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Tuesday, November 2, 2021

Scalable RNA Isoform Sequencing using Intramolecular Multiplexed cDNAs

While RNA-sequencing has dramatically accelerated our understanding of biology, quantitation and discovery of full-length RNA isoforms resulting from alternative splicing remain poorly resolved. Alternative splicing is a core regulatory process that modulates the structure, expression, and localization of expressed proteins through differential exon and/or UTR splicing during transcript maturation. Beyond being an integral component of cellular development and homeostatic maintenance, RNA splicing is implicated in a wide range of pathologies with hallmark isoforms being linked to cardiovascular, neurological, and immunological diseases. Current limitations in isoform quantitation and discovery arise from the inability of existing sequencing platforms to scalably sequence full-length…

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Thursday, October 28, 2021

Allele-Specific, Isoform-Resolution Single-Cell RNA-Seq Analysis Using Long-Read Sequencing on Concatenated Single-Cell Molecules

In this talk, Dr. Elizabeth Tseng demonstrates a throughput increase for the scIso-Seq method by concatenating single-cell molecules, increasing yield a minimum of 6-fold per SMRT Cell 8M. She explains the bioinformatics workflow for analyzing concatenated scIso-Seq data, which begins with de-concatenation, followed by tagging of UMI and barcode information that can be processed by the isoseq3 pipeline for deduplication. Reads are then aligned against the reference genome, followed by SQANTI3 for transcript classification against a reference annotation (ex: GENCODE) which produces an isoform-level sparse matrix to be analyzed with single-cell tools such as Seurat. She also shows how to…

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Thursday, September 23, 2021

Getting the Most Out of Your Breeding Program with DNA and RNA Sequencing

Understanding genome sequences and how they evolved is critical for harnessing that evolutionary process for agricultural improvement. Whether asking questions about the gain/loss of genes, the role of structural variation on phenotypic diversity, or identifying favorable alleles in exotic and wild species, DNA and RNA sequencing have proven to be extremely valuable tools for breeding programs all over the world. This panel-style webinar brings together three plant biologists to talk about their journeys into sequencing plant genomes and how these efforts have helped push plant breeding forward.

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Thursday, August 19, 2021

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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Thursday, August 19, 2021

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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Thursday, August 19, 2021

Application Brief: Single-cell RNA sequencing with HiFi reads – Best Practices

With PacBio single-cell RNA sequencing using the Iso-Seq method, you can now distinguish between alternative transcript isoforms at the single-cell level. The highly accurate long reads (HiFi reads) can span the entire 5′ to 3′ end of a transcript, allowing a high-resolution view of isoform diversity and revealing cell-to-cell heterogeneity without the need for assembly.

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Tuesday, June 1, 2021

Isoform sequencing: Unveiling the complex landscape of the eukaryotic transcriptome on the PacBio RS II.

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths. Here, we describe the Isoform Sequencing (Iso-Seq) method using long reads generated by the PacBio RS II. We sequenced rat heart and lung RNA using the Clontech® SMARTer® cDNA preparation kit followed by size selection using agarose gel. Additionally, we tested the BluePippin™ device from Sage Science for efficiently extracting longer transcripts = 3 kb. Post-sequencing,…

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Tuesday, June 1, 2021

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark…

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