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.
Genomic variation beyond single nucleotide variants, including structural variation (SV), copy number variants (CNV), and repeat expansions, plays a significant role in rare disease. However, current technologies require multiple tests to fully access these variants, resulting in complex testing algorithms and the potential for missed diagnoses. Long-read genome sequencing offers the ability to accurately detect SNV, CNV, SV, and expansions with a single test. This presentation will highlight the integrated analyses offered by HiFi sequencing, using case examples demonstrating the potential for a unified test.
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…
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.
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…
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…
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…
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…
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.…
The COVID-19 pandemic has brought new focus and resources to pathogen surveillance of all kinds. HiFi sequencing, which combines high accuracy, long read lengths, and single-molecule sequencing, is unique in its ability to provide comprehensive, high-resolution views of pathogens — either as isolates or as part of complex systems. Join Meredith Ashby (PacBio) and Haley Fiske (Illumina) as they discuss pathogen sequencing in the COVID era. Meredith Ashby (PacBio) - HiFi Sequencing for the COVID Era: High-Resolution Pathogen Surveillance Haley Fiske (Illumina) - Unlocking the Power of Genomics for Pathogen Detection and Surveillance
In this talk, Dr. Meredith Course presents her research on uncovering a 69-bp human-specific tandem repeat expansion in the final intron of WDR7. Larger repeat copy number is significantly associated with sporadic ALS cases, suggesting that it plays a role in disease susceptibility. Long-read sequencing reveals remarkable internal nucleotide variation, which was harnessed to determine the evolutionary origin of the expansion, its mechanism of replication, and its current state in modern-day humans. Each copy of the repeat has been determined to be able to form microRNAs and aggregate in cells and may sequester ALS-related RNA-binding proteins.
In this talk, Dr. Stephanie Tome describes using PacBio Single Molecule, Real-Time (SMRT) Sequencing to precisely measure large CTG repeat size and identify sequence interruptions of expanded allele to understand clinical and genetic variability in DM1 patients, sequencing several DM1 patients with CTG repeat expansion ranging from 130 to > 1000 CTG repeats on the Sequel I and II Systems from amplicons. She obtained more than 77% full DM1 reads per sample, with >70% of the reads from expanded alleles. The data includes long reads in the expected size range for all samples, including DM1 patients with more than 1000…
In this talk, Dr. Zachary McEachin describes how No-Amp targeted sequencing enables sequencing analysis of multiple (at least 15) Ataxia-related repeat expansion loci in one assay, presenting example sequencing results with selected samples from Ataxia patients and patients with unknown diagnostics. He demonstrates how PacBio HiFi sequencing with the No-Amp approach could provide repeat expansion and sequence interruption information at the target loci that is not available with PCR or southern-blot based diagnostics assays.
Spinocerebellar ataxia type 10 (SCA10) is a rare autosomal-dominant disorder caused by an expanded intronic pentanucleotide repeat in the ATXN10 gene. This repeat expansion when fully penetrant can be typically expected between 850 and 4500 repeats or 4.25 to 22.5 kb. Current diagnostic assays using PCR-based methods or Southern blotting cannot accurately size or resolve the genomic structure of the ATXN10 repeat. In this talk, Dr. Birgitt Schuele elaborates that CRISPR-Cas9 enrichment/single molecule real time (SMRT) sequencing technology and optical mapping now allow for accurate sizing of the repeat expansion, repeat composition, and can resolve somatic mosaicism, which are critical…
In this talk, Dr. Flora Tassone focuses on the identification of alternative splicing isoforms at the FMR1 locus (both sense and antisense direction) in individual carriers of the FMR1 premutation allele, using the SMRT Sequencing approach. The characterization of a transcriptional profile could help to define a biomarker for monitoring disease progression and development.