FLASH: fast length adjustment of short reads to improve genome assemblies.
Ontology highlight
ABSTRACT:
PROVIDER: S-EPMC3198573 | BioStudies |
REPOSITORIES: biostudies
Similar Datasets
Project description:BACKGROUND:Chloroplasts are organelles that conduct photosynthesis in plant and algal cells. The information chloroplast genome contained is widely used in agriculture and studies of evolution and ecology. Correctly assembling chloroplast genomes can be challenging because the chloroplast genome contains a pair of long inverted repeats (10-30?kb). Typically, it is simply assumed that the gross structure of the chloroplast genome matches the most commonly observed structure of two single-copy regions separated by a pair of inverted repeats. The advent of long-read sequencing technologies should remove the need to make this assumption by providing sufficient information to completely span the inverted repeat regions. Yet, long-reads tend to have higher error rates than short-reads, and relatively little is known about the best way to combine long- and short-reads to obtain the most accurate chloroplast genome assemblies. Using Eucalyptus pauciflora, the snow gum, as a test case, we evaluated the effect of multiple parameters, such as different coverage of long-(Oxford nanopore) and short-(Illumina) reads, different long-read lengths, different assembly pipelines, with a view to determining the most accurate and efficient approach to chloroplast genome assembly. RESULTS:Hybrid assemblies combining at least 20x coverage of both long-reads and short-reads generated a single contig spanning the entire chloroplast genome with few or no detectable errors. Short-read-only assemblies generated three contigs (the long single copy, short single copy and inverted repeat regions) of the chloroplast genome. These contigs contained few single-base errors but tended to exclude several bases at the beginning or end of each contig. Long-read-only assemblies tended to create multiple contigs with a much higher single-base error rate. The chloroplast genome of Eucalyptus pauciflora is 159,942?bp, contains 131 genes of known function. CONCLUSIONS:Our results suggest that very accurate assemblies of chloroplast genomes can be achieved using a combination of at least 20x coverage of long- and short-reads respectively, provided that the long-reads contain at least ~5x coverage of reads longer than the inverted repeat region. We show that further increases in coverage give little or no improvement in accuracy, and that hybrid assemblies are more accurate than long-read-only or short-read-only assemblies.
| S-EPMC6311037 | BioStudies
Project description:For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set.
| S-EPMC3223268 | BioStudies
Project description:Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.
| S-EPMC5479803 | BioStudies
Project description:<h4>Background</h4>Shotgun metagenomes are often assembled prior to annotation of genes which biases the functional capacity of a community towards its most abundant members. For an unbiased assessment of community function, short reads need to be mapped directly to a gene or protein database. The ability to detect genes in short read sequences is dependent on pre- and post-sequencing decisions. The objective of the current study was to determine how library size selection, read length and format, protein database, e-value threshold, and sequencing depth impact gene-centric analysis of human fecal microbiomes when using DIAMOND, an alignment tool that is up to 20,000 times faster than BLASTX.<h4>Results</h4>Using metagenomes simulated from a database of experimentally verified protein sequences, we find that read length, e-value threshold, and the choice of protein database dramatically impact detection of a known target, with best performance achieved with longer reads, stricter e-value thresholds, and a custom database. Using publicly available metagenomes, we evaluated library size selection, paired end read strategy, and sequencing depth. Longer read lengths were acheivable by merging paired ends when the sequencing library was size-selected to enable overlaps. When paired ends could not be merged, a congruent strategy in which both ends are independently mapped was acceptable. Sequencing depths of 5 million merged reads minimized the error of abundance estimates of specific target genes, including an antimicrobial resistance gene.<h4>Conclusions</h4>Shotgun metagenomes of DNA extracted from human fecal samples sequenced using the Illumina platform should be size-selected to enable merging of paired end reads and should be sequenced in the PE150 format with a minimum sequencing depth of 5 million merge-able reads to enable detection of specific target genes. Expecting the merged reads to be 180-250?bp in length, the appropriate e-value threshold for DIAMOND would then need to be more strict than the default. Accurate and interpretable results for specific hypotheses will be best obtained using small databases customized for the research question.
| S-EPMC7041091 | BioStudies
Project description:BACKGROUND: Despite the short length of their reads, micro-read sequencing technologies have shown their usefulness for de novo sequencing. However, especially in eukaryotic genomes, complex repeat patterns are an obstacle to large assemblies. PRINCIPAL FINDINGS: We present a novel heuristic algorithm, Pebble, which uses paired-end read information to resolve repeats and scaffold contigs to produce large-scale assemblies. In simulations, we can achieve weighted median scaffold lengths (N50) of above 1 Mbp in Bacteria and above 100 kbp in more complex organisms. Using real datasets we obtained a 96 kbp N50 in Pseudomonas syringae and a unique 147 kbp scaffold of a ferret BAC clone. We also present an efficient algorithm called Rock Band for the resolution of repeats in the case of mixed length assemblies, where different sequencing platforms are combined to obtain a cost-effective assembly. CONCLUSIONS: These algorithms extend the utility of short read only assemblies into large complex genomes. They have been implemented and made available within the open-source Velvet short-read de novo assembler.
| S-EPMC2793427 | BioStudies
Project description:Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis.Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs.Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.
| S-EPMC3091720 | BioStudies
Project description:BACKGROUND:Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. RESULTS:We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. CONCLUSIONS:Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.
| S-EPMC6325685 | BioStudies
Project description: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.
| S-EPMC3285633 | BioStudies
Project description:Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit-and revealed many critical ecosystem roles for viruses-microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data ('enhanced' viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a 'living document' to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.
| S-EPMC8018248 | BioStudies
Project description:Genome assembly depends critically on read length. Two recent technologies, from Pacific Biosciences (PacBio) and Oxford Nanopore, produce read lengths >20 kb, which yield de novo genome assemblies with vastly greater contiguity than those based on Sanger, Illumina, or other technologies. However, the very high error rates of these two new technologies (?15% per base) makes assembly imprecise at repeats longer than the read length and computationally expensive. Here we show that the contiguity and quality of the assembly of these noisy long reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in Illumina reads (which account for ?95% of the distinct k-mers) are deemed sequencing errors and ignored at the seed alignment step. By focusing on the ?5% of k-mers that are error free, read overlap sensitivity is dramatically increased. Of equal importance, the validation procedure can be extended to exclude repetitive k-mers, which prevents read miscorrection at repeats and further improves the resulting assemblies. We tested the k-mer validation procedure using one long-read technology (PacBio) and one assembler (MHAP/Celera Assembler), but it is very likely to yield analogous improvements with alternative long-read technologies and assemblers, such as Oxford Nanopore and BLASR/DALIGNER/Falcon, respectively.
2016-01-01 | S-EPMC5131822 | BioStudies