1. Current methods for high-throughput resequencing of custom targets Adam Gordon Nickerson Lab, UW Genome Sciences WHI Genetics SIG call 3/26/14

2. Targeted sequencing: Why? Genome sequencing is decreasing in cost, but still expensive (and slow) Genotyping is cheap and quick, but is limited to a subset of human variation Targeted sequencing is a middle ground – High-throughput, cost-effective variant typing and discovery on genomic regions of interest 3 approaches in the Nickerson Lab: Custom Capture, Custom Amplicon, Molecular Inversion Probes (MIPs)

3. Barcoded library prep Custom Capture -- Method Pool wash captured DNA streptavidin bead cleanup elute Hyb. biotin probes Multiplexed sequencing

4. Custom Capture -- Details Price: $150-$250 per sample depending on target size and sample number Throughput: 24-96 samples per lane Strengths: – Highest quality data – ‘oldest’ method: robust protocols/software/data analysis pipelines Weaknesses: – Priciest of the 3 methods – Hybridization-based capture can have issues with complex / repetitive regions

5. PGRNseq: custom capture of pharmacogenetic targets Target: 84 PGx-associated genes (exons + UTRs + 1.5kb upstream/downstream) – ~1 Mb total Price: $230 / sample (for ~200 samples) Plex Level Avg. # Reads (M) Avg. Unique Aligned Gb Avg. Mean Quality Score Avg. % Q30 Bases Avg. % Targets Hit Avg. Coverage Avg. % Targets at > 20x Avg. % Targets at > 40x 2x2416.71.3736.792.194.7247x94.893.4 PGRNseq testing: 32 diverse HapMap trios

6. Custom Amplicon -- Method

7. Custom Amplicon -- Details Price: $100-$200 per sample depending on target size and sample number Throughput: 24-96 samples per lane Strengths: – Cheaper than capture – Extension-ligation could potentially help with sequencing complex regions Weaknesses: – Newer than capture: not a lot of data out there yet – Data quality not quite as good as capture – PCR based: some regions cannot be designed

8. MIPs -- Method

11. MIPs -- Details Price: $50-$150 per sample depending on target size and sample number Throughput: 48-96+ samples per lane Strengths: – Highest-throughput of the 3 methods – Cost scales extremely well with sample number – Linked probe design reduces erroneous capture Weaknesses: – Design pipelines not as robust – Newer than capture: not a lot of data out there yet

12. PGx13: MIP-based capture of pharmacogenetic targets 13 highest priority, actionable PGx targets – Exons, UTRs, 1.5kb up/downstream – All variants in these genes with level 1 evidence (PharmGKB) – ~100 kB total Price: $130/sample (for ~200 samples) Avg. Depth849x Avg. Coding Depth908x Mean % Target > 20x91.4 Mean % Coding > 20x97.6 Mean PGRNseq Concordance (coding only) 99.4% Mean PGRNseq Concordance (Actionable Variants) 100% PGx13 testing: 32 diverse HapMap trios

13. Comparing all 3 methods: same sample, different capture Custom Amplicon* Custom Capture MIPs *this data from an older version of Illumina’s Custom Amp protocol max = 189x max = 2698x max = 1452x

14. Some regions are just plain difficult Sequence homology, repeat content, and %GC can hinder all capture methods Custom Amplicon* Custom Capture MIPs CYP2D6 Pseudogene

15. Comparing all 3 methods Highest quality data: Capture Cheapest overall: MIPs Highest throughput: MIPs Custom Amplicon potentially a middle ground, but data from most recent kit is scant