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Tuesday, June 1, 2021

Amplification-free targeted enrichment powered by CRISPR-Cas9 and long-read Single Molecule Real-Time (SMRT) Sequencing can efficiently and accurately sequence challenging repeat expansion disorders

Genomic regions with extreme base composition bias and repetitive sequences have long proven challenging for targeted enrichment methods, as they rely upon some form of amplification. Similarly, most DNA sequencing technologies struggle to faithfully sequence regions of low complexity. This has been especially trying for repeat expansion disorders such as Fragile-X disease, Huntington disease and various Ataxias, where the repetitive elements range from several hundreds of bases to tens of kilobases. We have developed a robust, amplification-free targeted enrichment technique, called No-Amp Targeted Sequencing, that employs the CRISPR-Cas9 system. In conjunction with SMRT Sequencing, which delivers long reads spanning the…

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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

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

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

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

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

AGBT Presentation: Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression

In this AGBT presentation from AGBT 2019, Jason Underwood, shares information about single-cell isoform sequencing (scIso-Seq), focusing on a collaborative project with the labs of Evan Eichler and Alex Pollen. For this effort, scientists used Drop-seq sample prep and then loaded cDNA products onto the Sequel System. Results from a barnyard experiment using mouse and human cells as well as from cerebral organoids demonstrated that this approach could deliver cell type-specific gene expression data. Underwood also presents data from the Sequel II System comparing chimp and human organoids, resulting in information about 14,000 unique genes with important insights for post-transcriptional…

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Friday, February 5, 2021

Webinar: Discover full-length RNA sequencing – No assembly required

In this webinar we present Single Molecule, Real-Time (SMRT) Sequencing and the Iso-Seq method, which allow you to generate full-length cDNA sequences — no assembly required — to characterize transcript isoforms within targeted genes or across an entire transcriptome. The presenters share how the Iso-Seq method: (1) Provides high quality, full-length transcript sequences of up to 15 kb; (2) Allows for one-day library prep on a single SMRT Cell 8M to comprehensively characterize a whole transcriptome; (3) Facilitates discovery of alternative splicing events, fusion gene detection, and allelic specific isoform detection; and (4) Enables discovery of potential cancer-specific isoforms in…

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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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Friday, February 5, 2021

User Group Meeting: Sequencing chemistry & application updates

To start Day 1 of the PacBio User Group Meeting, Jonas Korlach, PacBio CSO, provides an update on the latest releases and performance metrics for the Sequel II System. The longest reads generated on this system with the SMRT Cell 8M now go beyond 175,000 bases, while maintaining extremely high accuracy. HiFi mode, for example, uses circular consensus sequencing to achieve accuracy of Q40 or even Q50.

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Friday, February 5, 2021

PAG Conference: Phylogenetic insights into the endophyte symbiosis using PacBio ribosomal DNA sequencing

Jana U’Ren of the University of Arizona discusses the fungi that live inside of plants at a PacBio workshop at the PAG 2020 conference. U’Ren studies the biology and evolution of mycorrhizal fungi found in the photosynthetic tissue of plant leaves, which are grouped together functionally as endophytes. In this video, she shares some of her preliminary findings collecting and analyzing samples from Boreal forests around the world.

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Friday, February 5, 2021

AGBT Presentation: Studying CRISPR guide RNA specificity by amplification-free long-read sequencing

At AGBT 2020, Adam Ameur from Uppsala University discussed the use of long-read PacBio sequencing to detect off-target results from CRISPR/Cas9 gene editing studies. His team uses HiFi reads from the Sequel II System to perform whole genome sequencing and figure out exactly where guide RNAs bind. In one example using a human embryonic kidney cell line, they found 55 off-target sites for three guide RNAs. Ameur’s group has already generated preliminary data on results from editing living cells.

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Friday, February 5, 2021

AGBT Presentation: Generating high quality human reference assemblies with PacBio sequencing

Tina Graves-Lindsay from the McDonnell Genome Institute reports at AGBT 2020 on how her team is using PacBio sequencing to produce reference-grade human genome assemblies. With highly accurate HiFi reads, no error correction step is needed during the sequencing and analysis process, and they can produce reference-grade assemblies with half the sequence coverage needed before. They are now generating diploid assemblies and will be contributing to the human pangenome reference project.

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