 96

454 fusion primer plates

(workflow as above)ǂ

MR & HR plates commercially available, (VHR primers could be

added on by lab)

Storing plates at 4oC; need to store VHR primers in diff format (liquid) unless made

commercially available

Fluidigm Access Array™ǂLess sample

consumption & PCR reagent consumption

as above

Unrelated bone marrow donor

registry screening

"Medium" (MR)

"High" (HR) As above As above

A, B, C, DQB1, DRB1 (also get DRB3/4/5)

& DQA1, DPB1

8

14

40-48 (conservative est)"Very High"

(VHR)Clinical

& DPA1

22

Research

Primer Set ComparisonGS GType MR, HR with added VHR* amplicons

MR Primers HR Primers VHRCLASS 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

Primer Plate Layout for Very High Resolution Sequencing 9 loci, 22 primer pairs, 11 MIDs, 10 samples per set (3 plates)MR Plate

1 2 3 4 5 6 7 8 9 10 11 12A A2 A2 A2 A2 A2 A2 A2 A2 A2 A2 Neg (-) B A3 A3 A3 A3 A3 A3 A3 A3 A3 A3 Neg (-) C B2 B2 B2 B2 B2 B2 B2 B2 B2 B2 Neg (-) D B3 B3 B3 B3 B3 B3 B3 B3 B3 B3 Neg (-) E C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 Neg (-) F C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Neg (-) G DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 DQB1 E2 Neg (-) H DRB E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 DRB1 E2 Neg (-)

HR PlateA A4-5 A4-5 A4-5 A4-5 A4-5 A4-5 A4-5 A4-5 A4-5 A4-5 Neg (-) B B4 B4 B4 B4 B4 B4 B4 B4 B4 B4 Neg (-) C C4 C4 C4 C4 C4 C4 C4 C4 C4 C4 Neg (-) D DPB1 DPB1 DPB1 DPB1 DPB1 DPB1 DPB1 DPB1 DPB1 DPB1 Neg (-) E DQA1 DQA1 DQA1 DQA1 DQA1 DQA1 DQA1 DQA1 DQA1 DQA1 Neg (-) F DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 DQB1 E3 Neg (-) GH

VHR PlateA A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 Neg (-) B B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 Neg (-) C C1 C1 C1 C1 C1 C1 C1 C1 C1 C1 Neg (-) D B5 B5 B5 B5 B5 B5 B5 B5 B5 B5 Neg (-) E C5 C5 C5 C5 C5 C5 C5 C5 C5 C5 Neg (-) F C6-7 C6-7 C6-7 C6-7 C6-7 C6-7 C6-7 C6-7 C6-7 C6-7 Neg (-) G DPA1 DPA1 DPA1 DPA1 DPA1 DPA1 DPA1 DPA1 DPA1 DPA1 Neg (-) H DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 DRB E3 Neg (-)

454 Amplicon Sequencing File Generation

454 Pyrosequencing

Image Acquisition

Image Processing Image

Processed CWF

Signal Processing

PNG

Examine seq

Signal Processed CWF

Signal Processing

Signal Processing SFFFNA

(FASTA)

Consolidation (454 AVA software)

Consolidated FNA

(FASTA)

Genotype Report Genotyping

Sequence Export +

(Conexio Assign)

Conexio Assign ATF 454 InterfaceGenotypes automatically assigned , sequences visible

Conexio Assign ATF 454 InterfaceGenotype Report allele name format and output format can be chosen

Conexio Assign MPS v1.0 Genotyping ReportAll 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

GS GType HLA HR primers, Conexio Assign MPSv1.0References for gDNA; Noncoding sequence can be consideredHLA-A genotype: Ambiguity String includes A*03:01:01:02N

NC seq not activated

Null

GS GType HLA VHR primers, Conexio MPSv1.0Noncoding sequence is activated, ambiguity string greatly reducedHLA-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?

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?)

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 allelesWorks in Progress

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

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

GS GType HLA VHR primers, Conexio MPSv1.0New allele is identifiableDRB1*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

Reporting Sequences for New AlleleUsing info from Res Layers, manually harvest sequences and assemble 1 allele

“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

Gaps in IMGT database create ambiguity in typingGaps 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

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.

Acknowledgements

• Roche Molecular Systems– Henry Erlich

• Conexio Genomics– Damian Goodridge

We Innovate Healthcare

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Back-up slides

Ambiguity C*03:03/ 03:20NGS GType HLA HR primers

Ambiguity Resolution C*03:03/ 03:20NVHR primers