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Thursday, November 12, 2020

Application Note: Microbial multiplexing workflow on the Sequel System

Obtaining microbial genomes with the highest accuracy and contiguity is extremely important when exploring the functional impact of genetic and epigenetic variants on a genome-wide scale. A comprehensive view of the bacterial genome, including genes, regulatory regions, IS elements, phage integration sites, and base modifications is vital to understanding key traits such as antibiotic resistance, virulence, and metabolism. SMRT Sequencing provides complete genomes, often assembled into a single contig. Our streamlined microbial multiplexing procedure for the Sequel System, from library preparation to genome assembly, can be completed with less than 8 hours bench time. Starting with high-quality genomic DNA (gDNA),…

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Thursday, November 12, 2020

Application Note: Considerations for using the low and ultra-low DNA input workflows for whole genome sequencing

As the foundation for scientific discoveries in genetic diversity, sequencing data must be accurate and complete. With highly accurate long-read sequencing, or HiFi sequencing, there is no longer a compromise between read length and accuracy. HiFi sequencing enables some of the highest quality de novo genome assemblies available today as well as comprehensive variant detection in human samples. PacBio HiFi libraries constructed using our standard library workflows require at least 3 µg of DNA input per 1 Gb of genome length, or ~10 µg for a human sample. For some samples it is not possible to extract this amount of…

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Tuesday, October 27, 2020

ASHG PacBio Workshop: Latest product and application updates

In this ASHG 2020 PacBio Workshop Jonas Korlach, CSO, shares how the new PacBio Sequel IIe System makes highly accurate long-read sequencing easy and affordable so?all scientists can gain comprehensive views of human genomes and transcriptomes. He goes on to provide updates on the applications including human WGS for variant detection, de novo genome assembly, single-cell full-length RNA sequencing, and targeted sequencing using PCR and No-Amp methods.

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Sunday, October 25, 2020

Seminar: Optimizing eukaryotic de novo genome assembly with long-read sequencing

This seminar features great hands-on information and best practices for analyzing SMRT Sequencing data for eukaryotic genome assembly. Michael Schatz provides an overview of the assembly tools, provides recommendations for when to use each one, and discusses the challenges of short-read assemblies. James Gurtowski gives an in-depth overview of hybrid assemblies methods, where short read data are used used to correct errors in longer reads. Finally, Sergey Koren presents on chromosome-scale assembly, including the MinHash Alignment Process (MHAP) he developed to dramatically reduce the computational processing power required for genome assemblies.

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Sunday, October 25, 2020

Educational Video: Step 2 – de novo assembly of a genome

This video provides an overview of the techniques and steps of generating a de novo genome assembly with long-read sequencing data generated using PacBio Single Molecule, Real-Time (SMRT) Sequencing. In this video, a PacBio scientist covers the benefits of long reads when generating high-quality genome assemblies, the latest tools for creating assemblies, including HGAP, FALCON and FALCON-Unzip, how to polish and assess the quality of a genome assembly, and how to submit an assembly to NCBI.

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Sunday, October 25, 2020

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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Sunday, October 25, 2020

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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Sunday, October 25, 2020

Webinar: SMRT Sequencing applications for human genomics and medicine

In this webinar, Adam Ameur of SciLifeLab at Uppsala University shares how he uses Single Molecule, Real-Time (SMRT) Sequencing applications for medical diagnostics and human genetics research, including sequencing of single genes and de novo assembly of human genomes as well as a new method for detection of CRISPR-Cas9 off-targets.

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Sunday, October 25, 2020

Webinar: Beyond a single reference genome – The advantages of sequencing multiple individuals

Hear how scientists have used PacBio sequencing to develop pangenome collections and to study population genetics of plant and animal species to power their research. Learn about the advantages of sequencing multiple individuals to gain comprehensive views of genetic variation, and understand the speed, cost, and accuracy benefits of using highly accurate long reads (HiFi reads) to sequence your species of interest.

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Tuesday, April 21, 2020

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in…

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Tuesday, April 21, 2020

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and…

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Tuesday, April 21, 2020

Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting…

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