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Auto Tag: Genome assembly

July 7, 2019

Exploiting next-generation sequencing to solve the haplotyping puzzle in polyploids: a simulation study.

Haplotypes are the units of inheritance in an organism, and many genetic analyses depend on their precise determination. Methods for haplotyping single individuals use the phasing information available in next-generation sequencing reads, by matching overlapping single-nucleotide polymorphisms while penalizing post hoc nucleotide corrections made. Haplotyping diploids is relatively easy, but the complexity of the problem increases drastically for polyploid genomes, which are found in both model organisms and in economically relevant plant and animal species. Although a number of tools are available for haplotyping polyploids, the effects of the genomic makeup and the sequencing strategy followed on the accuracy of these methods have hitherto not been thoroughly evaluated.We developed the simulation pipeline haplosim to evaluate the performance of three haplotype estimation algorithms for polyploids: HapCompass, HapTree and SDhaP, in settings varying in sequencing approach, ploidy levels and genomic diversity, using tetraploid potato as the model. Our results show that sequencing depth is the major determinant of haplotype estimation quality, that 1?kb PacBio circular consensus sequencing reads and Illumina reads with large insert-sizes are competitive and that all methods fail to produce good haplotypes when ploidy levels increase. Comparing the three methods, HapTree produces the most accurate estimates, but also consumes the most resources. There is clearly room for improvement in polyploid haplotyping algorithms.

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July 7, 2019

Microbial metagenomics mock scenario-based sample simulation (M3S3).

Shotgun sequencing in increasingly applied in clinical microbiology for unbiased culture-independent diagnosis. While software solutions for metagenomics proliferate, integration of metagenomics in clinical care, requires method standardisation and validation. Virtual metagenomics samples could underpin validation by substituting real samples and thus we sought to develop a novel solution for simulation of metagenomics samples based on user-defined clinical scenarios.We designed the Microbial Metagenomics Mock Scenario-based Sample Simulation (M3S3) workflow, which allows users to generate virtual samples from raw reads or assemblies. The M3S3 output is a mock sample in FASTQ or FASTA format. M3S3 was tested by generating virtual samples for ten challenging infectious disease scenarios, involving a background matrix ‘spiked’ in silico with pathogens including mixtures. Replicate samples (seven per scenario) were used to represent different compositional ratios. Virtual samples were analysed using Taxonomer and Kraken db.The ten challenge scenarios were successfully applied, generating 80 samples. For all tested scenarios, the virtual samples showed sequence compositions as predicted from the user input. Spiked pathogen sequences were identified with the majority of the replicates and most exhibited acceptable abundance (deviation between expected and observed abundance of spiked pathogens), with slight differences observed between software tools.Despite demonstrated proof-of-concept, integration of clinical metagenomics in routine microbiology remains a substantial challenge. M3S3 is capable of producing virtual samples on-demand, simulating a spectrum of clinical diagnostic scenarios of varying complexity. The M3S3 tool can therefore support the development and validation of standardised metagenomics applications. Copyright © 2017. Published by Elsevier Ltd.

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July 7, 2019

ReMILO: reference assisted misassembly detection algorithm using short and long reads.

Contigs assembled from the second generation sequencing short reads may contain misassemblies, and thus complicate downstream analysis or even lead to incorrect analysis results. Fortunately, with more and more sequenced species available, it becomes possible to use the reference genome of a closely related species to detect misassemblies. In addition, long reads of the third generation sequencing technology have been more and more widely used, and can also help detect misassemblies.Here, we introduce ReMILO, a reference assisted misassembly detection algorithm that uses both short reads and PacBio SMRT long reads. ReMILO aligns the initial short reads to both the contigs and reference genome, and then constructs a novel data structure called red-black multipositional de Bruijn graph to detect misassemblies. In addition, ReMILO also aligns the contigs to long reads and find their differences from the long reads to detect more misassemblies. In our performance test on short read assemblies of human chromosome 14 data, ReMILO can detect 41.8-77.9% extensive misassemblies and 33.6-54.5% local misassemblies. On hybrid short and long read assemblies of S.pastorianus data, ReMILO can also detect 60.6-70.9% extensive misassemblies and 28.6-54.0% local misassemblies.The ReMILO software can be downloaded for free under Artistic License 2.0 from this site: https://github.com/songc001/remilo.baoe@bjtu.edu.cn.Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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July 7, 2019

