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Friday, July 19, 2019

Advantages of Single-Molecule Real-Time Sequencing in high-GC content genomes.

Next-generation sequencing has become the most widely used sequencing technology in genomics research, but it has inherent drawbacks when dealing with high-GC content genomes. Recently, single-molecule real-time sequencing technology (SMRT) was introduced as a third-generation sequencing strategy to compensate for this drawback. Here, we report that the unbiased and longer read length of SMRT sequencing markedly improved genome assembly with high GC content via gap filling and repeat resolution.

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Friday, July 19, 2019

A benchmark study on error assessment and quality control of CCS reads derived from the PacBio RS.

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the…

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Friday, July 19, 2019

A window into third-generation sequencing.

First- and second-generation sequencing technologies have led the way in revolutionizing the field of genomics and beyond, motivating an astonishing number of scientific advances, including enabling a more complete understanding of whole genome sequences and the information encoded therein, a more complete characterization of the methylome and transcriptome and a better understanding of interactions between proteins and DNA. Nevertheless, there are sequencing applications and aspects of genome biology that are presently beyond the reach of current sequencing technologies, leaving fertile ground for additional innovation in this space. In this review, we describe a new generation of single-molecule sequencing technologies (third-generation…

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Friday, July 19, 2019

Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms – 20 nanograms) for single- molecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

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Friday, July 19, 2019

Error correction and assembly complexity of single molecule sequencing reads.

Third generation single molecule sequencing technology is poised to revolutionize genomics by en- abling the sequencing of long, individual molecules of DNA and RNA. These technologies now routinely produce reads exceeding 5,000 basepairs, and can achieve reads as long as 50,000 basepairs. Here we evaluate the limits of single molecule sequencing by assessing the impact of long read sequencing in the assembly of the human genome and 25 other important genomes across the tree of life. From this, we develop a new data-driven model using support vector regression that can accurately predict assembly performance. We also present a novel hybrid…

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Friday, July 19, 2019

Detecting DNA modifications from SMRT sequencing data by modeling sequence context dependence of polymerase kinetic.

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering…

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Friday, July 19, 2019

Identification of restriction-modification systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and associated methylome analysis.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome…

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Friday, July 19, 2019

PBHoney: identifying genomic variants via long-read discordance and interrupted mapping.

As resequencing projects become more prevalent across a larger number of species, accurate variant identification will further elucidate the nature of genetic diversity and become increasingly relevant in genomic studies. However, the identification of larger genomic variants via DNA sequencing is limited by both the incomplete information provided by sequencing reads and the nature of the genome itself. Long-read sequencing technologies provide high-resolution access to structural variants often inaccessible to shorter reads.We present PBHoney, software that considers both intra-read discordance and soft-clipped tails of long reads (>10,000 bp) to identify structural variants. As a proof of concept, we identify four…

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Friday, July 19, 2019

Pacific Biosciences sequencing technology for genotyping and variation discovery in human data.

Pacific Biosciences technology provides a fundamentally new data type that provides the potential to overcome some limitations of current next generation sequencing platforms by providing significantly longer reads, single molecule sequencing, low composition bias and an error profile that is orthogonal to other platforms. With these potential advantages in mind, we here evaluate the utility of the Pacific Biosciences RS platform for human medical amplicon resequencing projects.We evaluated the Pacific Biosciences technology for SNP discovery in medical resequencing projects using the Genome Analysis Toolkit, observing high sensitivity and specificity for calling differences in amplicons containing known true or false SNPs.…

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Friday, July 19, 2019

Amplification and thrifty single-molecule sequencing of recurrent somatic structural variations.

Deletion of tumor-suppressor genes as well as other genomic rearrangements pervade cancer genomes across numerous types of solid tumor and hematologic malignancies. However, even for a specific rearrangement, the breakpoints may vary between individuals, such as the recurrent CDKN2A deletion. Characterizing the exact breakpoints for structural variants (SVs) is useful for designating patient-specific tumor biomarkers. We propose AmBre (Amplification of Breakpoints), a method to target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre validates SVs called by whole-exome/genome sequencing and hybridization arrays. AmBre involves a PCR-based approach to amplify the DNA segment containing an…

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Friday, July 19, 2019

Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations.

Next-generation sequencing (NGS) technologies enable new insights into the diversity of virus populations within their hosts. Diversity estimation is currently restricted to single-nucleotide variants or to local fragments of no more than a few hundred nucleotides defined by the length of sequence reads. To study complex heterogeneous virus populations comprehensively, novel methods are required that allow for complete reconstruction of the individual viral haplotypes. Here, we show that assembly of whole viral genomes of ~8600 nucleotides length is feasible from mixtures of heterogeneous HIV-1 strains derived from defined combinations of cloned virus strains and from clinical samples of an HIV-1…

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Friday, July 19, 2019

Genome reference and sequence variation in the large repetitive central exon of human MUC5AC.

Despite modern sequencing efforts, the difficulty in assembly of highly repetitive sequences has prevented resolution of human genome gaps, including some in the coding regions of genes with important biological functions. One such gene, MUC5AC, encodes a large, secreted mucin, which is one of the two major secreted mucins in human airways. The MUC5AC region contains a gap in the human genome reference (hg19) across the large, highly repetitive, and complex central exon. This exon is predicted to contain imperfect tandem repeat sequences and multiple conserved cysteine-rich (CysD) domains. To resolve the MUC5AC genomic gap, we used high-fidelity long PCR…

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Friday, July 19, 2019

Going beyond five bases in DNA sequencing.

DNA sequencing has provided a wealth of information about biological systems, but thus far has focused on the four canonical bases, and 5-methylcytosine through comparison of the genomic DNA sequence to a transformed four-base sequence obtained after treatment with bisulfite. However, numerous other chemical modifications to the nucleotides are known to control fundamental life functions, influence virulence of pathogens, and are associated with many diseases. These modifications cannot be accessed with traditional sequencing methods. In this opinion, we highlight several emerging single-molecule sequencing techniques that have the potential to directly detect many types of DNA modifications as an integral part…

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Friday, July 19, 2019

Long-read, whole-genome shotgun sequence data for five model organisms.

Single molecule, real-time (SMRT) sequencing from Pacific Biosciences is increasingly used in many areas of biological research including de novo genome assembly, structural-variant identification, haplotype phasing, mRNA isoform discovery, and base-modification analyses. High-quality, public datasets of SMRT sequences can spur development of analytic tools that can accommodate unique characteristics of SMRT data (long read lengths, lack of GC or amplification bias, and a random error profile leading to high consensus accuracy). In this paper, we describe eight high-coverage SMRT sequence datasets from five organisms (Escherichia coli, Saccharomyces cerevisiae, Neurospora crassa, Arabidopsis thaliana, and Drosophila melanogaster) that have been publicly released…

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Friday, July 19, 2019

Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives…

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