2023 ACMG Annual Clinical Genetics Meeting Digital Edition: Poster Gallery-HiFi reads provide accurate detection of variants and DNA methylation in challenging regions of the genome

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HiFi long-read DNA sequencing provides accurate detection of variants and DNA methylation in challenging regions of the genome. Approximately 5% of protein-coding genes in the human genome have difficult-to-map exons, but HiFi reads can access these regions by connecting the homologous exon to neighboring unique sequences. HiFi sequencing also allows for phasing of variants into haplotypes and simultaneous detection of DNA methylation.<br /><br />The research conducted on the Genome in a Bottle trio samples used HiFi reads to achieve high mappability, high read quality, and the ability to detect methylation. Variant calling accuracy exceeded 99.9% for single nucleotide variants (SNVs), 99.3% for indels, and 95% for structural variants. Phasing and direct DNA methylation detection allowed for the detection of parental imprinting in certain genes.<br /><br />During HiFi sequencing, DNA is mechanically sheared to an average size of 15-20 kb, and a library is prepared using the SMRTbell prep kit 3.0. The circularized DNA is then sequenced by the polymerase in multiple passes, with each pass aligning the read, trimming adapters, and calling consensus and 5mC status to generate a HiFi read. HiFi reads have an average accuracy of 99.9% and can be 100 times longer than short-reads.<br /><br />The results of the study showed that HiFi sequencing on the PacBio Revio system produced high-quality data with high read accuracy and the ability to call variants and detect methylation. HiFi reads successfully mapped across regions that were difficult to map with short-read data. Phased HiFi reads were also able to reveal large structural variations.<br /><br />In conclusion, HiFi long-read DNA sequencing allows for accurate variant calling and DNA methylation detection, particularly in difficult-to-map genes. The use of HiFi sequencing is predicted to lead to the discovery of additional disease genes and variants that were previously beyond the reach of next-generation sequencing.

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Presenting Author - Greg Young, MS; Co-Author - Aaron Wenger, PhD; Co-Author - Matthew Boitano, BS; Co-Author - Armin Toepfer, PhD; Co-Author - Christine Lambert, PhD; Co-Author - Primo Baybayan, PhD; Co-Author - Emilia Mollova, PhD;

Meta Tag

Epigenetics

Genome sequencing

Imprinting

Methylation

Sequencing

Variant Detection

Co-Author Aaron Wenger, PhD

Co-Author Matthew Boitano, BS

Co-Author Armin Toepfer, PhD

Co-Author Christine Lambert, PhD

Co-Author Primo Baybayan, PhD

Co-Author Emilia Mollova, PhD

Presenting Author Greg Young, MS

Keywords

HiFi long-read DNA sequencing

variants

DNA methylation

challenging regions

genome

exons

phasing

methylation detection

variant calling accuracy

structural variations