Scientific posters

Comparing the concatenated (16S MAS-Seq) to non-concatenated full-length 16S datasets, we found no bias in community compositions and were able to assign up to ~90 – 99% of denoised reads to species. In addition, on the highly complex ZymoBIOMICS Fecal Reference with TruMatrix Technology (D6323) sample, we found 16S MAS-Seq to have high correlation to taxonomic abundances estimated from shotgun metagenomics sequencing using the same sample, emphasizing that it’s possible to get shotgun metagenome taxonomic resolution at amplicon sequencing costs with full-length 16S HiFi sequencing. Furthermore, with 16S MAS-Seq, researchers may now multiplex more samples to reduce cost/sample or to profile each sample deeper with more reads/sample.

Highly accurate long reads can overcome many of the obstacles associated with metagenome assembly. PacBio HiFi sequencing of metagenomic samples with the Sequel IIe or Revio systems regularly produces reads 8–15 kb in size with a median QV ranging from 30–45 (99.9–99.99% accuracy). Here, we present the newest version of the HiFi-MAG-Pipeline (v2.0), a comprehensive workflow that automates major steps including binning, quality filtering, and taxonomic identification.

We recently published a benchmarking study of several taxonomic profiling/classification methods for long-read datasets1. Here, we outline the experimental design and key findings of our study. To improve accessibility to top-performing tools, we also developed comprehensive workflows for 1) sourmash2-4 and 2) Diamond & MEGAN-LR5,6 and describe them here.

While whole-genome sequencing allows identification of clinically relevant variants in ~90% of the genome, there exist difficult regions that remain challenging for short read sequencing. We developed Paraphase1, a HiFi- based informatics method that accurately genotypes highly homologous genes and applied it to resolve hundreds of homologous regions across the genome.

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).

Improved throughput and cost of long- read sequencing, driven by recent technological advances of the PacBio Revio system, enables investigation of whole human genomes across larger populations. To support the growing capabilities of long-read sequencing, high throughput (HT) sample and library preparation solutions are necessary. We present a fully automated HT DNA extraction, shearing, and library preparation workflow for human whole blood samples for PacBio HiFi sequencing.

PacBio HiFi reads (99.9% accuracy, 15-20 kb) enable comprehensive variant detection in human genomes, extending to repetitive regions of the genome not accessible with short-read WGS (srWGS) or WES (srWES). HiFi reads match or surpass srWGS for single nucleotide variant and small indel (<50 bp) detection while also improving detection of structural variants (SVs, ≥50 bp), with recall far exceeding that of srWGS. Here we apply HiFi-WGS to 10 probands with unexplained hearing loss who had previously undergone srWES and srWGS with a negative result.

The use of metagenome-assembled genomes (MAGs) in microbial surveillance has increased in popularity. Metagenome assembly can allow for fine-scale resolution of epidemiological tracing and improve recovery of antimicrobial resistance (AMR) genes. Highly accurate PacBio HiFi reads can dramatically improve metagenome assembly, particularly when used with assemblers like hifiasm-meta, and can produce complete genomes. We present HiFi-MAG-Pipeline, a comprehensive workflow that identifies and extracts high-quality MAGs from long-read metagenomic assemblies.

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.

Understanding the quality of rAAV vectors by assessing the presence of cellular impurities such as truncations, chimeras, and host genome integrations are all essential for ensuring the purity and efficacy of the vector. While next-generation sequencing has been applied for assessing the quality of rAAV vectors, short read length technologies cannot sequence the full-length AAV molecules or accurately distinguish or quantify mixed populations that might contain both scAAV and ssAAV. PacBio SMRT sequencing is a long-read sequencing technology that can produce reads of >Q20 (>99%) for insert sizes of 10–20 kb and longer, which easily cover the entirety of typical rAAV constructs. To demonstrate that PacBio long-read sequencing can accurately characterize AAV constructs, we purchased scAAV and ssAAV constructs from commercial vendors. We created sequencing libraries from both pure (scAAV or ssAAV-only) vectors, as well as an artificially mixed (scAAV with ssAAV) population. The long-read data is able to characterize the proportion of vector genomes for truncation (full-length or partial), purity, and structure (self-complementary or single-strand). To summarize, we show that long-read sequencing has the ability to accurately and more comprehensively assess the potency and purity of rAAV vectors, making it a valuable tool for the quality assessment of rAAV genome populations.

Sarcomas are a broad group of soft tissue and bone cancers that can be difficult to treat leading to a high mortality rate. Sarcomas comprise two broad genomic classes: (1) simple karyotypes, where a single oncogenic structural variant (SV) clonally expands a subtype that is diagnostic and relevant to tumor burden tracking; and (2) complex karyotypes, with genomic instability resulting in heterogeneous cellular subtypes. Fusion genes have been discovered in one third of all sarcomas and are drivers of disease progression. PacBio full-length RNA sequencing (Iso-Seq method) detects fusion transcripts. However, to date, there are few dedicated long-read fusion detection tools, and none take advantage of the high read accuracy from Iso-Seq data. We present pbfusion, a fusion detection tool designed specifically for PacBio Iso-Seq data and apply it to twelve sarcoma samples. We demonstrate that pbfusion accurately identifies known and putative fusion genes (e.g.,TFE3-ASPSCR1). Simulation studies show that pbfusion is more sensitive than the alternative long-read fusion detection software while maintaining similar specificity.

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. The PacBio Revio system will enable researchers to meet the throughput demands for agrigenomics while maintaining best-in-class quality.

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.

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.