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Friday, February 26, 2021

A workflow for the comprehensive detection and prioritization of variants in human genomes with PacBio HiFi reads

PacBio HiFi reads (minimum 99% accuracy, 15-25 kb read length) have emerged as a powerful data type for comprehensive variant detection in human genomes. The HiFi read length extends confident mapping and variant calling to repetitive regions of the genome that are not accessible with short reads. Read length also improves detection of structural variants (SVs), with recall exceeding that of short reads by over 30%. High read quality allows for accurate single nucleotide variant and small indel detection, with precision and recall matching that of short reads. While many tools have been developed to take advantage of these qualities…

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Friday, February 26, 2021

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has been applied successfully to assemble genomes and detect structural variants. However, due to high raw-read error rates (10-15%), it has remained difficult to call small variants from long reads. Recent improvements in library preparation and sequencing chemistry have increased length, accuracy, and throughput of PacBio circular consensus sequencing (CCS) reads, resulting in 15-20kb reads with average read quality above 99%. Materials and Methods: We sequenced a library from human reference sample HG002 to 18-fold coverage on the PacBio Sequel II with two SMRT Cells 8M. The CCS algorithm was used to generate highly accurate (average 99.9%)…

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Friday, February 26, 2021

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has revealed more than 20,000 structural variants spanning over 12 Mb in a healthy human genome. Short-read sequencing fails to detect most structural variants but has remained the more effective approach for small variants, due to 10-15% error rates in long reads, and copy-number variants (CNVs), due to lack of effective long-read variant callers. The development of PacBio highly accurate long reads (HiFi reads) with read lengths of 10-25 kb and quality >99% presents the opportunity to capture all classes of variation with one approach.Methods: We sequence the Genome in a Bottle benchmark sample HG002 and an…

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Friday, February 5, 2021

Webinar: Sequence with Confidence – Introducing the Sequel II System

In this webinar, Jonas Korlach, Chief Scientific Officer, PacBio provides an overview of the features and the advantages of the new Sequel II System. Kiran Garimella, Senior Computational Scientist, Broad Institute of MIT and Harvard University, describes his work sequencing humans with HiFi reads enabling discovery of structural variants undetectable in short reads. Luke Tallon, Scientific Director, Genomics Resource Center, Institute for Genome Sciences, University of Maryland School of Medicine, covers the GRC’s work on bacterial multiplexing, 16S microbiome profiling, and shotgun metagenomics. Finally, Shane McCarthy, Senior Research Associate, University of Cambridge, focuses on the scaling and affordability of high-quality…

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Friday, February 5, 2021

Webinar: Variant calling and de novo genome assembly with PacBio HiFi reads

In this webinar, Sarah Kingan, Staff Scientist, PacBio, presents recent work on de novo genome assembly using PacBio HiFi reads. She highlights the benefits of HiFi data for base level accuracy, haplotype phasing, and ease of computation. And in samples ranging from human to plants, she benchmarks various tools for HiFi assembly and phasing, including the newly extended FALCON-Unzip assembler. Subsequently, Andrew Carroll, Genomics Product Lead, GoogleAI, explores how the GoogleAI team retrained DeepVariant, a highly accurate SNP and Indel caller, for PacBio HiFi data. The resulting DeepVariant models achieve comparable accuracies to short-read methods with the additional benefit of…

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Friday, February 5, 2021

Webinar: Best practices for working with PacBio tools (SMRT Link & developmental) on the command line

This webinar, presented by Roberto Lleras, provides the best practices for working with PacBio tools (SMRT Link and Developmental) on the command line. Please access this file for step-by-step instructions for installing SMRT Analysis tools and pbbioconda in order to analyze SMRT Sequencing data on the command line: https://www.pacb.com/wp-content/uploads/Bioinformatics-Webinar-Session-1-Getting-Started.pdf

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Friday, February 5, 2021

User Group Meeting: No Assembly Required – Making the most of Iso-Seq data

In this PacBio User Group Meeting presentation, PacBio scientist Kristin Mars speaks about recent updates, such as the single-day library prep that’s now possible with the Iso-Seq Express workflow. She also notes that one SMRT Cell 8M is sufficient for most Iso-Seq experiments for whole transcriptome sequencing at an affordable price.

