SAM/BAM format v1.5 extensions for de novo assemblies

Cock, Peter J. A.; Bonfield, JK; Chevreux, Bastien; Li, H

doi:10.1101/020024

Cited by 14 publications

(12 citation statements)

References 14 publications

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“…Concatenated assemblies were cleaned of Illumina artifact low-complexity (long homopolymer) contigs with prinseq-lite v0.20.4 ( 70 ), removing contigs consisting of greater than 80% one nucleotide. Mixed-assembled reads were then mapped back to their original metagenomes using bowtie2 v2.2.6 ( 71 ), and sam files were converted to bam files and sorted using samtools v1.5 ( 72 ) and deduplicated with picard v2.10.3 ( https://github.com/broadinstitute/picard.git ). Mixed-assembly contigs were binned into putative MAGs with metabat2 v2.12.1 ( 73 ), and bins were validated with CheckM ( 74 ), hmmer v3.1b2 ( 75 ), and pplacer v1.1 alpha19 ( 76 ).…”

Section: Methodsmentioning

confidence: 99%

Streamlined and Abundant Bacterioplankton Thrive in Functional Cohorts

et al. 2020

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While fastidious microbes can be abundant and ubiquitous in their natural communities, many fail to grow axenically in laboratories due to auxotrophies or other dependencies. To overcome auxotrophies, these microbes rely on their surrounding cohort. A cohort may consist of kin (ecotypes) or more distantly related organisms (community) with the cooperation being reciprocal or nonreciprocal and expensive (Black Queen hypothesis) or costless (by-product). These metabolic partnerships (whether at single species population or community level) enable dominance by and coexistence of these lineages in nature. Here we examine the relevance of these cooperation models to explain the abundance and ubiquity of the dominant fastidious bacterioplankton of a dimictic mesotrophic freshwater lake. Using both culture-dependent (dilution mixed cultures) and culture-independent (small subunit [SSU] rRNA gene time series and environmental metagenomics) methods, we independently identified the primary cohorts of actinobacterial genera “Candidatus Planktophila” (acI-A) and “Candidatus Nanopelagicus” (acI-B) and the proteobacterial genus “Candidatus Fonsibacter” (LD12). While “Ca. Planktophila” and “Ca. Fonsibacter” had no correlation in their natural habitat, they have the potential to be complementary in laboratory settings. We also investigated the bifunctional catalase-peroxidase enzyme KatG (a common good which “Ca. Planktophila” is dependent upon) and its most likely providers in the lake. Further, we found that while ecotype and community cooperation combined may explain “Ca. Planktophila” population abundance, the success of “Ca. Nanopelagicus” and “Ca. Fonsibacter” is better explained as a community by-product. Ecotype differentiation of “Ca. Fonsibacter” as a means of escaping predation was supported but not for overcoming auxotrophies. IMPORTANCE This study examines evolutionary and ecological relationships of three of the most ubiquitous and abundant freshwater bacterial genera: “Ca. Planktophila” (acI-A), “Ca. Nanopelagicus” (acI-B), and “Ca. Fonsibacter” (LD12). Due to high abundance, these genera might have a significant influence on nutrient cycling in freshwaters worldwide, and this study adds a layer of understanding to how seemingly competing clades of bacteria can coexist by having different cooperation strategies. Our synthesis ties together network and ecological theory with empirical evidence and lays out a framework for how the functioning of populations within complex microbial communities can be studied.

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Section: Methodsmentioning

confidence: 99%

Streamlined and Abundant Bacterioplankton Thrive in Functional Cohorts

et al. 2020

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“…Briefly, the draft genome was first indexed using BWA (version 0.7.15-r1140) [24] and the basecalled reads were aligned to the draft genome using BWA. SAMtools (version 1.6 using htslib 1.6) [25] was then used to sort and index the alignment. Nanopolish then computed the new consensus sequence in 50 kb blocks in parallel, which were then merged into the polished assembly.…”

Section: Long-read Basecalling De Novo Assembly and Genome Polishingmentioning

confidence: 99%

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation

et al. 2020

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Background: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genomes. Subsequent addition of highly accurate short reads to long-read assemblies further improves assembly quality. Using this hybrid approach, we assembled genomes for three Giardia isolates, two with published assemblies and one novel, to evaluate the improvement in genome quality gained from long reads. We then used the long reads to predict structural variants to examine this previously unexplored source of genetic variation in Giardia. Methods: With MinION reads for each isolate, we assembled genomes using several assemblers specializing in long reads. Assembly metrics, gene finding, and whole genome alignments to the reference genomes enabled direct comparison to evaluate the performance of the nanopore reads. Further improvements from adding Illumina reads to the long-read assemblies were evaluated using gene finding. Structural variants were predicted from alignments of the long reads to the best hybrid genome for each isolate and enrichment of key genes was analyzed using random genome sampling and calculation of percentiles to find thresholds of significance. Results: Our hybrid assembly method generated reference quality genomes for each isolate. Consistent with previous findings based on SNPs, examination of heterozygosity using the structural variants found that Giardia BGS was considerably more heterozygous than the other isolates that are from Assemblage A. Further, each isolate was shown to contain structural variant regions enriched for variant-specific surface proteins, a key class of virulence factor in Giardia. Conclusions: The ability to generate reference quality genomes from a single MinION run and a multiplexed MiSeq run enables future large-scale comparative genomic studies within the genus Giardia. Further, prediction of structural variants from long reads allows for more in-depth analyses of major sources of genetic variation within and between Giardia isolates that could have effects on both pathogenicity and host range.

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“…However, lack of a closed reference genome may cause biases in allelic proportions due to mapping errors. Updates to the sequence alignment format (SAM/BAM) to accommodate storing de novo sequence alignments can resolve this issue ( Cock et al, 2015 ). For reference-based SNP discovery, reads can fail to align to regions of high divergence ( Bertels et al, 2014 ) if the short read aligner is too stringent.…”

Section: Introductionmentioning

confidence: 99%

Best practices for evaluating single nucleotide variant calling methods for microbial genomics

et al. 2015

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Innovations in sequencing technologies have allowed biologists to make incredible advances in understanding biological systems. As experience grows, researchers increasingly recognize that analyzing the wealth of data provided by these new sequencing platforms requires careful attention to detail for robust results. Thus far, much of the scientific Communit’s focus for use in bacterial genomics has been on evaluating genome assembly algorithms and rigorously validating assembly program performance. Missing, however, is a focus on critical evaluation of variant callers for these genomes. Variant calling is essential for comparative genomics as it yields insights into nucleotide-level organismal differences. Variant calling is a multistep process with a host of potential error sources that may lead to incorrect variant calls. Identifying and resolving these incorrect calls is critical for bacterial genomics to advance. The goal of this review is to provide guidance on validating algorithms and pipelines used in variant calling for bacterial genomics. First, we will provide an overview of the variant calling procedures and the potential sources of error associated with the methods. We will then identify appropriate datasets for use in evaluating algorithms and describe statistical methods for evaluating algorithm performance. As variant calling moves from basic research to the applied setting, standardized methods for performance evaluation and reporting are required; it is our hope that this review provides the groundwork for the development of these standards.

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SAM/BAM format v1.5 extensions for de novo assemblies

Cited by 14 publications

References 14 publications

Streamlined and Abundant Bacterioplankton Thrive in Functional Cohorts

Streamlined and Abundant Bacterioplankton Thrive in Functional Cohorts

Benchmarking hybrid assemblies of Giardia and prediction of widespread intra-isolate structural variation

Best practices for evaluating single nucleotide variant calling methods for microbial genomics

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