1 HiFi sequencing identifies likely causal variants missed by short-reads William J Rowell (wrowell@pacb.com) COI: Employee and shareholder of PacBio.

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 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 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 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 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

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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 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