Video Gallery

We created the HiFiViral SARS-CoV-2 Kit for labs working on the front line of the COVID-19 pandemic — tracking and identifying the spread of novel variants in their communities. The kit is an easy-to-use, scalable, and highly accurate solution for sequencing the entire SARS-CoV-2 genome. The differentiated design approach is resilient to novel variants enabling comprehensive detection of all types of mutations.

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 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 two 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. In the talk, Dr. Sarah Kingan, demonstrates 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.

In this talk, Dr. Aaron Wenger, describes how 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.

In this talk, Dr. Elizabeth Tseng demonstrates a throughput increase for the scIso-Seq method by concatenating single-cell molecules, increasing yield a minimum of 6-fold per SMRT Cell 8M. She explains the bioinformatics workflow for analyzing concatenated scIso-Seq data, which begins with de-concatenation, followed by tagging of UMI and barcode information that can be processed by the isoseq3 pipeline for deduplication. Reads are then aligned against the reference genome, followed by SQANTI3 for transcript classification against a reference annotation (ex: GENCODE) which produces an isoform-level sparse matrix to be analyzed with single-cell tools such as Seurat. She also shows how to apply isoform-level phasing using a tool called IsoPhase, by first calling SNPs based on the deduplicated aligned reads, then using the full-length read information to identify the haplotypes, and finally assigning each isoform-haplotype its associated read counts. Since each read can be associated with its distinct cell type, this allows quantifying isoform expressions in an allele-specific and cell type-specific manner. Finally, she shows how this can be visualized using both standard genome browsers like IGV and long read-aware tools such as SWAN.

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. While numerous assays and genetic tests have been developed to interrogate pharmacogenes, several limitations exist, including lack of phasing information, and poor detection in complex regions with structural variants, pseudogenes, or gene conversions. In this talk, Dr. Nina Gonzaludo, 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, she discusses an amplicon strategy that resulted in ≥99.9% accuracy per sequencing run and >99% demultiplexed on target reads to CYP2D6. Additionally, results show improved haplotyping accuracy over orthogonal technologies in a set of 22 reference samples. Separately, she discusses 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.

The hereditary ataxias are a group of rare neurological diseases with similar symptoms. Many of these ataxic syndromes are caused by expansions of short tandem repeat (STR) in a number of different genes. Molecular genetic testing to accurately determine the genetic cause of known ataxias is often employed to support clinical diagnoses. We have recently developed an ataxia expansion panel using the PacBio No-Amp targeted sequencing approach to capture and sequence repeat expansion loci associated with fifteen ataxia diseases. The method utilizes CRISPR-Cas9 nuclease and pairs of guide RNAs to excise DNA fragments containing the repeat sequences within ataxia genes. This approach eliminates PCR amplification artifacts, amplification bias, and preserves native DNA for base modification detection. In this study, we sequenced samples with known or unknown diagnosis for ataxia with the No-Amp targeted sequencing panel utilizing PacBio highly accurate long reads – HiFi reads. The high accuracy of HiFi reads provides both certainty in sizing of the repeat expansion and repeat sequence interruption within the expansion sequences. Sequencing results demonstrate the potential of using this repeat expansion panel for eventual genetic testing. As additional ataxia, and related neurological diseases, caused by STR expansions are discovered and studied, the No-Amp targeted sequencing panel could be expanded to include additional targets. The ability to multiplex samples from different patients also makes the method a potentially cost-effective option for molecular genetic screening in the future.

With the September 2021 closing of PacBio’s acquisition of Omniome, PacBio intends to become the first company to offer both long-read and short-read sequencing platforms. What does this mean for customers? How is PacBio leadership thinking about delivering a differentiated set of products and applications into high-growth clinical markets? In this intimate conversation with genomics leaders, Christian Henry, and Richard Shen, they share their vision for the future as a combined company.

While RNA-sequencing has dramatically accelerated our understanding of biology, quantitation and discovery of full-length RNA isoforms resulting from alternative splicing remain poorly resolved. Alternative splicing is a core regulatory process that modulates the structure, expression, and localization of expressed proteins through differential exon and/or UTR splicing during transcript maturation. Beyond being an integral component of cellular development and homeostatic maintenance, RNA splicing is implicated in a wide range of pathologies with hallmark isoforms being linked to cardiovascular, neurological, and immunological diseases. Current limitations in isoform quantitation and discovery arise from the inability of existing sequencing platforms to scalably sequence full-length mRNAs – short-read approaches are unable to span most successive splice sites while long-read approaches are constrained by limited throughput. To enable scalable full-length RNA sequencing, we have developed the method Multiplexed Arrays sequencing (MAS-seq) which maximizes the sequencing potential of isoforms on the PacBio platform. Through the use of deoxy-uracil digestion followed by barcode-directed ligation of cDNAs, MAS-seq generates long multiplexed cDNA arrays with a length distribution that allows for both accurate consensus sequencing and optimal capacity utilization of PacBio sequencers. In combination with upstream artifact depletion measures, MAS-seq boosts the sequencing throughput to approximately 40 million full-length transcripts per SMRT Cell 8M, a >20-fold increase compared to PacBio’s scIso-Seq workflow. We sequenced synthetic RNA isoform standards (Lexogen) and demonstrate a 99.8% isoform identification accuracy using MAS-seq, far exceeding the 56.8% accuracy of Smart-seq3, the best-in-class short-read isoform identification protocol. MAS-seq-enabled single-cell RNA isoform sequencing of tumor infiltrating CD8+ T cells robustly identifies the canonical CD45 (PTPRC) isoforms associated with the range of observed T cell states – a finding additionally validated at the protein level via CITE-seq. Further, we demonstrate the impact of the long-read sequencing throughput gains on single-cell isoform analysis enabled by MAS-seq, providing a 44% increase in single-cell clustering capacity (adjusted Rand index) and a 34-fold gain in identifying differentially spliced genes amongst the CD8+ T cell subtypes. MAS-seq is a streamlined and cost-effective approach that enables scalable bulk and single-cell RNA isoform sequencing.

Many neurological diseases result from expansion of unstable variable nucleotide tandem repeats (VNTRs) that influence gene transcription of neighboring genes. In this talk, Dr. Henne Holstege presents research that investigated VNTRs across several genomes including a 115-year-old cognitively healthy individual. She and her group found that the genes that contained most VNTRs, of which PTPRN2 and DLGAP2 are the most prominent examples, were found to be predominantly expressed in the brain and associated with a wide variety of neurological disorders.

PacBio Vice President of Segment Marketing, Dr. Jennifer Stone, demonstrates 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.