Microbial sequence typing in the genomic era.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is changing the field of microbial genomics research. NGS allows for a more comprehensive analysis of the diversity, structure and composition of microbial genes and genomes compared to the traditional automated Sanger capillary sequencing at a lower cost. NGS strategies have expanded the versatility of standard and widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments (MLST, MLVA, rMLST and cgMLST). NGS can now accommodate variation in thousands or millions of sequences from selected amplicons to full genomes (WGS, NGMLST and HiMLST). To extract signals from high-dimensional NGS data and make valid statistical inferences, novel analytic and statistical techniques are needed. In this review, we describe standard and new approaches for microbial sequence typing at gene and genome levels and guidelines for subsequent analysis, including methods and computational frameworks. We also present several applications of these approaches to some disciplines, namely genotyping, phylogenetics and molecular epidemiology. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Genomic insights into Photobacterium damselae subsp. damselae strain KC-Na-1, isolated from the finless porpoise (Neophocaena asiaeorientalis)

Photobacterium damselae subsp. damselae (PDD) is a marine bacterium that can infect a variety of marine animals and humans. Although this bacterium has been isolated from several stranded dolphins and whales, its pathogenic role in cetaceans is still unclear. In this study, we report the complete genome of PDD strain KC-Na-1 isolated from a finless porpoise (Neophocaena asiaeorientalis) rescued from the South Sea (Republic of Korea). The sequenced genome comprised two chromosomes and four plasmids. Among the recently identified major virulence factors in PDD, only phospholipase (plpV) was found in strain KC-Na-1. Interestingly, two genes homologous to Vibrio thermostable direct hemolysin (tdh) and its transcriptional regulator toxR, which are known virulence factors associated with Vibrio parahaemolyticus, were encoded on the plasmid pPDD-Na-1-3. Based on these results, strain KC-Na-1 may have potential pathogenicity in humans and other marine animals and also could act as a potential virulent strain. To the best of our knowledge, this is the first report of the complete genome sequence of P. damselae.

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July 7, 2019

New high copy tandem repeat in the content of the chicken W chromosome.

The content of repetitive DNA in avian genomes is considerably less than in other investigated vertebrates. The first descriptions of tandem repeats were based on the results of routine biochemical and molecular biological experiments. Both satellite DNA and interspersed repetitive elements were annotated using library-based approach and de novo repeat identification in assembled genome. The development of deep-sequencing methods provides datasets of high quality without preassembly allowing one to annotate repetitive elements from unassembled part of genomes. In this work, we search the chicken assembly and annotate high copy number tandem repeats from unassembled short raw reads. Tandem repeat (GGAAA)n has been identified and found to be the second after telomeric repeat (TTAGGG)n most abundant in the chicken genome. Furthermore, (GGAAA)n repeat forms expanded arrays on the both arms of the chicken W chromosome. Our results highlight the complexity of repetitive sequences and update data about organization of sex W chromosome in chicken.

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July 7, 2019

Cupriavidus malaysiensis sp. nov., a novel poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumulating bacterium isolated from the Malaysian environment.

Bacterial classification on the basis of a polyphasic approach was conducted on three poly(3 hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] accumulating bacterial strains that were isolated from samples collected from Malaysian environments; Kulim Lake, Sg. Pinang river and Sg. Manik paddy field. The Gram-negative, rod-shaped, motile, non-sporulating and non-fermenting bacteria were shown to belong to the genus Cupriavidus of the Betaproteobacteria on the basis of their 16S rRNA gene sequence analyses. The sequence similarity value with their near phylogenetic neighbour, Cupriavidus pauculus LMG3413T, was 98.5%. However, the DNA-DNA hybridization values (8-58%) and ribotyping analysis both enabled these strains to be differentiated from related Cupriavidus species with validly published names. The RiboPrint patterns of the three strains also revealed that the strains were genetically related even though they displayed a clonal diversity. The major cellular fatty acids detected in these strains included C15:0 ISO 2OH/C16:1 ?7c, hexadecanoic (16:0) and cis-11-octadecenoic (C18:1 ?7c). Their G+C contents ranged from 68.0  to 68.6 mol%, and their major isoprenoid quinone was Ubiquinone Q-8. Of these three strains, only strain USMAHM13 (= DSM 25816 = KCTC 32390) was discovered to exhibit yellow pigmentation that is characteristic of the carotenoid family. Their assembled genomes also showed that the three strains were not identical in terms of their genome sizes that were 7.82, 7.95 and 8.70 Mb for strains USMAHM13, USMAA1020 and USMAA2-4, respectively, which are slightly larger than that of Cupriavidus necator H16 (7.42 Mb). The average nucleotide identity (ANI) results indicated that the strains were genetically related and the genome pairs belong to the same species. On the basis of the results obtained in this study, the three strains are considered to represent a novel species for which the name Cupriavidus malaysiensis sp. nov. is proposed. The type strain of the species is USMAA1020T (= DSM 19416T = KCTC 32390T).

