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Tuesday, June 1, 2021

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.

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Tuesday, June 1, 2021

Whole gene sequencing of KIR-3DL1 with SMRT Sequencing and the distribution of allelic variants in different ethnic groups

The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However, it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. SMRT (Single Molecule Real-Time) Sequencing generates average long reads of 10 to 15 kb and allows us to obtain in-phase long…

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Tuesday, June 1, 2021

From RNA to full-length transcripts: The PacBio Iso-Seq method for transcriptome analysis and genome annotation

A single gene may encode a surprising number of proteins, each with a distinct biological function. This is especially true in complex eukaryotes. Short- read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. The PacBio Isoform Sequencing (Iso-Seq™) method employs long reads to sequence transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for transcript reconstruction and inference. These long reads result in complete, unambiguous information about alternatively spliced exons, transcriptional…

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Tuesday, June 1, 2021

Targeted sequencing using a long-read sequencing technology

Targeted sequencing employing PCR amplification is a fundamental approach to studying human genetic disease. PacBio’s Sequel System and supporting products provide an end-to-end solution for amplicon sequencing, offering better performance to Sanger technology in accuracy, read length, throughput, and breadth of informative data. Sample multiplexing is supported with three barcoding options providing the flexibility to incorporate unique sample identifiers during target amplification or library preparation. Multiplexing is key to realizing the full capacity of the 1 million individual reactions per Sequel SMRT Cell. Two analysis workflows that can generate high-accuracy results support a wide range of amplicon sizes in two…

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Tuesday, June 1, 2021

Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts

The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information may be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment.1 The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, high-quality cDNA sequences, allowing the unambiguous determination of splice variants, identifying potential biomarkers and yielding new insights into gene fusion events. Recent improvements…

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Tuesday, June 1, 2021

High-throughput SMRT Sequencing of clinically relevant targets

Targeted sequencing with Sanger as well as short read based high throughput sequencing methods is standard practice in clinical genetic testing. However, many applications beyond SNP detection have remained somewhat obstructed due to technological challenges. With the advent of long reads and high consensus accuracy, SMRT Sequencing overcomes many of the technical hurdles faced by Sanger and NGS approaches, opening a broad range of untapped clinical sequencing opportunities. Flexible multiplexing options, highly adaptable sample preparation method and newly improved two well-developed analysis methods that generate highly-accurate sequencing results, make SMRT Sequencing an adept method for clinical grade targeted sequencing. The…

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Tuesday, June 1, 2021

Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving…

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Friday, February 5, 2021

Webinar: SMRT Sequencing applications in plant and animal sciences: an overview

In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains an overview of several applications: whole-genome sequencing for de novo assembly; transcript isoform sequencing (Iso-Seq) method for genome annotation; targeted sequencing solutions; and metagenomics and microbial interactions. High-level workflows and best practices are discussed for key applications.

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Friday, February 5, 2021

Webinar: A paradigm shift in HLA sequencing: from exons to high-resolution allele-level HLA yyping

Human MHC class I genes HLA-A, -B, -C, and class II genes HLA -DR, -DQ, and -DP play a critical role in the immune system as primary factors responsible for organ transplant rejection. Additionally, the HLA genes are important targets for clinical and drug sensitivity research because of their direct or linkage-based association with several diseases, including cancer, and autoimmune diseases. HLA genes are highly polymorphic, and their diversity originates from exonic combinations as well as recombination events. With full-length gene sequencing, a significant increase of new alleles in the HLA database is expected, stressing the need for high-resolution sequencing.…

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Friday, February 5, 2021

Webinar: Using Iso-Seq analysis to build a better annotation

Long-read sequencing technologies like Iso-Seq analysis present researchers with a powerful tool for probing the transcriptomes of many species. The ability to sequence transcripts from end-to-end has revealed transcription complexity on a scale that was previously impossible. This sequence rich information has also improved our ability to predict transcript functions and biotypes. Researchers can now use Iso-Seq analysis to discover transcript models in almost any species with an accuracy on par with human and mouse annotations. In this webinar, Richard Kuo discusses the core concepts behind Iso-Seq analysis and how to use it to improve or build a new transcriptome…

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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

Webinar: A HiFi View – Sequencing the gut microbiome with highly accurate long reads

In this webinar, Dr. Ashby gives attendees a brief update on PacBio’s metagenomics solutions on the Sequel II System. Then, Dr. Ma, University of Maryland School of Medicine, discusses her work using long read sequencing to identify high-resolution microbial biomarkers associated with leaky gut syndrome in premature infants. Finally, Dr. Weinstock, The Jackson Laboratory, talks about the potential of highly accurate long reads to enable strain-level resolution of the human gut microbiome by resolving intraspecies variation in multiple copies of the 16S gene.

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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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