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HiFi sequencing identifies likely causal variants missed by short-reads.

HiFi sequencing identifies likely causal variants missed by short-reads.

Presented during "W09 Structural chromosomal variation in cytogenomics" at ESHG 2023.

William Rowell

June 11, 2023

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  1. 1 HiFi sequencing identifies likely causal variants missed by short-reads

    William J Rowell ([email protected]) COI: Employee and shareholder of PacBio.
  2. 2 Background • ~60% of pediatric hearing loss cases have

    genetic cause • 124 known non-syndromic hearing loss genes, ~400 syndromic forms of hearing loss • srWES explains ~40% of pediatric hearing loss cases • PacBio HiFi reads (99.9% accuracy, 15-20 kb) enable comprehensive variant detection in human genomes Approach • 10 probands with unexplained hearing; negative after srWES and srWGS • Prepare 12-15 kb WGS libraries and sequence to ~24- to 32-fold depth on Sequel II system • Call, filter, and annotate variants with a variety of computational tools according to PacBio best practices
  3. 3 Identification of inversion interrupting MITF 530 kb MITF SAMMSON

    MDFIC2 PBSV BCH-06 HiFi haplotype 1 link supplementary alignments BCH-06 HiFi haplotype 1 BCH-06 HiFi haplotype 2 BCH-06 HiFi unphased reads INV - 403kb
  4. 4 Identification of compound heterozygous pathogenic variants in OTOA Segmental

    duplication: chr16:21,729,104-21,797,508 (OTOA 3’ exons) → chr16:22,545,231-22,613,938 (OTOAP1) 11 kb DeepVariant PBSV BCH-02 HiFi haplotype 1 BCH-02 HiFi haplotype 2 OTOA exon 22 exon 24 A>G missense Segmental Duplications
  5. 5 Identification of compound heterozygous pathogenic variants in STRC Segmental

    duplication: chr15:43,558,938-43,658,153 (PP1P5K1/CKMT1B/STRC/CATSPER2) → chr15:43,658,492-43,749,512 (PPIP5K1P1/CKMT1A/STRCP1/CATSPER2P1) PPIP5K1 CKMT1B STRC CATSPER2 CN1 - 104 kb DeepVariant HiFiCNV BCH-01 HiFi 120 kb G>A stop Segmental Duplications
  6. 6 Visualization of STRC & STRCP1 haplotypes identified by Paraphase

    More on Paraphase at poster P18.025.A STRC STRC hap1 variants BCH-01 STRC haplotype 1 23 kb STRCP1 hap1 variants STRCP1 hap2 variants BCH-01 STRCP1 haplotype 1 BCH-01 STRCP1 haplotype 2 G>A stop
  7. 7 www.pacb.com Research use only. Not for use in diagnostic

    procedures. © 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. Information in this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party products. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, and Onso are trademarks of PacBio.
  8. 8 PacBio HiFi WGS identifies potential causal variants not found

    by short-read sequencing (P16.011.D) William J Rowell*1, Shelby Redfield2, Cillian Nolan1, J Matthew Holt1, Xiao Chen1, Cairbre Fanslow1, Eirini Maria Lampraki1, Christine Lambert1, Chris Saunders1, Margaret A Kenna2,3, Eliot Shearer2,3, Michael A Eberle1 1. Pacific Biosciences, Menlo Park, United States 2. Boston Children's Hospital, Otolaryngology & Communication Enhancement, Boston, United States 3. Harvard Medical School, Otolaryngology Head and Neck Surgery, Boston, United States COI: Employees and shareholders of PacBio.
  9. 9 Computational tools drive discovery pbsv* Paraphase Methylation TRGT/TRVZ HiPhase**

    >28M CpG sites 5mC included* N50=493kb DeepVariant >22,000 SVs Sequence, repeat length, methylation >300 genes Compute power CNVs HiFiCNV Advanced CNV calling SNPs / small indels SVs Tandem Repeats Segmental duplications Phasing Discovery power Comprehensive WGS workflow available for AWS, GCP, Azure or on-prem HPC *Developing new 2nd analysis for SVs and 5mC **HiPhase replacing WhatsHap ~4.5M SNVs and indels