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July 7, 2019

De novo mutations resolve disease transmission pathways in clonal malaria

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However, these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here, we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a combination of sequencing, library preparation, and genotyping methods that have been optimized for accuracy in low-complexity genomic regions, we have detected de novo mutations that distinguish nominally identical parasites from clonal lineages. Despite its slower evolutionary rate compared with bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2?years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

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July 7, 2019

Comparative genomic analysis of Lactobacillus plantarum GB-LP4 and identification of evolutionarily divergent genes in high-osmolarity environment.

Lactobacillus plantarum is one of the widely-used probiotics and there have been a large number of advanced researches on the effectiveness of this species. However, the difference between previously reported plantarum strains, and the source of genomic variation among the strains were not clearly specified. In order to understand further on the molecular basis of L. plantarum on Korean traditional fermentation, we isolated the L. plantarum GB-LP4 from Korean fermented vegetable and conducted whole genome assembly. With comparative genomics approach, we identified the candidate genes that are expected to have undergone evolutionary acceleration. These genes have been reported to associate with the maintaining homeostasis, which are generally known to overcome instability in external environment including low pH or high osmotic pressure. Here, our results provide an evolutionary relationship between L. plantarum species and elucidate the candidate genes that play a pivotal role in evolutionary acceleration of GB-LP4 in high osmolarity environment. This study may provide guidance for further studies on L. plantarum.

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July 7, 2019

Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D.

Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of second-generation sequencing. Saccharomyces cerevisiae strain CEN.PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as the mitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN.PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5′ UTR and 3′ UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms.

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July 7, 2019

Assembly, annotation, and comparative genomics in PATRIC, the All Bacterial Bioinformatics Resource Center.

In the “big data” era, research biologists are faced with analyzing new types that usually require some level of computational expertise. A number of programs and pipelines exist, but acquiring the expertise to run them, and then understanding the output can be a challenge.The Pathosystems Resource Integration Center (PATRIC, www.patricbrc.org ) has created an end-to-end analysis platform that allows researchers to take their raw reads, assemble a genome, annotate it, and then use a suite of user-friendly tools to compare it to any public data that is available in the repository. With close to 113,000 bacterial and more than 1000 archaeal genomes, PATRIC creates a unique research experience with “virtual integration” of private and public data. PATRIC contains many diverse tools and functionalities to explore both genome-scale and gene expression data, but the main focus of this chapter is on assembly, annotation, and the downstream comparative analysis functionality that is freely available in the resource.

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July 7, 2019

RepLong: de novo repeat identification using long read sequencing data.

The identification of repetitive elements is important in genome assembly and phylogenetic analyses. The existing de novo repeat identification methods exploiting the use of short reads are impotent in identifying long repeats. Since long reads are more likely to cover repeat regions completely, using long reads is more favorable for recognizing long repeats.In this study, we propose a novel de novo repeat elements identification method namely RepLong based on PacBio long reads. Given that the reads mapped to the repeat regions are highly overlapped with each other, the identification of repeat elements is equivalent to the discovery of consensus overlaps between reads, which can be further cast into a community detection problem in the network of read overlaps. In RepLong, we first construct a network of read overlaps based on pair-wise alignment of the reads, where each vertex indicates a read and an edge indicates a substantial overlap between the corresponding two reads. Secondly, the communities whose intra connectivity is greater than the inter connectivity are extracted based on network modularity optimization. Finally, representative reads in each community are extracted to form the repeat library. Comparison studies on Drosophila melanogaster and human long read sequencing data with genome-based and short-read-based methods demonstrate the efficiency of RepLong in identifying long repeats. RepLong can handle lower coverage data and serve as a complementary solution to the existing methods to promote the repeat identification performance on long-read sequencing data.The software of RepLong is freely available at https://github.com/ruiguo-bio/replong.ywsun@szu.edu.cn or zhuzx@szu.edu.cn.Supplementary data are available at Bioinformatics online.

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July 7, 2019

Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.

Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.

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