Improving Human Health through Genomics with Highly Accurate Long Reads

HiFi sequencing allows researchers around world the ability to see more, and to do more. With highly accurate long-read sequencing, researchers can see beyond the ordinary in the pursuit of improving human health.

During this year's ASHG workshop, we shared our excitement about what's in store for long-read sequencing and human health in the pursuit of unlocking great discoveries.

PacBio Vice President of Segment Marketing, Jennifer Stone, demonstrated how HiFi sequencing is changing the game in human genetics by sharing some of the exciting milestones and seminal publications our technology has produced this year.

Jennifer was joined by:

Emily Farrow, Children’s Mercy, Kansas City, who shared how long-read genome sequencing offers the ability to detect SNV, CNS, and SV expansions with a single test

Aziz Al'Khafaji, Broad Institute, who shared how PacBio sequencing is working to unravel the obstacles of alternative splicing in the pursuit of full-length RNA isoforms linked to cardiovascular, neurological, and immunological diseases

Henne Holstege, Amsterdam University Medical Center, who discussed her work with centenarians and long-read sequencing to identify predominantly expressed VNTRs in the brains of patients who have been diagnosed with Alzheimer's

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Workshop Speakers

Scalable RNA Isoform Sequencing using Intramolecular Multiplexed cDNAs

Aziz Al’Khafaji, Ph.D.
Postdoctoral Associate, Broad Institute

Integrated Rare Disease Genomics using Long-Read Genome Sequencing

Emily G. Farrow, Ph.D., CGC
Director, Laboratory Operations - Genomic Medicine Center, Children’s Mercy Kansas City

Uncovering Neurological Disorders Through an Examination of VNTRs

Henne Holstege, Ph.D.
Assistant Professor, Amsterdam University Medical Center

HiFi Sequencing: See What You’ve Been Missing

Jennifer Stone, Ph.D.
Vice President, Segment Marketing, PacBio

PACBIO FIRESIDE CHAT

With Long Reads and Short Reads, the Possibilities are Endless

With the September closing of PacBio’s acquisition of Omniome, PacBio intends to become the first company to offer both long-read and short-read sequencing platforms. During this 30-minute intimate conversation with genomics leaders, Christian Henry and Richard Shen, they share their vision for the future as a combined company.

Christian Henry
President and CEO, PacBio

Richard Shen
President, Omniome

Pacbio colab theater talks

Using Long-Read Sequencing for Haplotyping and Phasing of PGx Alleles and Allele-Specific RNA-Seq Analysis on Concatenated Single-Cell Molecules

Pharmacogenomics (PGx) utilizes genomic information to assess an individual’s response to certain medications and can be used to predict adverse drug reactions or decreased efficacy. This talk describes amplicon and targeted enrichment capture SMRT Sequencing workflows that generate HiFi reads for high resolution of PGx alleles. To fully resolve CYP2D6, a highly polymorphic gene in a region with extensive homology, we discuss an amplicon strategy that resulted in ≥99.9% accuracy per sequencing run and >99% demultiplexed on target reads to CYP2D6. Results show improved haplotyping accuracy over orthogonal technologies in a set of 22 reference samples. Also discussed are preliminary results from a panel enrichment strategy for high resolution genotyping and phasing of 43 clinically significant pharmacogenes. These approaches can be leveraged as cost-effective and highly accurate methods for advancing PGx research and discovery. Finally, recent studies have shown that the scIso-Seq (single-cell isoform sequencing) method using PacBio revealed cell type-specific splicing events not observable with short reads, while the high accuracy (>99.9%) of the PacBio HiFi reads allowed direct linkage with cell barcodes from matching short read data. We demonstrate a further throughput increase for the scIso-Seq method by concatenating single-cell molecules, increasing yield a minimum of 6-fold per SMRT Cell 8M. We also explain the bioinformatics workflow for analyzing concatenated scIso-Seq data.

