Scientific posters

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.

Advancements in sequencing technologies have made metagenomic analyses of complex microbial samples routine and accessible. Mock communities of known composition are often run in parallel to allow for accurate data evaluation and to facilitate cross-study and inter-lab comparisons, yet they lack the microbial diversity of real-world samples. The ZymoBIOMICS Fecal Reference with TruMatrix Technology (D6323) is a highly diverse pooled human fecal reference that provides a truly complex alternative to mock communities. However, the microbial content of this standard is only partially characterized, and species level composition remains underexplored. Here, we explore the content of this sample using highly accurate long-read sequencing.

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.

Targeted resequencing allows for high-resolution characterization of gene panels at a scale and cost that is more accessible than whole genome sequencing. While long-read PacBio HiFi sequencing has been shown to accurately and comprehensively interrogate complex clinically actionable loci, such as pharmacogenomic targets, studies have been primarily focused on single genes using PCR amplicon- based methods. We describe a method to leverage Twist Bioscience target enrichment probes for the design of custom gene panels sequenced with HiFi reads.

Both the genome and epigenome contribute to inherited disease. While genome sequencing has been applied at large scale, epigenome sequencing remains more difficult and expensive and less frequently used. Here we extend PacBio HiFi sequencing to simultaneously generate accurate genomes and epigenomes with a single library prep and sequencing experiment.

A heteroduplex is a double-stranded sequence comprised of two non-complementary strands that can form during PCR. These mixed-template artifacts produce misleading results in downstream analysis, e.g., false haplotypes during diplotyping. Unlike short-read technologies, PacBio Single- Molecule Real-Time sequencing produces strand-level base calls. Heteroduplex signatures can be directly observed and corrected using the stranded sub-read data. Our new method is integrated in the circular consensus sequence algorithm which generates accurate HiFi data from sub-reads.

The CYP2D6 locus is well known for its importance to pharmacogenetics as well as for its high diversity and complex genomic setting. Resolving individual alleles at this locus using short-read sequencing technologies requires inference-based methods due to ambiguous mapping in the presence of highly homologous pseudogenes. In contrast, long-range sequencing with PacBio HiFi reads directly resolves and phases a wide range of complicated and difficult genetic loci without inference. We present a novel bioinformatics workflow using PacBio HiFi reads which enables rapid and precise diplotyping and star(*)-allele classification of CYP2D6.