Scientific posters

We developed Paraphase1, a HiFi-based informatics method that accurately genotypes highly homologous genes, and applied it to resolve ten clinically relevant challenging genes. Here we show detailed results in three genomic regions, revealing new population-wide biological and evolutionary insights.

HiFi long-read DNA sequencing provides increased mappability, accessing many of the difficult regions by connecting the homologous exon to neighboring unique sequences. Additionally, HiFi sequencing allows phasing of variants into haplotypes and detects DNA methylation to provide simultaneous characterization of the epigenome.

We demonstrate the use of the Twist Alliance Long-Read PGx panel for use with PacBio HiFi sequencing systems, with an analysis pipeline that includes primary analysis (on-instrument), bioinformatics tools for secondary and tertiary analysis, star allele calling with Pangu and PharmCAT, and interpretation and reporting with PharmCAT and custom EHR clinical decision support (CDS) (e.g. Epic Best Practice Alerts).

Legacy low-cost technologies can be biased towards known variants and/or populations and may result in data that are incomplete or difficult to phase, potentially affecting phenotype prediction and subsequent clinical utility. With long-read PacBio HiFi sequencing and Twist Bioscience hybrid capture technology, we describe a pre-designed PGx panel that is comprehensive and cost-efficient, allowing for scalable application to precision medicine research programs.

Understanding cell heterogeneity at the isoform level is critical for both basic and disease research. Short reads can only capture gene-level information, while other longread technologies lack the accuracy for accurate unique molecular identifiers (UMI) and cell barcode (CBC) identification. PacBio HiFi reads sequence full-length RNA isoforms along with single-cell barcode and UMI information, revealing unprecedented insight into singlecell biology.

Climate change and the rapidly growing global population are increasingly straining world food production. Long-read sequencing is being used in large-scale agricultural initiatives to help improve crop yields and combat pests and disease to meet increased agricultural demand. DNA extraction was automated on the Kingfisher DNA purification systems with PacBio-developed DNA extraction reagents and protocols.

PacBio sequencing technology delivers the most complete, accurate, continuous genomes, and has been used as the core technology in many biodiversity, conservation, and agrigenomics initiatives. Here we present significant advancements in our workflow that further facilitate sequencing efforts by offering methods for DNA isolation and an enhanced size selection for the library prep process. These improvements were applied to a diversity of plant, insect, and animal samples and sequenced on the new Revio system, generating more than 90 Gb of data from each library.

There are many challenges involved with metagenome assembly, including the presence of multiple species, uneven species abundances, and conserved genomic regions that are shared across species. Highly accurate long reads offer clear advantages over short reads and can overcome many of the obstacles associated with metagenome assembly. PacBio HiFi sequencing of metagenomic samples with the Sequel IIe system regularly produces reads 8–15 kb in size with a median QV ranging from 30–45 (99.9–99.99% accuracy).

The CYP2D6 locus is known for its importance to pharmacogenetics as well as for its high diversity and complexity. SNPs, indels, gene duplications, hybrid genes, gene conversion, and large deletions are common at this locus. Resolving and phasing individual alleles without imputation requires long and highly accurate reads. We demonstrate a novel CYP2D6 typing tool, Pangu, for accurately assigning star allele diplotypes from PacBio HiFi reads. Unambiguous calls are made using multiple input data types from HiFi long-read sequencing, including whole genome sequencing (WGS), hybridization-based target enrichment, and long-range amplicon sequencing.

Unencumbered by generations of selective pressure, de novo mutations (DNMs) are more likely to be deleterious than inherited variants and account for much of the variation driving rare disease. HiFi reads are useful for detecting DNMs because of their accuracy and length.

Sample preparation workflows for long-read sequencing often contain bottlenecks in extraction, size selection, shearing, and library preparation that limit sample throughput, add cost, and create variability in data yields and quality. We present a high-throughput, fully automated 96-sample workflow that can be used to sequence and analyze a variety of human sample types and thus support growing numbers of large-scale population genomics and clinical research studies in a manner that rivals standard NGS workflows in simplicity.

PacBio HiFi sequencing provides the most accurate and complete characterization of human genomes. Sequencing observes a polymerase in real time as it incorporates fluorescently labeled nucleotides to synthesize a DNA strand. Kinetic signatures including pulse width and interpulse duration correlate with chemical modifications to the canonical DNA bases (Fig. 1), including the 5 methylcytosine (5mC) modification without bisulfite treatment. This kinetic information is independent of the fluorescence intensity used for base calling.

Long-read shotgun metagenomic sequencing is gaining in popularity and offers many advantages over short-read sequencing. The higher information content in long reads is useful for taxonomic profiling, where the main goal is to identify the species present in a microbiome sample (typically bacteria, archaea, fungi, viruses) and their relative abundances. The development of long-read specific tools for taxonomic profiling is accelerating, yet there is a lack of consensus regarding their relative performance. We performed a critical benchmarking study using five long-read methods and four popular short-read methods1. We applied these tools to several mock community datasets generated using PacBio HiFi sequencing or Oxford Nanopore Technology (ONT) sequencing, and Illumina data.

There are many challenges involved with metagenome assembly, including the presence of multiple species, uneven species abundances, and conserved genomic regions that are shared across species. Highly accurate long reads offer clear advantages over short reads and can overcome many of the obstacles associated with metagenome assembly. PacBio HiFi sequencing of metagenomic samples with the Sequel IIe system regularly produces reads 8–15 kb in size with a median QV ranging from 30 – 45 (99.9–99.99% accuracy). With the development of new metagenome assembly algorithms specific to HiFi reads (hifiasm-meta, metaFlye), it is now possible to reconstruct full metagenome assembled genomes (MAGs) for many high abundance species. These MAGs are often composed of a single circular contig, representing high-quality complete bacterial genomes. However, discontiguous assemblies still occur for lower abundance taxa, and post-assembly tools are required to identify MAGs in this category. Here, we present the HiFi-MAG-Pipeline, a comprehensive workflow for processing long-read metagenome assemblies.

Long-read shotgun metagenomic sequencing is gaining in popularity and offers many advantages over short-read sequencing. The higher information content in long reads is useful for taxonomic profiling, where the main goal is to identify the species present in a microbiome sample (typically bacteria, archaea, fungi, viruses) and their relative abundances. The development of long-read specific tools for taxonomic profiling is accelerating, yet there is a lack of consensus regarding their relative performance. We performed a critical benchmarking study using five long-read methods and four popular short-read methods. We applied these tools to several mock community datasets generated using PacBio HiFi sequencing or Oxford Nanopore Technology (ONT) sequencing, and Illumina data.