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July 7, 2019

Long single-molecule reads can resolve the complexity of the influenza virus composed of rare, closely related mutant variants

As a result of a high rate of mutations and recombination events, an RNA-virus exists as a heterogeneous “swarm” of mutant variants. The long read length offered by single-molecule sequencing technologies allows each mutant variant to be sequenced in a single pass. However, high error rate limits the ability to reconstruct heterogeneous viral population composed of rare, related mutant variants. In this paper, we present 2SNV, a method able to tolerate the high error-rate of the single-molecule protocol and reconstruct mutant variants. 2SNV uses linkage between single nucleotide variations to efficiently distinguish them from read errors. To benchmark the sensitivity of 2SNV, we performed a single-molecule sequencing experiment on a sample containing a titrated level of known viral mutant variants. Our method is able to accurately reconstruct clone with frequency of 0.2 % and distinguish clones that differed in only two nucleotides distantly located on the genome. 2SNV outperforms existing methods for full-length viral mutant reconstruction. The open source implementation of 2SNV is freely available for download at http://?alan.?cs.?gsu.?edu/?NGS/???q=?content/?2snv.

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July 7, 2019

Structural variation detection using next-generation sequencing data: A comparative technical review.

Structural variations (SVs) are mutations in the genome of size at least fifty nucleotides. They contribute to the phenotypic differences among healthy individuals, cause severe diseases and even cancers by breaking or linking genes. Thus, it is crucial to systematically profile SVs in the genome. In the past decade, many next-generation sequencing (NGS)-based SV detection methods have been proposed due to the significant cost reduction of NGS experiments and their ability to unbiasedly detect SVs to the base-pair resolution. These SV detection methods vary in both sensitivity and specificity, since they use different SV-property-dependent and library-property-dependent features. As a result, predictions from different SV callers are often inconsistent. Besides, the noises in the data (both platform-specific sequencing error and artificial chimeric reads) impede the specificity of SV detection. Poorly characterized regions in the human genome (e.g., repeat regions) greatly impact the reads mapping and in turn affect the SV calling accuracy. Calling of complex SVs requires specialized SV callers. Apart from accuracy, processing speed of SV caller is another factor deciding its usability. Knowing the pros and cons of different SV calling techniques and the objectives of the biological study are essential for biologists and bioinformaticians to make informed decisions. This paper describes different components in the SV calling pipeline and reviews the techniques used by existing SV callers. Through simulation study, we also demonstrate that library properties, especially insert size, greatly impact the sensitivity of different SV callers. We hope the community can benefit from this work both in designing new SV calling methods and in selecting the appropriate SV caller for specific biological studies. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

The draft genome of MD-2 pineapple using hybrid error correction of long reads.

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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July 7, 2019

Plasmid dynamics in KPC-positive Klebsiella pneumoniae during long-term patient colonization.

Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms.In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. Copyright © 2016 Conlan et al.

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July 7, 2019

Complete genome sequence of a Klebsiella pneumoniae strain carrying blaNDM-1 on a multidrug resistance plasmid

Here, we report the genome sequence of a blaNDM-1-positive Klebsiella pneumoniae AATZP isolate cultured from a perirectal surveillance swab collected upon admission of a patient to the NIH Clinical Center in 2014. Genome sequencing of this isolate revealed three plasmids, including one carrying the blaNDM-1 gene encoding resistance to carbapenems. Copyright © 2016 Conlan et al.

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July 7, 2019

Microsatellite length scoring by Single Molecule Real Time Sequencing – Effects of sequence structure and PCR regime.

Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.

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July 7, 2019

Challenges, solutions, and quality metrics of personal genome assembly in advancing precision medicine.

Even though each of us shares more than 99% of the DNA sequences in our genome, there are millions of sequence codes or structure in small regions that differ between individuals, giving us different characteristics of appearance or responsiveness to medical treatments. Currently, genetic variants in diseased tissues, such as tumors, are uncovered by exploring the differences between the reference genome and the sequences detected in the diseased tissue. However, the public reference genome was derived with the DNA from multiple individuals. As a result of this, the reference genome is incomplete and may misrepresent the sequence variants of the general population. The more reliable solution is to compare sequences of diseased tissue with its own genome sequence derived from tissue in a normal state. As the price to sequence the human genome has dropped dramatically to around $1000, it shows a promising future of documenting the personal genome for every individual. However, de novo assembly of individual genomes at an affordable cost is still challenging. Thus, till now, only a few human genomes have been fully assembled. In this review, we introduce the history of human genome sequencing and the evolution of sequencing platforms, from Sanger sequencing to emerging “third generation sequencing” technologies. We present the currently available de novo assembly and post-assembly software packages for human genome assembly and their requirements for computational infrastructures. We recommend that a combined hybrid assembly with long and short reads would be a promising way to generate good quality human genome assemblies and specify parameters for the quality assessment of assembly outcomes. We provide a perspective view of the benefit of using personal genomes as references and suggestions for obtaining a quality personal genome. Finally, we discuss the usage of the personal genome in aiding vaccine design and development, monitoring host immune-response, tailoring drug therapy and detecting tumors. We believe the precision medicine would largely benefit from bioinformatics solutions, particularly for personal genome assembly.

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July 7, 2019

SAR11 bacteria linked to ocean anoxia and nitrogen loss.

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.

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July 7, 2019

Representing genetic variation with synthetic DNA standards.

The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of synthetic DNA standards, termed ‘sequins’, that emulate human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis. Here we use this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We validate the design and performance of sequin standards by comparison to examples in the NA12878 reference genome, and we demonstrate their utility during the detection and quantification of variants. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

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July 7, 2019

DBG2OLC: Efficient assembly of large genomes using long erroneous reads of the third generation sequencing technologies.

