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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

SMRT Leiden: Ultra-fast comparative genomics of complex genomes at population scale

In this SMRT Leiden 2020 Online Virtual Event presentation, Erwin Datema of KeyGene shares his work on using high-throughput, accurate long-read sequencing technologies, such as PacBio HiFi sequencing, to drastically reduced the investment required to generate high-quality genome sequences. As a result, they have shifted away from the reference-centric view of the genome, and entered the pan-genome era. Here, Datema highlights some of the breakthrough algorithmic innovations KeyGene has developed to generate and analyze population-scale pan-genomes for plant genomes of all complexities and sizes.

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Friday, February 5, 2021

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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Friday, February 5, 2021

SMRT Leiden: The impact of polishing on Darwin Tree of Life chromosome-level lepidopteran genome assemblies built with PacBio HiFi and Illumina Hi-C reads

In this SMRT Leiden 2020 Online Virtual Event presentation, Marcela Uliano da Silva of Wellcome Sanger Institute shares her work using CCS data combined with HiC reads to assemble chromosome-level genomes for Lepidoptera. This was done as part of the The Darwin Tree of Life Project which is assembling high-quality genomes for all eukaryotic species in Britain and Ireland. Find data: https://github.com/darwintreeoflife/darwintreeoflife.data

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Friday, February 5, 2021

Educational Video: Variant Detection with HiFi Reads – Understanding Results from the precisionFDA Truth Challenge

In this short video, Aaron Wenger, a Principal Scientist at PacBio, explains what highly accurate long reads, or HiFi reads, are and how they help to detect all variant types including single nucleotide, indels, and structural variants. He goes on to recap the precisionFDA Truth Challenge V2 which used Genome in a Bottle (GIAB) benchmarks to evaluate various sequencing technologies. In the 2020 challenge, when ranked for accuracy, PacBio HiFi reads delivered the highest precision and recall in all categories.

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Friday, February 5, 2021

ASHG PacBio Workshop: Applications of third generation sequencing in unsolved disease

In this ASHG 2020 PacBio Workshop Emily Farrow of Children’s Mercy Kansas City, shares how the incorporation of long-read sequencing into the Genomic Answers for Kids research study is increasing diagnostic yields through the identification of novel genetic variation. Emily highlights several cases in which PacBio HiFi sequencing was able to provide insights where short-read sequencing alone was inconclusive, due to limitations stemming from repetitive regions and large structural variants.

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Friday, February 5, 2021

Video Poster: Accurate, comprehensive variant calling in difficult-to-map genes using HiFi reads

Introduction: Around 5% (1,168) of protein-coding genes in the human genome contain an exon that is difficult to map with typical next-generation sequencing (NGS) read lengths due to homologous pseudogenes or segmental duplications. Among the difficult-to-map genes are 193 with known medical relevance, including CYP2D6, GBA, SMN1/2, and VWF. Long-read DNA sequencing provides increased mappability, accessing many of the difficult-to-map regions by connecting the homologous exon to neighboring unique sequence. Until recently, the read-level accuracy of long-read sequencing had made it challenging to accurately call small variants. The recently developed HiFi reads from the PacBio Sequel II System provide both…

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Friday, February 5, 2021

Webinar: Bioinformatics lunch & learn – A quick guide to metagenomic analysis with PacBio HiFi reads

In this webinar, scientists from PacBio share how using Single Molecule, Real-Time (SMRT) Sequencing, you can generate highly accurate long reads – HiFi reads – with 99% accuracy (Q20) and read lengths of 10 kb or more. This high resolution of each single molecule enables species or strain-level profiling of complex populations in both targeted and shotgun sequencing experiments. Genome assemblies are more cost effective than ever before when sequencing metagenomics samples with the Sequel II System.

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Friday, February 5, 2021

Video Poster: Long-read sequencing of the SARS-CoV-2 genome and the human immune repertoire

COVID-19 is caused by the infection of SARS-CoV-2, a member of the coronavirus family. Complete and accurate sequencing of the SARS-CoV-2 genome enables discovery and epidemiological tracing of mutations that may be important for antiviral and vaccine research. A complementary approach, sequencing the patients’ immune repertoire, allows for detection of neutralizing antibodies and understanding variation in the adaptive immune response. PacBio’s SMRT Sequencing uses circular consensus sequencing that can generate long, highly accurate (HiFi) reads. We find that a tiled multiplex PCR amplicon approach of ~1-2 kb fragments achieves a balanced tradeoff between ease of library preparation and robustness to…

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Friday, February 5, 2021

ASHG CoLab: PacBio HiFi reads for comprehensive characterization of genomes and single-cell isoform expression

In this ASHG 2020 CoLab presentation hear Principal Scientists, Aaron Wenger and Elizabeth Tseng share how highly accurate long reads (HiFi reads) provide comprehensive variant detection for both genomes and transcriptomes. Aaron Wenger describes how new improvements in protocols and analysis methods have increased scalability and accuracy of variant calling. As demonstrated in the precisionFDA Truth Challenge V2, HiFi reads (>99% accurate, 15 kb – 20 kb) now outperform short reads for single nucleotide and structural variant calling and match for small indels. This includes calling >30,000 small variants and >10,000 structural variants missed by short reads, many in medically…

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Friday, February 5, 2021

Video Poster: Full-length RNA sequencing of Alzheimer brain sample using long reads reveals complex alternative splicing patterns

Long-read mRNA sequencing such as PacBio’s Iso-Seq method offer high-throughput transcriptome profiling that circumvents the transcript assembly problem by sequencing full-length cDNA. The Iso-Seq method has emerged as the most reliable technology for fully characterizing isoforms and, in turn, help shed light on underlying disease mechanisms. Here, we have utilized the Iso-Seq method to sequence an Alzheimer’s disease whole brain?sample. This is a devastating neurodegenerative disease that affects ~44 million people worldwide, making it the most common form of dementia. Studies looking into disease mechanism have shown that changes in gene expression due to alternative splicing likely contribute to the…

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