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Current methods for high-throughput resequencing of custom targets

Current methods for high-throughput resequencing of custom targets. Adam Gordon Nickerson Lab, UW Genome Sciences WHI Genetics SIG call 3/26/14. Targeted sequencing: Why?. Genome sequencing is decreasing in cost, but still expensive (and slow)

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Current methods for high-throughput resequencing of custom targets

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  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. Custom Capture -- Method Pool Barcoded libraryprep Hyb. biotinprobes streptavidinbead cleanup wash elute Multiplexed sequencing captured DNA

  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) 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

  9. MIPs -- Method

  10. 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) PGx13 testing: 32 diverse HapMap trios

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

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

  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

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