1. HLA Analysis and Next Generation Sequencing Henry Erlich, Ph.D. Cherie Holcomb, Ph.D. Roche Molecular Systems picture placeholder NGS and EFI, May 14, 2013

2. Library preparation strategies for NGS for HLA typing Amplify gene (multiple Kb)/cleave and ligate universal 454 adaptors/emPCR/sequence Sequence capture (NimbleGen)/cleave and ligate universal 454 adaptors/emPCR/sequence Amplify exons (and introns) with MID primers/emPCR/sequence (“fusion” or Fluidigm system primers) Sequencing HLA genes vs. HLA typing by sequencing

3. Incompletely Extended PCR Product 5’ 3’ Schematic of PCR Crossover Formation Increased probability in long amplicon PCR Crossover Product 5’ 3’ 5’ Template 1 PCR cycle “n” 5’ 3’3’ Template 2 PCR cycle “n+1” 3

4. The Benefits of Clonal Sequencing for HLA Genotyping Sets phase for linked polymorphisms within amplicon (reduces ambiguity) Allows amplification and sequencing of multiple members of multi-gene family (ie DRB1, DRB3, DRB4, DRB5) with generic (DRB) primers Allows for the separation of co-amplified sequences (pseudo- genes, related genes as “noise”) from target sequence (“signal”) Allows for the “digital” analysis (counting sequence reads) of mixtures and, with “deep” sequencing, for the detection of rare variants

5. Phasing across amplicons Exon 2 seq 1 Exon 2 seq 2 Exon 3 seq 1 Exon 3 seq 2 2-1, 3-1 2-1, 3-2 2-2, 3-2 2-1, 3-2 Note: Assumes all HLA class I alleles in IMGT/HLA database have exon 2 and exon 3 sequences. If one of the 4 combinations is absent from the sequence database, an unambiguous inference of phase can be made. Physical direct phasing can be accomplished with longer amplicons or “bridging “amplicons.

6. NGS Applications for HLA Sequencing High Resolution: many amplicons/many exons per gene High Throughput: many samples per run Deep Sequencing: many reads per sample for detection of rare variants; analysis of mixtures

7. Neurosis is the inability to tolerate ambiguity -Sigmund Freud