The highly anticipated transition from next generation sequencing (NGS) to third generation sequencing (3GS) has been difficult primarily due to high error rates and excessive sequencing cost. The high error rates make the assembly of long erroneous reads of large genomes challenging because existing software solutions are often overwhelmed by error correction tasks. Here we report a hybrid assembly approach that simultaneously utilizes NGS and 3GS data to address both issues. We gain advantages from three general and basic design principles: (i) Compact representation of the long reads leads to efficient alignments. (ii) Base-level errors can be skipped; structural errors need to be detected and corrected. (iii) Structurally correct 3GS reads are assembled and polished. In our implementation, preassembled NGS contigs are used to derive the compact representation of the long reads, motivating an algorithmic conversion from a de Bruijn graph to an overlap graph, the two major assembly paradigms. Moreover, since NGS and 3GS data can compensate for each other, our hybrid assembly approach reduces both of their sequencing requirements. Experiments show that our software is able to assemble mammalian-sized genomes orders of magnitude more quickly than existing methods without consuming a lot of memory, while saving about half of the sequencing cost.

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July 7, 2019

Comparative genomics and transcriptomics of Pichia pastoris.

Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species. Generation of comparative high-quality genomes and transcriptomes will enable meaningful comparisons between the organisms, and potentially inform distinct biotechnological utilies for each species.Here, we present a comprehensive and standardized comparative analysis of the genomic features of the three most commonly used strains comprising the tradename Pichia: K. pastoris wild-type, K. phaffii wild-type, and K. phaffii GS115. We used a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies to achieve over 1000X coverage of each genome. Construction of individual genomes was then performed using as few as seven individual contigs to create gap-free assemblies. We found substantial syntenic rearrangements between the species and characterized a linear plasmid present in K. phaffii. Comparative analyses between K. phaffii genomes enabled the characterization of the mutational landscape of the GS115 strain. We identified and examined 35 non-synonomous coding mutations present in GS115, many of which are likely to impact strain performance. Additionally, we investigated transcriptomic profiles of gene expression for both species during cultivation on various carbon sources. We observed that the most highly transcribed genes in both organisms were consistently highly expressed in all three carbon sources examined. We also observed selective expression of certain genes in each carbon source, including many sequences not previously reported as promoters for expression of heterologous proteins in yeasts.Our studies establish a foundation for understanding critical relationships between genome structure, cultivation conditions and gene expression. The resources we report here will inform and facilitate rational, organism-wide strain engineering for improved utility as a host for protein production.

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July 7, 2019

An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly.

High density genetic linkage maps that are extensively anchored to assembled genome sequences of the organism in question are extremely useful in gene discovery. To facilitate this process in perennial ryegrass (Lolium perenne L.), a high density single nucleotide polymorphism (SNP)- and presence/absence variant (PAV)-based genetic linkage map has been developed in an F2 mapping population that has been used as a reference population in numerous studies. To provide a reference sequence to which to align genotyping by sequencing (GBS) reads, a shotgun assembly of one of the grandparents of the population, a tenth-generation inbred line, was created using Illumina-based sequencing.The assembly was based on paired-end Illumina reads, scaffolded by mate pair and long jumping distance reads in the range of 3-40?kb, with >200-fold initial genome coverage. A total of 169 individuals from an F2 mapping population were used to construct PstI-based GBS libraries tagged with unique 4-9 nucleotide barcodes, resulting in 284 million reads, with approx. 1·6 million reads per individual. A bioinformatics pipeline was employed to identify both SNPs and PAVs. A core genetic map was generated using high confidence SNPs, to which lower confidence SNPs and PAVs were subsequently fitted in a straightforward binning approach.The assembly comprises 424?750 scaffolds, covering 1·11 Gbp of the 2·5 Gbp perennial ryegrass genome, with a scaffold N50 of 25 212?bp and a contig N50 of 3790?bp. It is available for download, and access to a genome browser has been provided. Comparison of the assembly with available transcript and gene model data sets for perennial ryegrass indicates that approx. 570 Mbp of the gene-rich portion of the genome has been captured. An ultra-high density genetic linkage map with 3092 SNPs and 7260 PAVs was developed, anchoring just over 200?Mb of the reference assembly.The combined genetic map and assembly, combined with another recently released genome assembly, represent a significant resource for the perennial ryegrass genetics community.© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019

Assemblytics: a web analytics tool for the detection of variants from an assembly.

Assemblytics is a web app for detecting and analyzing variants from a de novo genome assembly aligned to a reference genome. It incorporates a unique anchor filtering approach to increase robustness to repetitive elements, and identifies six classes of variants based on their distinct alignment signatures. Assemblytics can be applied both to comparing aberrant genomes, such as human cancers, to a reference, or to identify differences between related species. Multiple interactive visualizations enable in-depth explorations of the genomic distributions of variants.http://assemblytics.com, https://github.com/marianattestad/assemblytics CONTACT: mnattest@cshl.eduSupplementary information: Supplementary data are available at Bioinformatics online.© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Whole-genome sequencing recommendations

Recent technological developments have revolutionized the way we perform genetic analyses. In particular whole-genome sequencing provides access to the entire genetic makeup of an individual, and it is now an affordable approach for many research groups. As a consequence genome sequencing is pervading many fields of biological research. Sequencing technologies are evolving rapidly and so do their applications. Here we provide a first primer on whole-genome sequencing, focusing on two of the most popular applications: (1) de novo genome sequencing, in which the objective is obtaining a high-quality genome assembly that can serve as a reference for a species or variety, and (2) genome resequencing, when there is an available reference genome and the objective is to map sequence variation of an individual or a set of individuals. It is not our intention to provide a comprehensive overview of current methodologies that will likely soon become obsolete, but rather focus on general principles that will have a more general applicability.

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