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Wednesday, October 21, 2020

Application Brief: Long-read RNA sequencing – Best Practices

With Single Molecule, Real-Time (SMRT) Sequencing and the Sequel Systems, you can easily and affordably sequence complete transcript isoforms in genes of interest or across the entire transcriptome. The Iso-Seq method allows users to generate full-length cDNA sequences up to 10 kb in length — with no assembly required — to confidently characterize full-length transcript isoforms.

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Wednesday, October 21, 2020

Informational Guide: What’s the value of sequencing full-length RNA transcripts?

The study of genomics has revolutionized our understanding of science, but the field of transcriptomics grew with the need to explore the functional impacts of genetic variation. While different tissues in an organism may share the same genomic DNA, they can differ greatly in what regions are transcribed into RNA and in their patterns of RNA processing. By reviewing the history of transcriptomics, we can see the advantages of RNA sequencing using a full-length transcript approach become clearer.

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Wednesday, May 13, 2020

PacBio Workshop: Understanding the biology of genomes with HiFi sequencing

The utility of new highly accurate long reads, or HiFi reads, was first demonstrated for calling all variant types in human genomes. It has since been shown that HiFi reads can be used to generate contiguous, complete, and accurate human genomes, even in repeat structures such as centromeres and telomeres. In this virtual workshop scientists from PacBio as well as Tina Graves-Lindsay from the McDonnell Genome Institute at Washington University share the many improvements we’ve made to HiFi sequencing in the past year, tools that take advantage of HiFi data for variant detection and assembly, and examples in numerous genomics…

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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

An improved pig reference genome sequence to enable pig genetics and genomics research

The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model with high anatomical and immunological similarity to humans. The draft reference genome (Sscrofa10.2) represented a purebred female pig from a commercial pork production breed (Duroc), and was established using older clone-based sequencing methods. The Sscrofa10.2 assembly was incomplete and unresolved redundancies, short range order and orientation errors and associated misassembled genes limited its utility. We present two highly contiguous chromosome-level genome assemblies created with more recent long read technologies and a whole genome shotgun strategy, one for the same Duroc female (Sscrofa11.1) and…

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Tuesday, April 21, 2020

Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline

Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal…

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Tuesday, April 21, 2020

A quick guide for student-driven community genome annotation.

High quality gene models are necessary to expand the molecular and genetic tools available for a target organism, but these are available for only a handful of model organisms that have undergone extensive curation and experimental validation over the course of many years. The majority of gene models present in biological databases today have been identified in draft genome assemblies using automated annotation pipelines that are frequently based on orthologs from distantly related model organisms and usually have minor or major errors. Manual curation is time consuming and often requires substantial expertise, but is instrumental in improving gene model structure…

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Tuesday, April 21, 2020

Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data.

Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were…

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Monday, March 30, 2020

ASHG PacBio Workshop: The Iso-Seq method for discovering alternative splicing in human diseases

In this ASHG workshop presentation, Elizabeth Tseng of PacBio showed how the Iso-Seq method can be used to discover disease-associated alternative splicing. Because this approach to isoform sequencing yields accurate, full-length transcripts requiring no assembly, it’s ideal for disease studies that need a more comprehensive picture of alternative splicing activity. Tseng offered several published examples of how the Iso-Seq method has been used for everything from single-gene studies to whole-transcriptome studies, and also detailed how the latest Sequel System chemistry recovers more genes and produces more usable reads.

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Monday, March 30, 2020

PAG Conference: Iso-Seq analysis for plant & animal genomes – annotation evaluation & phasing

In this presentation, Elizabeth Tseng explains how PacBio’s full-length RNA Sequencing using the Iso-Seq method can characterize full-length transcripts without the need for computational transcript assembly. The Iso-Seq method is fully supported bioinformatically through PacBio’s SMRT Analysis software that outputs high-quality, full-length transcript sequences that can be used for genome annotation and novel gene discovery. Elizabeth shows that the highly accurate reads can be used to discover allelic-specific isoform expressions in transcriptome data.

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Monday, March 30, 2020

Webinar: Smoking out structural variants in the cannabis genome

In this webinar, Sarah Kingan, Staff Scientist, PacBio, and Kevin McKernan, Founder and Chief Science Officer, Medicinal Genomics, describe their work assembling the most comprehensive and complete cannabis genome of a Type II (THCA and CBDA producing) plant. They also share the latest advances in cannabis genomics, including how PacBio long-read sequencing enables high-quality genomics research in plants, annotation of the cannabis reference genome using full-length transcript sequencing, and new insights into cannabinoid synthesis across different types of cannabis plants.

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Wednesday, February 26, 2020

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with…

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Wednesday, February 26, 2020

A complete solution for full-length transcript sequencing using the PacBio Sequel II System

Long read mRNA sequencing methods such as PacBio’s Iso-Seq method offers high-throughput transcriptome profiling in prokaryotic and eukaryotic cells. By avoiding the transcript assembly problem and instead sequencing full-length cDNA, Iso-Seq has emerged as the most reliable technology for annotating isoforms and, in turn, improving proteome predictions in a wide variety of organisms. Improvements in library preparation, sequencing throughput, and bioinformatics has enabled the Iso-Seq method to be complete solution for transcript characterization. The Iso-Seq Express kit is a one-day library prep requiring 60-300 ng of total RNA. The PacBio Sequel II system produces 4-5 million full-length reads, sufficient to…

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