1. A next generation HLA sequencing method provides robust reliable class II typing of an HSCT cohort
Anajane G. Smith1, 2
Shalini E. Pereira1, 2
Dan E. Geraghty1, 2
John A. Hansen1
1 Fred Hutchinson Cancer Research Center, Seattle, WA
2 Scisco Genetics, Inc., Seattle, WA

2. Challenges of current HLA SBT
The vast array of HLA alleles often generates ambiguous typing results.
Inability to assign the phase of sequence polymorphisms.
DRB1*11:01, 13:01 or,
DRB1*11:04, 13:02 or,
16 other possible allele combinations.
Polymorphism exists outside of exon 2, the traditionally typed class II antigen recognition site (ARS).
DRB1*12:01/12:06/12:10/12:17 (exon 3 and signal peptide).

3. NG-HLA sequencing procedure
Locus specific PCR amplification of genomic DNA.
DRB1/B3/B4/B5, DQB1, DQA1, DPB1: exons 2 and 3.
4 primer pairs for DRB genes.
3 primer pairs for DQB1.
2 primer pairs each for DQA1 and DPB1.
1 primer pair for DPA1 exon 2.
Pool all amplicons into a single aliquot per sample.
Barcode PCR to attach a unique label on each sample.
Pool all reactions in a single tube.
Load onto the MiSeq sequencing device.

4. NGS analysis tools
MiSeq paired end reads matched with “.fasta” sequences.
Phase between exons determined by coincidence with established databases.
Statistics for the likelyhood of each allele match.
Number of paired end reads with a perfect allele match.
Number of paired end reads with a single mismatch.
Number of unique pairs for a given type.
Consistency between independently derived exon data.
Aggregate quality values of base calls at each position.

5. Legacy DRB1-DQB1 typing Typing technology evolved over 20 years.
DRB1 by SSOP and SBT.
DQB1 by SSOP, SBT, and serology.
SSOP typed for the alleles known at the time.
Assessed exon 2 polymorphism (ARS).
Reported at the 4 digit allele level or with letter codes.
DRB1*11:01:01/11:01:02 = DRB1*11:01.
DRB1*12:01/12:06/12:10/12:17 = DRB1*12DUKV.

6. Study Population Fred Hutchinson Cancer Research Center
HSCT recipients and related or unrelated donors.
86% Caucasian.
14% Asian/AFA/Hispanic/Native American.
2,605 DNA specimens plus 83 no DNA controls.

7. Legacy versus NGS DRB1-DQB1
Alleles compared at the 4 digit level of legacy typing.
Discordant allele resolution.
Original clinical typing records.
NG raw sequencing data.
96% of the typing results were concordant.
47 (0.5%) of NGS allele assignments were discordant.
350 (3.5%) of legacy allele assignments were discordant.

8. Legacy discordant: DRB1-DQB1
34 discordant alleles due to exon 2 polymorphism
Alleles not known when SSOP typing was done.
Early SSO probe problems.
316 discordant alleles due to exon 3 polymorphism.
Legacy DRB1*14:01 versus NGS DRB1*14:54.
Legacy DQB1*03:01 versus NGS DQB1*03:19.
Legacy DQB1*02:01 versus NGS DQB1*02:02.
Above includes 9 new allele sequences by NGS.

9. NGS discordant: DRB1-DQB1
27 due to low read sequences.
One allele not assigned in heterozygous samples.
Setting limits for acceptable data is critical for accuracy.
20 of analysis software errors.
Extra allele assigned in homozygous samples.
Errors resolved with software improvements.

10. 166 class II alleles identified by NGS
Total: Number of alleles identified at each locus.
New: Novel allele sequences (n=9).

11. Summary
Our study demonstrates the robust reliability of next generation sequencing for HLA typing.
99.5% accuracy of NGS.
Identification of novel allele sequences.
NGS eliminates the complex, time-consuming testing required for resolution of ambiguous typing results.
This technology has been extended to HLA-class I exons 2, 3, and 4 (see poster 1004-LBP).

12. Conclusion
Next generation sequencing, with fully phased typing by parallel sequencing of individual transcripts, is no longer next, it is now the technology for HLA typing.

13. Acknowledgements John A. Hansen Dan E. Geraghty Chul-Woo Pyo
Wyatt Nelson
Shalini E. Pereira
Sandy Warnock
Maggie Sprague
Entire staff, past and present, of the FHCRC/SCCA Clinical Immunogenetics Laboratory for the legacy typing.