Background: Alternative splicing expands the repertoire of gene functions and is a signature for different cell populations. Here we characterize the transcriptome of human bone marrow subpopulations including progenitor cells to understand their contribution to homeostasis and pathological conditions such as atherosclerosis and tumor metastasis. To obtain full-length transcript structures, we utilized long reads in addition to RNA-seq for estimating isoform diversity and abundance. Method: Freshly harvested, viable human bone marrow tissues were extracted from discarded harvesting equipment and separated into total bone marrow (total), lineage-negative (lin-) progenitor cells and differentiated cells (lin+) by magnetic bead sorting with antibodies to…
As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but…
The comprehensive characterization of cancer genomes and epigenomes for understanding drug resistance remains an important challenge in the field of oncology. For example, PC-9, a non-small cell lung cancer (NSCL) cell line, contains a deletion mutation in exon 19 (DelE746A750) of EGRF that renders it sensitive to erlotinib, an EGFR inhibitor. However, sustained treatment of these cells with erlotinib leads to drug-tolerant cell populations that grow in the presence of erlotinib. However, the resistant cells can be resensitized to erlotinib upon treatment with methyltransferase inhibitors, suggesting a role of epigenetic modification in development of drug resistance. We have characterized for…
The Genome in a Bottle Consortium is developing the reference materials, reference methods , and reference data n
In addition to the genome and transcriptome, epigenetic information is essential to understand biological processes and their regulation, and their misregulation underlying disease. Traditionally, epigenetic DNA modifications are detected using upfront sample preparation steps such as bisulfite conversion, followed by sequencing. Bisulfite sequencing has provided a wealth of knowledge about human epigenetics, however it does not access the entire genome due to limitations in read length and GC- bias of the sequencing technologies used. In contrast, Single Molecule, Real-Time (SMRT) DNA Sequencing is unique in that it can detect DNA base modifications as part of the sequencing process. It can…
Purpose: Clinical laboratories, research laboratories and technology developers all need DNA samples with reliably known genotypes in order to help validate and improve their methods. The Genome in a Bottle Consortium (genomeinabottle.org) has been developing Reference Materials with high-accuracy whole genome sequences to support these efforts.Methodology: Our pilot reference material is based on Coriell sample NA12878 and was released in May 2015 as NIST RM 8398 (tinyurl.com/giabpilot). To minimize bias and improve accuracy, 11 whole-genome and 3 exome data sets produced using 5 different technologies were integrated using a systematic arbitration method [1]. The Genome in a Bottle Analysis Group…
The sensitivity, speed, and reduced cost associated with Next-Generation Sequencing (NGS) technologies have made them indispensable for the molecular profiling of cancer samples. For effective use, it is critical that the NGS methods used are not only robust but can also accurately detect 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. Here we characterize SMRT…
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…
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.
Target enrichment capture methods allow scientists to rapidly interrogate important genomic regions of interest for variant discovery, including SNPs, gene isoforms, and structural variation. Custom targeted sequencing panels are important for characterizing heterogeneous, complex diseases and uncovering the genetic basis of inherited traits with more uniform coverage when compared to PCR-based strategies. With the increasing availability of high-quality reference genomes, customized gene panels are readily designed with high specificity to capture genomic regions of interest, thus enabling scientists to expand their research scope from a single individual to larger cohort studies or population-wide investigations. Coupled with PacBio® long-read sequencing, these…
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…
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…
In this AGBT 2017 talk, PacBio CSO Jonas Korlach provided a technology roadmap for the Sequel System, including plans the continue performance and throughput increases through early 2019. Per SMRT Cell throughput of the Sequel System is expected to double this year and again next year. Together with a new higher-capacity SMRT Cell expected to be released by the end of 2018, these improvements result in a ~30-fold increase or ~150 Gb / SMRT Cell allowing a real $1000 real de novo human genome assembly. Also discussed: Additional application protocol improvements, new chemistry and software updates, and a look at…
Howard Jacob, Chief Genomics Officer at the HudsonAlpha Institute for Biotechnology, explored the role of genomics in diagnosing rare diseases. In this podcast he shared his views on the economics of clinical sequencing and how long-read sequencing is advancing the ability to sequence an individual’s genome –de novo– and use structural variant calling to make clinical diagnoses. He concluded with the hurdles limiting adoption of clinical sequencing and his vision for the future of genomic medicine.
SMRT Sequencing is a DNA sequencing technology characterized by long read lengths and high consensus accuracy, regardless of the sequence complexity or GC content of the DNA sample. These characteristics can be harnessed to address medically relevant genes, mRNA transcripts, and other genomic features that are otherwise difficult or impossible to resolve. I will describe examples for such new clinical research in diverse areas, including full-length gene sequencing with allelic haplotype phasing, gene/pseudogene discrimination, sequencing extreme DNA contexts, high-resolution pharmacogenomics, biomarker discovery, structural variant resolution, full-length mRNA isoform cataloging, and direct methylation detection.