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Auto Tag: De novo assembly

July 19, 2019

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms.

Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.

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July 19, 2019

Characterizing and measuring bias in sequence data.

DNA sequencing technologies deviate from the ideal uniform distribution of reads. These biases impair scientific and medical applications. Accordingly, we have developed computational methods for discovering, describing and measuring bias.We applied these methods to the Illumina, Ion Torrent, Pacific Biosciences and Complete Genomics sequencing platforms, using data from human and from a set of microbes with diverse base compositions. As in previous work, library construction conditions significantly influence sequencing bias. Pacific Biosciences coverage levels are the least biased, followed by Illumina, although all technologies exhibit error-rate biases in high- and low-GC regions and at long homopolymer runs. The GC-rich regions prone to low coverage include a number of human promoters, so we therefore catalog 1,000 that were exceptionally resistant to sequencing. Our results indicate that combining data from two technologies can reduce coverage bias if the biases in the component technologies are complementary and of similar magnitude. Analysis of Illumina data representing 120-fold coverage of a well-studied human sample reveals that 0.20% of the autosomal genome was covered at less than 10% of the genome-wide average. Excluding locations that were similar to known bias motifs or likely due to sample-reference variations left only 0.045% of the autosomal genome with unexplained poor coverage.The assays presented in this paper provide a comprehensive view of sequencing bias, which can be used to drive laboratory improvements and to monitor production processes. Development guided by these assays should result in improved genome assemblies and better coverage of biologically important loci.

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July 19, 2019

Returning to more finished genomes

Abstract Genomic data have become commonplace in most branches of the biological sciences and have fundamentally altered the way research is conducted. However, the predominance of short-read sequence data from second-generation sequencing technologies has commonly resulted in fragmented and partial genomic data characteristics. In this opinion, I will highlight how long, unbiased reads from single molecule, real-time (SMRT) sequencing now allow for a return to more contiguous and comprehensive views of genomes.

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July 19, 2019

The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development.

Programmed DNA rearrangements in the single-celled eukaryote Oxytricha trifallax completely rewire its germline into a somatic nucleus during development. This elaborate, RNA-mediated pathway eliminates noncoding DNA sequences that interrupt gene loci and reorganizes the remaining fragments by inversions and permutations to produce functional genes. Here, we report the Oxytricha germline genome and compare it to the somatic genome to present a global view of its massive scale of genome rearrangements. The remarkably encrypted genome architecture contains >3,500 scrambled genes, as well as >800 predicted germline-limited genes expressed, and some posttranslationally modified, during genome rearrangements. Gene segments for different somatic loci often interweave with each other. Single gene segments can contribute to multiple, distinct somatic loci. Terminal precursor segments from neighboring somatic loci map extremely close to each other, often overlapping. This genome assembly provides a draft of a scrambled genome and a powerful model for studies of genome rearrangement. Copyright © 2014 Elsevier Inc. All rights reserved.

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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 structural variants and two genomic features in a strain of Escherichia coli with PBHoney and validate them via de novo assembly. PBHoney is available for download at http://sourceforge.net/projects/pb-jelly/.Implementing two variant-identification approaches that exploit the high mappability of long reads, PBHoney is demonstrated as being effective at detecting larger structural variants using whole-genome Pacific Biosciences RS II Continuous Long Reads. Furthermore, PBHoney is able to discover two genomic features: the existence of Rac-Phage in isolate; evidence of E. coli’s circular genome.

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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 biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts.The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

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July 19, 2019

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1.

