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Friday, July 19, 2019

Efficient and accurate whole genome assembly and methylome profiling of E. coli.

With the price of next generation sequencing steadily decreasing, bacterial genome assembly is now accessible to a wide range of researchers. It is therefore necessary to understand the best methods for generating a genome assembly, specifically, which combination of sequencing and bioinformatics strategies result in the most accurate assemblies. Here, we sequence three E. coli strains on the Illumina MiSeq, Life Technologies Ion Torrent PGM, and Pacific Biosciences RS. We then perform genome assemblies on all three datasets alone or in combination to determine the best methods for the assembly of bacterial genomes.Three E. coli strains – BL21(DE3), Bal225, and…

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Friday, July 19, 2019

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens.

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens–Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n?=?44) and the complete gene of pvcsp (n?=?47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess…

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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

In vivo generation of DNA sequence diversity for cellular barcoding.

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia…

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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

The origin of the Haitian cholera outbreak strain.

Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti.We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of…

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Friday, July 19, 2019

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.

The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. We sequenced the genome of V. parahaemolyticus with…

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Friday, July 19, 2019

An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome.

Second generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio…

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Friday, July 19, 2019

Microsatellite marker discovery using single molecule real-time circular consensus sequencing on the Pacific Biosciences RS.

Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Here we describe microsatellite discovery on a Pacific Biosciences single molecule real-time sequencer. For the Greater White-fronted Goose (Anser albifrons), we identified 316 microsatellite loci in a single genome shotgun sequencing experiment. We found that the capability of handling large insert sizes and high quality circular consensus sequences provides an advantage over short read technologies for primer design. Combined with…

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Friday, July 19, 2019

The complete genome sequence of Escherichia coli EC958: a high quality reference sequence for the globally disseminated multidrug resistant E. coli O25b:H4-ST131 clone.

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific…

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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

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis.

More than 20% of the world’s population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the…

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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

Comparative genomic analysis and virulence differences in closely related Salmonella enterica serotype Heidelberg isolates from humans, retail meats, and animals.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. Recently, an antibiotic-resistant strain of this serovar was implicated in a large 2011 multistate outbreak resulting from consumption of contaminated ground turkey that involved 136 confirmed cases, with one death. In this study, we assessed the evolutionary diversity of 44 S. Heidelberg isolates using whole-genome sequencing (WGS) generated by the 454 GS FLX (Roche) platform. The isolates, including 30 with nearly indistinguishable (one band difference) Xbal pulsed-field gel electrophoresis patterns (JF6X01.0032, JF6X01.0058), were collected from various sources between 1982 and 2011 and included…

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