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Monday, April 16, 2018

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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Monday, April 16, 2018

Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

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Monday, April 16, 2018

The role of androgen receptor variant AR-V9 in prostate cancer

The expression of androgen receptor (AR) variants is a frequent, yet poorly-understood mechanism of clinical resistance to AR-targeted therapy for castration-resistant prostate cancer (CRPC). Among the multiple AR variants expressed in CRPC, AR-V7 is considered the most clinically-relevant AR variant due to broad expression in CRPC, correlations of AR-V7 expression with clinical resistance, and growth inhibition when AR-V7 is knocked down in CRPC models. Therefore, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The aim of this study was to understand whether other AR variants are co-expressed with AR-V7 and promote…

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Saturday, April 1, 2017

SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants

Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being…

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Saturday, April 1, 2017

Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel platform

Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. 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. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The…

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Saturday, April 16, 2016

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin-embedded tissues

Recent advances in next-generation sequencing have led to the 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 in that it allows 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. DNA samples extracted from FFPE often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and amplification. Additionally, the quality and quantity of the…

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Saturday, April 16, 2016

Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust, but capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we…

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Saturday, April 18, 2015

Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads.

While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. With PacBio’s long reads, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso- Seq protocol produces reads that originate from independent observations of single molecules, meaning no assembly is needed. Here, we sequenced the transcriptome of the human MCF-7 breast cancer cell line using the Clontech SMARTer® cDNA preparation kit and the PacBio RS II. Using PacBio Iso-Seq bioinformatics software, we…

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