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Scientific posters

ASM 2023  |  2023

Increasing throughput of full-length 16S sequencing using concatenation

Jacob Brandenburg1, Khi Pin Chua1, Siyuan Zhang1, Jason Underwood1, Minning Chin1, Wei-Shen Cheng1, Sian Loong Au1, Primo Baybayan1, Holly Ganz2, Guillaume Jospin2, Ye Tao3, Qin Lin3, Elizabeth Tseng1, Jeremy E Wilkinson1 1. PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 2. AnimalBiome, 400 29th St., Ste 101, Oakland, CA, USA 94609 3. Biozeron Biotechnology Co., Ltd., Shanghai, 201800 China

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.
ASM 2023  |  2023

Maximizing MAGs from long-read metagenomic assemblies: a post-assembly pipeline with completeness-aware binning

Daniel M. Portik & Jeremy E. Wilkinson PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025

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.
ASM 2023  |  2023

Taxonomic classification and profiling of long-read shotgun metagenomics datasets: best practices and new pipelines

Daniel J. Nasko1, Jeremy E. Wilkinson1, C. Titus Brown2, N. Tessa Pierce-Ward2, and Daniel M. Portik1 1. PacBio, 1305 O’Brien Drive, Menlo Park, California USA; 2. Department of Population Health and Reproduction, University of California Davis, Davis, California USA

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.
ESHG 2023  |  2023

Complete resolution of gene/paralog pairs with PacBio HiFi sequencing

Xiao Chen1, Emily Farrow2, Isabelle Thiffault2, Dalia Kasperaviciute3, Genomics England Research Consortium, Alexander Hoischen4, Christian Gilissen4, Tomi Pastinen2, Michael A Eberle1 1. PacBio, Menlo Park, CA, USA. 2. Children’s Mercy Kansas City, MO, USA. 3. Genomics England Ltd., London, UK. 4. Radboud University Medical Center, Nijmegen, The Netherlands.

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.
ESHG 2023  |  2023

From sample to star alleles: a long-read pharmacogenomics pipeline powered by Twist target enrichment and PacBio HiFi sequencing

N. Gonzaludo1, T. Han2, L. Arbiza2, A. Souppe1, C. Lambert1, S. Zhang1, P. Baybayan1, H. Ferraro1, S. Kingan1, B. Li3, K. Sangkuhl3, M. Woon3, R. Whaley3, M. Whirl-Carrillo3, Y. Yang3, T. E. Klein3, S. A. Scott3, J. Harting1 1 PacBio, Menlo Park, CA, 2 Twist Bioscience, South San Francisco, CA, 3 Stanford University, Stanford, CA

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).
ESHG 2023  |  2023

High throughput multiomic analysis for human genomics on PacBio Revio system

Julian Rocha1, Jeffrey Burke1, Renee Fedak1, Duncan Kilburn1, Deborah Moine1, Enrique Bayo Iglesias2,Dominik Laubscher2, Birgit Ottenwälder2, Suzanne Dee1, Heather Ferrao1, Kelvin J Liu1 1. PacBio, MP, USA; 2. Hamilton, Bonaduz, Switzerland

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.
ESHG 2023  |  2023

PacBio HiFi WGS identifies potential causal variants not found by short read sequencing

William Rowell*1, Shelby Redfield2, Cillian Nolan1, J. Matthew Holt1, Cairbre Fanslow1, Eirini Maria Lampraki1, Christine Lambert1, Chris Saunders1, Margaret A. Kenna2, 3, Eliot Shearer2, 3, Michael Eberle11Pacific Biosciences, Menlo Park, United States, 2 Boston Children's Hospital, Otolaryngology & Communication Enhancement, Boston, United States, 3Harvard Medical School, Otolaryngology Head and Neck Surgery, Boston, United States

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.
APHL 2023  |  2023

Maximizing MAGs from long-read metagenomic assemblies

Portik, Daniel M. and Wilkinson, Jeremy E. and Dahlen, Trang

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.
Microbiology Society Annual Conference 2023  |  2023

High MAG recovery and precision species profiling of a pooled human gut microbiome reference using PacBio HiFi sequencing

Kaur, Kirren and Wilkinson, Jeremy E. and Ashby, Meredith and Zhang, Siyuan and Chua, Khi Pin and Locken, Kris and Tang, Shuiquan and Farthing, Brett and Weinstein, Michael and Carlin, Martha and Cano, Raul and Langford, Kyle and Auch, Benjamin and Liachko, Ivan and Portik, Daniel M.

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.
ASGCT 2023  |  2023

Full-length rAAV sequencing for mixture population characterization using highly accurate long reads

Tseng, Elizabeth and Dhillon, Harsharan and Volden, Roger

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.
AACR 2023  |  2023

pbfusion: Detecting fusion genes and other transcriptional abnormalities from PacBio Iso-Seq data Abstract No. 9285

Roger Volden1 , Zev Kronenberg1 , Aaron Gillmor2 , Ted Verhey2 , Michael Monument2 , Donna Senger2 , Harsharan Dhillon1 , Jason Underwood1 , Khi Pin Chua1 , Elizabeth Tseng1 , Daniel Baker1 , Primo Baybayan1 , Michael A. Eberle1 , Jonas Korlach1 , Sorana Morrissy2 1 PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 2 Charbonneau Cancer Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada

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.
AGBT-Ag 2023  |  2023

Ultra-high throughput multi-omic analysis for agrigenomics on PacBio Revio system

Jeffrey Burke1, Charlotte Harris2, Renee Fedak1, Victor Llaca2, Gina Zastrow-Hayes2, Kelvin Liu1 1 PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 and 2 Corteva Agriscience, 8325 NW 62nd Ave, Johnston, IA 50131

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.
ACMG 2023  |  2023

HiFi reads provide accurate detection of variants and DNA methylation in challenging regions of the genome

Young, Gregory and Wenger, Aaron M. and Boitano, Matthew and Toepfer, Armin and Lambert, Christine and Baybayan, Primo and Mollova, Emilia

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.
ACMG 2023  |  2023

Highly scalable pharmacogenomic panel testing with hybrid capture and long-read sequencing

N. Gonzaludo1 , S. Kingan1 , J. Harting1 , P. Baybayan1 , T. Han2 , L. Arbiza2 , B. Li3, Y. Yang3,4 , N. Hammond4 , S. A. Scott3,4 1 PacBio, Menlo Park, CA, 2 Twist Bioscience, South San Francisco, CA, 3 Stanford University, Stanford, CA, 4 Clinical Genomics Laboratory, Stanford Medicine, Palo Alto, CA

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.
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