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Asset Tag: Short-read sequencing limitations

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 for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.

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July 19, 2019  |  

Whole genome complete resequencing of Bacillus subtilis natto by combining long reads with high-quality short reads.

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.

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July 19, 2019  |  

Comparison of genome sequencing technology and assembly methods for the analysis of a GC-rich bacterial genome.

Improvements in technology and decreases in price have made de novo bacterial genomic sequencing a reality for many researchers, but it has created a need to evaluate the methods for generating a complete and accurate genome assembly. We sequenced the GC-rich Caulobacter henricii genome using the Illumina MiSeq, Roche 454, and Pacific Biosciences RS II sequencing systems. To generate a complete genome sequence, we performed assemblies using eight readily available programs and found that builds using the Illumina MiSeq and the Roche 454 data produced accurate yet numerous contigs. SPAdes performed the best followed by PANDAseq. In contrast, the Celera assembler produced a single genomic contig using the Pacific Biosciences data after error correction with the Illumina MiSeq data. In addition, we duplicated this build using the Pacific Biosciences data with HGAP2.0. The accuracy of these builds was verified by pulsed-field gel electrophoresis of genomic DNA cut with restriction enzymes.

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July 19, 2019  |  

Assessing structural variation in a personal genome-towards a human reference diploid genome.

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

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July 19, 2019  |  

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage f-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

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July 19, 2019  |  

The complete genome sequence of the murine pathobiont Helicobacter typhlonius.

Immuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing.The genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island (PAIs) containing components of type IV secretion system, virulence-associated proteins and cag PAI protein. We compared the genome of H. typhlonius to those of the murine pathobiont H. hepaticus and human pathobiont H. pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori.The complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease.

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July 19, 2019  |  

An incomplete understanding of human genetic variation.

Deciphering the genetic basis of human disease requires a comprehensive knowledge of genetic variants irrespective of their class or frequency. Although an impressive number of human genetic variants have been catalogued, a large fraction of the genetic difference that distinguishes two human genomes is still not understood at the base-pair level. This is because the emphasis has been on single-nucleotide variation as opposed to less tractable and more complex genetic variants, including indels and structural variants. The latter, we propose, will have a large impact on human phenotypes but require a more systematic assessment of genomes at deeper coverage and alternate sequencing and mapping technologies. Copyright © 2016 by the Genetics Society of America.

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July 19, 2019  |  

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies. Copyright © 2016. Published by Elsevier B.V.

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July 19, 2019  |  

Towards precision medicine.

There is great potential for genome sequencing to enhance patient care through improved diagnostic sensitivity and more precise therapeutic targeting. To maximize this potential, genomics strategies that have been developed for genetic discovery – including DNA-sequencing technologies and analysis algorithms – need to be adapted to fit clinical needs. This will require the optimization of alignment algorithms, attention to quality-coverage metrics, tailored solutions for paralogous or low-complexity areas of the genome, and the adoption of consensus standards for variant calling and interpretation. Global sharing of this more accurate genotypic and phenotypic data will accelerate the determination of causality for novel genes or variants. Thus, a deeper understanding of disease will be realized that will allow its targeting with much greater therapeutic precision.

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July 19, 2019  |  

Variation and evolution in the glutamine-rich repeat region of Drosophila argonaute-2.

RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. Copyright © 2016 Palmer and Obbard.

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July 7, 2019  |  

First complete genome sequence of Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 13311 (NCTC 74), a reference strain of multidrug resistance, as achieved by use of PacBio Single-Molecule Real-Time technology.

We report the first complete genomic sequence of Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 13311, the leading food-borne pathogen and a reference strain used in drug resistance studies. De novo assembly with PacBio sequencing completed its chromosome and one plasmid. They will accelerate the investigation into multidrug resistance in Salmonella Typhimurium. Copyright © 2014 Terabayashi et al.

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July 7, 2019  |  

The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV.

Aerobic methanotrophs can grow in hostile volcanic environments and use methane as their sole source of energy. The discovery of three verrucomicrobial Methylacidiphilum strains has revealed diverse metabolic pathways used by these methanotrophs, including mechanisms through which methane is oxidized. The basis of a complete understanding of these processes and of how these bacteria evolved and are able to thrive in such extreme environments partially resides in the complete characterization of their genome and its architecture.In this study, we present the complete genome sequence of Methylacidiphilum fumariolicum SolV, obtained using Pacific Biosciences single-molecule real-time (SMRT) sequencing technology. The genome assembles to a single 2.5 Mbp chromosome with an average GC content of 41.5%. The genome contains 2,741 annotated genes and 314 functional subsystems including all key metabolic pathways that are associated with Methylacidiphilum strains, including the CBB pathway for CO2 fixation. However, it does not encode the serine cycle and ribulose monophosphate pathways for carbon fixation. Phylogenetic analysis of the particulate methane mono-oxygenase operon separates the Methylacidiphilum strains from other verrucomicrobial methanotrophs. RNA-Seq analysis of cell cultures growing in three different conditions revealed the deregulation of two out of three pmoCAB operons. In addition, genes involved in nitrogen fixation were upregulated in cell cultures growing in nitrogen fixing conditions, indicating the presence of active nitrogenase. Characterization of the global methylation state of M. fumariolicum SolV revealed methylation of adenines and cytosines mainly in the coding regions of the genome. Methylation of adenines was predominantly associated with 5′-m6ACN4GT-3′ and 5′-CCm6AN5CTC-3′ methyltransferase recognition motifs whereas methylated cytosines were not associated with any specific motif.Our findings provide novel insights into the global methylation state of verrucomicrobial methanotroph M. fumariolicum SolV. However, partial conservation of methyltransferases between M. fumariolicum SolV and M. infernorum V4 indicates potential differences in the global methylation state of Methylacidiphilum strains. Unravelling the M. fumariolicum SolV genome and its epigenetic regulation allow for robust characterization of biological processes that are involved in oxidizing methane. In turn, they offer a better understanding of the evolution, the underlying physiological and ecological properties of SolV and other Methylacidiphilum strains.

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July 7, 2019  |  

Characterization of structural variants with single molecule and hybrid sequencing approaches.

Structural variation is common in human and cancer genomes. High-throughput DNA sequencing has enabled genome-scale surveys of structural variation. However, the short reads produced by these technologies limit the study of complex variants, particularly those involving repetitive regions. Recent ‘third-generation’ sequencing technologies provide single-molecule templates and longer sequencing reads, but at the cost of higher per-nucleotide error rates.We present MultiBreak-SV, an algorithm to detect structural variants (SVs) from single molecule sequencing data, paired read sequencing data, or a combination of sequencing data from different platforms. We demonstrate that combining low-coverage third-generation data from Pacific Biosciences (PacBio) with high-coverage paired read data is advantageous on simulated chromosomes. We apply MultiBreak-SV to PacBio data from four human fosmids and show that it detects known SVs with high sensitivity and specificity. Finally, we perform a whole-genome analysis on PacBio data from a complete hydatidiform mole cell line and predict 1002 high-probability SVs, over half of which are confirmed by an Illumina-based assembly. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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