52 research outputs found
SLIQ: Simple Linear Inequalities for Efficient Contig Scaffolding
Scaffolding is an important subproblem in "de novo" genome assembly in which
mate pair data are used to construct a linear sequence of contigs separated by
gaps. Here we present SLIQ, a set of simple linear inequalities derived from
the geometry of contigs on the line that can be used to predict the relative
positions and orientations of contigs from individual mate pair reads and thus
produce a contig digraph. The SLIQ inequalities can also filter out unreliable
mate pairs and can be used as a preprocessing step for any scaffolding
algorithm. We tested the SLIQ inequalities on five real data sets ranging in
complexity from simple bacterial genomes to complex mammalian genomes and
compared the results to the majority voting procedure used by many other
scaffolding algorithms. SLIQ predicted the relative positions and orientations
of the contigs with high accuracy in all cases and gave more accurate position
predictions than majority voting for complex genomes, in particular the human
genome. Finally, we present a simple scaffolding algorithm that produces linear
scaffolds given a contig digraph. We show that our algorithm is very efficient
compared to other scaffolding algorithms while maintaining high accuracy in
predicting both contig positions and orientations for real data sets.Comment: 16 pages, 6 figures, 7 table
Genome Assembly: Novel Applications by Harnessing Emerging Sequencing Technologies and Graph Algorithms
Genome assembly is a critical first step for biological discovery. All current sequencing technologies share the fundamental limitation that segments read from a genome are much shorter than even the smallest genomes. Traditionally, whole- genome shotgun (WGS) sequencing over-samples a single clonal (or inbred) target chromosome with segments from random positions. The amount of over-sampling is known as the coverage. Assembly software then reconstructs the target. So called next-generation (or second-generation) sequencing has reduced the cost and increased throughput exponentially over first-generation sequencing. Unfortunately, next-generation sequences present their own challenges to genome assembly: (1) they require amplification of source DNA prior to sequencing leading to artifacts and biased coverage of the genome; (2) they produce relatively short reads: 100bp- 700bp; (3) the sizeable runtime of most second-generation instruments is prohibitive for applications requiring rapid analysis, with an Illumina HiSeq 2000 instrument requiring 11 days for the sequencing reaction.
Recently, successors to the second-generation instruments (third-generation) have become available. These instruments promise to alleviate many of the down- sides of second-generation sequencing and can generate multi-kilobase sequences. The long sequences have the potential to dramatically improve genome and transcriptome assembly. However, the high error rate of these reads is challenging and has limited their use. To address this limitation, we introduce a novel correction algorithm and assembly strategy that utilizes shorter, high-identity sequences to correct the error in single-molecule sequences. Our approach achieves over 99% read accuracy and produces substantially better assemblies than current sequencing strategies.
The availability of cheaper sequencing has made new sequencing targets, such as multiple displacement amplified (MDA) single-cells and metagenomes, popular. Current algorithms assume assembly of a single clonal target, an assumption that is violated in these sequencing projects. We developed Bambus 2, a new scaffolder that works for metagenomics and single cell datasets. It can accurately detect repeats without assumptions about the taxonomic composition of a dataset. It can also identify biological variations present in a sample. We have developed a novel end-to-end analysis pipeline leveraging Bambus 2. Due to its modular nature, it is applicable to clonal, metagenomic, and MDA single-cell targets and allows a user to rapidly go from sequences to assembly, annotation, genes, and taxonomic info. We have incorporated a novel viewer, allowing a user to interactively explore the variation present in a genomic project on a laptop.
Together, these developments make genome assembly applicable to novel targets while utilizing emerging sequencing technologies. As genome assembly is critical for all aspects of bioinformatics, these developments will enable novel biological discovery
MetAMOS: A modular and open source metagenomic assembly and analysis pipeline
© 2013 Treangen et al. We describe MetAMOS, an open source and modular metagenomic assembly and analysis pipeline. MetAMOS represents an important step towards fully automated metagenomic analysis, starting with next-generation sequencing reads and producing genomic scaffolds, open-reading frames and taxonomic or functional annotations. MetAMOS can aid in reducing assembly errors, commonly encountered when assembling metagenomic samples, and improves taxonomic assignment accuracy while also reducing computational cost. MetAMOS can be downloaded from: https://github.com/treangen/MetAMOS
A Reference-Free Algorithm for Computational Normalization of Shotgun Sequencing Data
Deep shotgun sequencing and analysis of genomes, transcriptomes, amplified
single-cell genomes, and metagenomes has enabled investigation of a wide range
of organisms and ecosystems. However, sampling variation in short-read data
sets and high sequencing error rates of modern sequencers present many new
computational challenges in data interpretation. These challenges have led to
the development of new classes of mapping tools and {\em de novo} assemblers.