We performed whole-genome analyses of DNA methylation in Shewanella oneidensis MR-1 to examine its possible role in regulating gene expression and other cellular processes. Single-molecule real-time (SMRT) sequencing revealed extensive methylation of adenine (N6mA) throughout the genome. These methylated bases were located in five sequence motifs, including three novel targets for type I restriction/modification enzymes. The sequence motifs targeted by putative methyltranferases were determined via SMRT sequencing of gene knockout mutants. In addition, we found that S. oneidensis MR-1 cultures grown under various culture conditions displayed different DNA methylation patterns. However, the small number of differentially methylated sites could not be directly linked to the much larger number of differentially expressed genes under these conditions, suggesting that DNA methylation is not a major regulator of gene expression in S. oneidensis MR-1. The enrichment of methylated GATC motifs in the origin of replication indicates that DNA methylation may regulate genome replication in a manner similar to that seen in Escherichia coli. Furthermore, comparative analyses suggest that many Gammaproteobacteria, including all members of the Shewanellaceae family, may also utilize DNA methylation to regulate genome replication.

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July 19, 2019

Entering the era of bacterial epigenomics with single molecule real time DNA sequencing.

DNA modifications, such as methylation guide numerous critical biological processes, yet epigenetic information has not routinely been collected as part of DNA sequence analyses. Recently, the development of single molecule real time (SMRT) DNA sequencing has enabled detection of modified nucleotides (e.g. 6mA, 4mC, 5mC) in parallel with acquisition of primary sequence data, based on analysis of the kinetics of DNA synthesis reactions. In bacteria, genome-wide mapping of methylated and unmethylated loci is now feasible. This technological advance sets the stage for comprehensive, mechanistic assessment of the effects of bacterial DNA methyltransferases (MTases)-which are ubiquitous, extremely diverse, and largely uncharacterized-on gene expression, chromosome structure, chromosome replication, and other fundamental biological processes. SMRT sequencing also enables detection of damaged DNA and has the potential to uncover novel DNA modifications. Copyright © 2013 Elsevier Ltd. All rights reserved.

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July 19, 2019

Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a novel method for quantifying individual genome editing outcomes at any site of interest using single molecule real time (SMRT) DNA sequencing. We show that this approach can be applied at various loci, using multiple engineered nuclease platforms including TALENs, RNA guided endonucleases (CRISPR/Cas9), and ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach facilitates the evaluation of new gene editing technologies and permits sensitive quantification of editing outcomes in almost every experimental system used.

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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 nine isolates associated with the 2011 outbreak. Additionally, we determined the complete sequence for the chromosome and three plasmids from a clinical isolate associated with the 2011 outbreak using the Pacific Biosciences (PacBio) system. Using single-nucleotide polymorphism (SNP) analyses, we were able to distinguish highly clonal isolates, including strains isolated at different times in the same year. The isolates from the recent 2011 outbreak clustered together with a mean SNP variation of only 17 SNPs. The S. Heidelberg isolates carried a variety of phages, such as prophage P22, P4, lambda-like prophage Gifsy-2, and the P2-like phage which carries the sopE1 gene, virulence genes including 62 pathogenicity, and 13 fimbrial markers and resistance plasmids of the incompatibility (Inc)I1, IncA/C, and IncHI2 groups. Twenty-one strains contained an IncX plasmid carrying a type IV secretion system. On the basis of the recent and historical isolates used in this study, our results demonstrated that, in addition to providing detailed genetic information for the isolates, WGS can identify SNP targets that can be utilized for differentiating highly clonal S. Heidelberg isolates.

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July 19, 2019

Evolutionary dynamics of Vibrio cholerae O1 following a single-source introduction to Haiti.

