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Sunday, October 25, 2020

ASHG PacBio Workshop: Going beyond the $1,000 genome? – the future of high quality de novo human genomes, epigenomes and transcriptomes?

Jonas Korlach, Chief Scientific Officer at PacBio, discussed the technology waves that have followed the initial human genome sequencing project, where we are today, and where we are going. Today, we are in what Korlach calls the 4th wave, where more comprehensive whole-genome re-sequencing is occurring, and we are nearing the 5th, when we will actually be able to free ourselves from reference genomes and sequence everything de novo.

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Sunday, October 25, 2020

ASHG Virtual Poster: Long range phasing of cardiac disease genes using new long read sequencing technologies

Alex Dainis, a graduate student in Euan Ashley’s lab at Stanford University, presents her ASHG 2015 poster on haplotyping for genes linked to hypertrophic cardiomyopathy. Using the Iso-Seq method with SMRT Sequencing, she sequenced full transcripts of two genes of interest, generating data on 150 different isoforms. Rare variants, which could not be found with other technologies, were associated with haplotypes.

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Sunday, October 25, 2020

AGBT Roche and PacBio Workshop: Towards precision medicine

Euan Ashley speaks about precision medicine and said clinical-grade analysis has been limited by complex regions in the human genome. His key theme,”Precision medicine needs to be accurate medicine,” was illustrated with several examples where short-read sequencing or traditional clinical sequencing methods failed to be accurate. Also included: targeted RNA sequencing and gene phasing with long-read sequencing.

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Sunday, October 25, 2020

ASHG Virtual Poster: Alternative splicing in FMR1 premutations carriers

In this ASHG 2016 virtual poster, Flora Tassone from UC Davis describes her study of the molecular mechanisms linked to fragile X syndrome and associated disorders, such as FXTAS. She is using SMRT Sequencing to resolve the FMR1 gene in premutation carriers because it’s the only technology that can generate full-length transcripts with the causative CGG repeat expansion. Plus: direct confirmation of predicted isoform configurations.

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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, October 21, 2020

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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Wednesday, February 26, 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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Wednesday, February 26, 2020

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin embedded tissues using adaptive focused acoustics by Covaris.

Recent advances in next-generation sequencing have led to an increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage for direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. FFPE samples often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and target enrichment. Additionally, the quality and quantity of the recovered DNA vary depending on…

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Wednesday, February 26, 2020

Full-length cDNA sequencing of alternatively spliced isoforms provides insight into human diseases.

The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans. Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete…

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Wednesday, February 26, 2020

Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey…

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