These algorithms are challenged by the continued improvement in sequencing
throughput. We here describe digital normalization, a single-pass computational
algorithm that systematizes coverage in shotgun sequencing data sets, thereby
decreasing sampling variation, discarding redundant data, and removing the
majority of errors. Digital normalization substantially reduces the size of
shotgun data sets and decreases the memory and time requirements for {\em de
novo} sequence assembly, all without significantly impacting content of the
generated contigs. We apply digital normalization to the assembly of microbial
genomic data, amplified single-cell genomic data, and transcriptomic data. Our
implementation is freely available for use and modification
A new strategy for better genome assembly from very short reads
<p>Abstract</p> <p>Background</p> <p>With the rapid development of the next generation sequencing (NGS) technology, large quantities of genome sequencing data have been generated. Because of repetitive regions of genomes and some other factors, assembly of very short reads is still a challenging issue.</p> <p>Results</p> <p>A novel strategy for improving genome assembly from very short reads is proposed. It can increase accuracies of assemblies by integrating <it>de novo </it>contigs, and produce comparative contigs by allowing multiple references without limiting to genomes of closely related strains. Comparative contigs are used to scaffold <it>de novo </it>contigs. Using simulated and real datasets, it is shown that our strategy can effectively improve qualities of assemblies of isolated microbial genomes and metagenomes.</p> <p>Conclusions</p> <p>With more and more reference genomes available, our strategy will be useful to improve qualities of genome assemblies from very short reads. Some scripts are provided to make our strategy applicable at <url>http://code.google.com/p/cd-hybrid/</url>.</p
Assessment of Metagenomic Assembly Using Simulated Next Generation Sequencing Data
Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available
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Reconstructing an ancestral genotype of two hexachlorocyclohexane-degrading Sphingobium species using metagenomic sequence data.
Over the last 60 years, the use of hexachlorocyclohexane (HCH) as a pesticide has resulted in the production of >4 million tons of HCH waste, which has been dumped in open sinks across the globe. Here, the combination of the genomes of two genetic subspecies (Sphingobium japonicum UT26 and Sphingobium indicum B90A; isolated from two discrete geographical locations, Japan and India, respectively) capable of degrading HCH, with metagenomic data from an HCH dumpsite (∼450 mg HCH per g soil), enabled the reconstruction and validation of the last-common ancestor (LCA) genotype. Mapping the LCA genotype (3128 genes) to the subspecies genomes demonstrated that >20% of the genes in each subspecies were absent in the LCA. This includes two enzymes from the 'upper' HCH degradation pathway, suggesting that the ancestor was unable to degrade HCH isomers, but descendants acquired lin genes by transposon-mediated lateral gene transfer. In addition, anthranilate and homogentisate degradation traits were found to be strain (selectively retained only by UT26) and environment (absent in the LCA and subspecies, but prevalent in the metagenome) specific, respectively. One draft secondary chromosome, two near complete plasmids and eight complete lin transposons were assembled from the metagenomic DNA. Collectively, these results reinforce the elastic nature of the genus Sphingobium, and describe the evolutionary acquisition mechanism of a xenobiotic degradation phenotype in response to environmental pollution. This also demonstrates for the first time the use of metagenomic data in ancestral genotype reconstruction, highlighting its potential to provide significant insight into the development of such phenotypes
Computational tools for viral metagenomics and their application in clinical research
AbstractThere are 100 times more virions than eukaryotic cells in a healthy human body. The characterization of human-associated viral communities in a non-pathological state and the detection of viral pathogens in cases of infection are essential for medical care and epidemic surveillance. Viral metagenomics, the sequenced-based analysis of the complete collection of viral genomes directly isolated from an organism or an ecosystem, bypasses the “single-organism-level” point of view of clinical diagnostics and thus the need to isolate and culture the targeted organism. The first part of this review is dedicated to a presentation of past research in viral metagenomics with an emphasis on human-associated viral communities (eukaryotic viruses and bacteriophages). In the second part, we review more precisely the computational challenges posed by the analysis of viral metagenomes, and we illustrate the problem of sequences that do not have homologs in public databases and the possible approaches to characterize them
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