Prior to the epidemic that emerged in Haiti in October of 2010, cholera had not been documented in this country. After its introduction, a strain of Vibrio cholerae O1 spread rapidly throughout Haiti, where it caused over 600,000 cases of disease and >7,500 deaths in the first two years of the epidemic. We applied whole-genome sequencing to a temporal series of V. cholerae isolates from Haiti to gain insight into the mode and tempo of evolution in this isolated population of V. cholerae O1. Phylogenetic and Bayesian analyses supported the hypothesis that all isolates in the sample set diverged from a common ancestor within a time frame that is consistent with epidemiological observations. A pangenome analysis showed nearly homogeneous genomic content, with no evidence of gene acquisition among Haiti isolates. Nine nearly closed genomes assembled from continuous-long-read data showed evidence of genome rearrangements and supported the observation of no gene acquisition among isolates. Thus, intrinsic mutational processes can account for virtually all of the observed genetic polymorphism, with no demonstrable contribution from horizontal gene transfer (HGT). Consistent with this, the 12 Haiti isolates tested by laboratory HGT assays were severely impaired for transformation, although unlike previously characterized noncompetent V. cholerae isolates, each expressed hapR and possessed a functional quorum-sensing system. Continued monitoring of V. cholerae in Haiti will illuminate the processes influencing the origin and fate of genome variants, which will facilitate interpretation of genetic variation in future epidemics.Vibrio cholerae is the cause of substantial morbidity and mortality worldwide, with over three million cases of disease each year. An understanding of the mode and rate of evolutionary change is critical for proper interpretation of genome sequence data and attribution of outbreak sources. The Haiti epidemic provides an unprecedented opportunity to study an isolated, single-source outbreak of Vibrio cholerae O1 over an established time frame. By using multiple approaches to assay genetic variation, we found no evidence that the Haiti strain has acquired any genes by horizontal gene transfer, an observation that led us to discover that it is also poorly transformable. We have found no evidence that environmental strains have played a role in the evolution of the outbreak strain.

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July 19, 2019

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51?N-6-methyladenines and 152?N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5a as a host bacterium to construct a shotgun library.This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.

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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 followed by single molecule real-time (SMRT) sequencing. This technology yielded long sequence reads and robust coverage that allowed for de novo sequence assembly spanning the entire repetitive region. Furthermore, we used SMRT sequencing of PCR amplicons covering the central exon to identify genetic variation in four individuals. The results demonstrated the presence of segmental duplications of CysD domains, insertions/deletions (indels) of tandem repeats, and single nucleotide variants. Additional studies demonstrated that one of the identified tandem repeat insertions is tagged by nonexonic single nucleotide polymorphisms. Taken together, these data illustrate the successful utility of SMRT sequencing long reads for de novo assembly of large repetitive sequences to fill the gaps in the human genome. Characterization of the MUC5AC gene and the sequence variation in the central exon will facilitate genetic and functional studies for this critical airway mucin.

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July 19, 2019

Recently published Streptomyces genome sequences.

Many readers of this journal will need no introduction to the bacterial genus Streptomyces, which includes several hundred species, many of which produce biotechnologically useful secondary metabolites. The last 2 years have seen numerous publications describing Streptomyces genome sequences (Table?1), mostly as short genome announcements restricted to just 500 words and therefore allowing little description and analysis. Our aim in this current manuscript is to survey these recent publications and to dig a little deeper where appropriate. The genus Streptomyces is now one of the most highly sequenced, with 19 finished genomic sequences (Table?2) and a further 125 draft assemblies available in the GenBank database as of 3rd of May 2014; by the time this is published, no doubt there will be more. The reasons given for sequencing this latest crop of Streptomyces include production of industrially important enzymes, degradation of lignin, antibiotic production, rapid growth and halo-tolerance and an endophytic lifestyle (Table?1).

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July 19, 2019

The genome sequence of African rice (Oryza glaberrima) and evidence for independent domestication.

The cultivation of rice in Africa dates back more than 3,000 years. Interestingly, African rice is not of the same origin as Asian rice (Oryza sativa L.) but rather is an entirely different species (i.e., Oryza glaberrima Steud.). Here we present a high-quality assembly and annotation of the O. glaberrima genome and detailed analyses of its evolutionary history of domestication and selection. Population genomics analyses of 20 O. glaberrima and 94 Oryza barthii accessions support the hypothesis that O. glaberrima was domesticated in a single region along the Niger river as opposed to noncentric domestication events across Africa. We detected evidence for artificial selection at a genome-wide scale, as well as with a set of O. glaberrima genes orthologous to O. sativa genes that are known to be associated with domestication, thus indicating convergent yet independent selection of a common set of genes during two geographically and culturally distinct domestication processes.

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