Elizabeth Tseng, Ph.D.
Associate Director, Product Marketing, PacBio

Nina Gonzaludo, Ph.D.
Sr. Manager, PGx & HLA Market Development, PacBio

PACBIO LIGHTNING TALKS

Methylation Detection with PacBio HiFi Sequencing - Presented by: Aaron Wenger, Ph.D.

PacBio HiFi reads (15 kb - 25 kb, >99.9% accuracy) provide the most complete view of human genetic variation, including small variants in difficult-to-map regions and structural variants genome-wide. Further, PacBio sequencing simultaneously detects epigenetic modifications without requiring a specialized library preparation step like bisulfite conversion. This ability is commonly used to characterize epigenetic marks in bacterial genomes. Recent improvements in read length and data analysis have extended the ability to include the 5mC methylation that is present at CpG sites in human genomes. Using a deep learning model that integrates sequencing kinetics and base context, the accuracy of 5mC detection in humans for individual HiFi reads is around 80%. Combining multiple reads, the concordance to EMseq and bisulfite sequencing reaches around 90%. The single-molecule resolution of methylation, together with phasing from accurate long reads, allows the detection of allele-specific methylation patterns such as parental imprinting. This ability to detect bases and modifications allows HiFi sequencing to provide the most complete genome and epigenomes with a single technology and library preparation.

HiFiViral: A Novel Method for Surveillance of SARS-CoV-2 that is Robust Across Sample Input Quantities and the Evolution of New Variants - Presented by: Sarah B Kingan, Ph.D.

The COVID-19 pandemic continues to be a major global epidemiological challenge with the ongoing emergence of new strain lineages that are more contagious, more virulent, drug resistant and in some cases evade vaccine-induced immunity. In response, the HiFiViral SARS-CoV-2 kit (PacBio; Menlo Park, California) was developed as a scalable solution for the Sequel II and Sequel IIe Systems. Unlike amplicon sequencing, the HiFiViral SARS-CoV-2 kit uses tiled probes, resulting in robust genome coverage across varying viral input quantities despite the presence of new variants. The use of highly accurate long reads, or HiFi reads, enables comprehensive variant detection, including single nucleotide variants, indels, and structural variants, as well as phasing of variants if multiple strains are present in samples. The fully kitted solution contains all reagents needed for viral enrichment and barcoding up to 384 samples, which can be pooled in a single SMRTbell library and run on a single SMRT Cell 8M. The add-only workflow requires 2 days in the lab with overnight sequencing and analysis and may be automated to achieve a turnaround time of 24 hours. SMRT Link analysis includes variant calling, HiFi read depth plots, the detection of multiple strains in samples, and consensus sequences ready for submission to public databases. Here, we demonstrate performance across a broad range of sample Ct, the accuracy of our variant calling method for viral controls, including variants of concern, and the ability to detect multiple variants down to 20% minor frequency. HiFiViral for SARS-CoV-2 is a cost effective, convenient, and accurate method for viral sequencing, well-suited for scalable surveillance of a rapidly evolving virus to inform public health decision making.

Aaron Wenger, Ph.D.
Associate Director, Product Marketing, PacBio

Sarah B Kingan, Ph.D.
Senior Staff Product Manager, PacBio

Presentations + Posters

Title	First Author
Towards isoform resolution single-cell transcriptomics for clinical applications using highly accurate long-read sequencing	E. Tseng
Long-read amplicon sequencing of the polymorphic CYP2D6 locus	L. Zhu
Simplified and robust library construction for high-throughput HiFi sequencing for human variant detection	H. Dhillon
Resolving complex pathogenic alleles using HiFi sequencing for long-range amplicon data with a new clustering algorithm	J. Harting
Targeting clinically significant dark regions of the human genome with high-accuracy, long-read sequencing	I. McLaughlin
Development and optimization of a 43-gene pharmacogenomic panel using enrichment-based capture and PacBio HiFi sequencing	D. Portik

Title	First Author
A high-resolution panel for uncovering repeat expansions that cause ataxias	Y. Tsai

PacBio HiFi Sequencing:
See Beyond the Ordinary