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Friday, February 5, 2021

User Group Meeting: Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

In this PacBio User Group Meeting presentation, PacBio scientist Meredith Ashby shared several examples of analysis — from full-length 16S sequencing to shotgun sequencing — showing how SMRT Sequencing enables accurate representation for metagenomics and microbiome characterization, in some cases even without fully assembling genomes. New updates will provide users with a dedicated microbial assembly pipeline, optimized for all classes of bacteria, as well as increased multiplexing on the Sequel II System, now with 48 validated barcoded adapters. That throughput could reduce the cost of microbial analysis substantially.

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Friday, February 5, 2021

User Group Meeting: Lowering input requirements

To start Day 2 of the PacBio User Group Meeting, Jonas Korlach, PacBio CSO, provides an update on lowering DNA input amounts for SMRT Sequencing workflows. Updates include a more robust shearing method, a revised AMPure size selection, and introduction of multiplexing low input samples. Finally, the use of HiFi sequencing with low input results in a more complete genome assembly. Jonas closes by mentioning that the low DNA input protocol is now available and further advancements to lower input requirements even more will open opportunities for different samples, such as cancer needle biopsies.

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Friday, February 5, 2021

Webinar: Amplicon sequencing with confidence – High-fidelity, long-read PacBio sequencing solutions

In this webinar, Lori Aro and Cheryl Heiner of PacBio describe how high-throughput amplicon sequencing using Single Molecule, Real-Time (SMRT) Sequencing and the Sequel System allows for the easy and cost-effective generation of high-fidelity, long reads from amplicons ranging in size from several hundred base pairs to 20 kb. Topics covered include the latest advances in SMRT Sequencing performance for detection of all variant types even in difficult to sequence regions of the genome, multiplexing options to increase throughput and improve efficiency, and examples of amplicon sequencing of clinically relevant targets.

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Friday, February 5, 2021

AGBT Presentation: The Sequel II System – The next evolution of SMRT Sequencing

In this AGBT presentation, Marty Badgett shares a look at the latest results from circular consensus sequencing (CCS) mode for highly accurate reads and data from our soon-to-be-released Sequel II System. As he demonstrates, CCS reads cover the same molecule many times, delivering high consensus accuracy despite noisy raw reads; on average, reaching 10 passes achieves Q30 accuracy. Badgett offers several examples where this is useful, such as pharmacogenomic gene analysis and resolving metagenomic communities. He also provides an update on the Iso-Seq method, which can now segregate transcripts into haplotype-specific alleles using a new tool called Iso-Phase.

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Friday, February 5, 2021

AGBT Presentation: HiFi long reads for comprehensive genomic analysis

In this AGBT presentation, Mike Hunkapiller shares insights on using highly accurate long (HiFi) reads generated in circular consensus sequencing (CCS) mode for comprehensive genomic analysis and provides examples such as the sequencing of a Genome in a Bottle reference sample, which concluded with Q48 accuracy, 18 Mb contigs, and clearly phased haplotypes.

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Friday, February 5, 2021

PAG Conference: The impact of highly accurate PacBio sequence data on the assembly of a tetraploid rose

In this presentation at PAG 2020, Bart Nijland of Genetwister Technologies explains how his team set out to make a haplotype-aware assembly of the highly complex tetraploid Rosa x hybrida L. genome in order to capture its full range of genetic variation. HiFi reads generated from PacBio’s Sequel II System have made it possible to parse out critical information from many of the plant’s parental genes.

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Friday, February 5, 2021

PAG Conference: Workshop introduction

In this introductory talk to our PAG 2020 workshop, PacBio Chief Scientific Officer Jonas Korlach presents the evolution of Single Molecule, Real-Time (SMRT) Sequencing technology over the past decade and highlights recent developments, including the Sequel II System performance and reliability

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