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Wednesday, January 6, 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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Wednesday, January 6, 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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Wednesday, January 6, 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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Wednesday, January 6, 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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Wednesday, January 6, 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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Wednesday, January 6, 2021

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, December 22, 2020

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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Tuesday, December 22, 2020

Allele-level sequencing and phasing of full-length HLA class I and II genes using SMRT Sequencing technology

The three classes of genes that comprise the MHC gene family are actively involved in determining donor-recipient compatibility for organ transplant, as well as susceptibility to autoimmune diseases via cross-reacting immunization. Specifically, Class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DQ and -DP are considered medically important for genetic analysis to determine histocompatibility. They are highly polymorphic and have thousands of alleles implicated in disease resistance and susceptibility. The importance of full-length HLA gene sequencing for genotyping, detection of null alleles, and phasing is now widely acknowledged. While DNA-sequencing-based HLA genotyping has become routine, only 7% of…

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Tuesday, December 22, 2020

Complete HIV-1 genomes from single molecules: Diversity estimates in two linked transmission pairs using clustering and mutual information.

We sequenced complete HIV-1 genomes from single molecules using Single Molecule, Real- Time (SMRT) Sequencing and derive de novo full-length genome sequences. SMRT sequencing yields long-read sequencing results from individual DNA molecules with a rapid time-to-result. These attributes make it a useful tool for continuous monitoring of viral populations. The single-molecule nature of the sequencing method allows us to estimate variant subspecies and relative abundances by counting methods. We detail mathematical techniques used in viral variant subspecies identification including clustering distance metrics and mutual information. Sequencing was performed in order to better understand the relationships between the specific sequences of…

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Tuesday, December 22, 2020

Rapid sequencing of HIV-1 genomes as single molecules from simple and complex samples.

Background: To better understand the relationships among HIV-1 viruses in linked transmission pairs, we sequenced several samples representing HIV transmission pairs from the Zambia Emory HIV Research Project (Lusaka, Zambia) using Single Molecule, Real-Time (SMRT) Sequencing. Methods: Single molecules were sequenced as full-length (9.6 kb) amplicons directly from PCR products without shearing. This resulted in multiple, fully-phased, complete HIV-1 genomes for each patient. We examined Single Genome Amplification (SGA) prepped samples, as well as samples containing complex mixtures of genomes. We detail mathematical techniques used in viral variant subspecies identification, including clustering distance metrics and mutual information, which were used…

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Tuesday, December 22, 2020

Single Molecule Real Time (SMRT) sequencing sensitively detects polyclonal and compound BCR-ABL in patients who relapse on kinase inhibitor therapy.

Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of…

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Tuesday, December 22, 2020

Next generation sequencing of full-length HIV-1 env during primary infection.

Background: The use of next generation sequencing (NGS) to examine circulating HIV env variants has been limited due to env’s length (2.6 kb), extensive indel polymorphism, GC deficiency, and long homopolymeric regions. We developed and standardized protocols for isolation, RT-PCR amplification, single molecule real-time (SMRT) sequencing, and haplotype analysis of circulating HIV-1 env variants to evaluate viral diversity in primary infection. Methodology: HIV RNA was extracted from 7 blood plasma samples (1 mL) collected from 5 subjects (one individual sampled and sequenced at 3 time points) in the San Diego Primary Infection Cohort between 3-33 months from their estimated date…

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Tuesday, December 22, 2020

Long-read, single-molecule applications for protein engineering.

The long read lengths of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases of sequence. This feature is particularly useful in the context of protein engineering, where large numbers of similar constructs are generated routinely to explore the effects of mutations on function and stability. We have developed a PCR-based barcoded sequencing method to generate high quality, full-length sequence data for batches of constructs generated in a common backbone. Individual barcodes are coupled to primers targeting a common region of the vector of interest. The amplified products are pooled into a single DNA library, and sequencing data…

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