Dataset Information

Stacks: building and genotyping Loci de novo from short-read sequences.

ABSTRACT: Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks can recover thousands of single nucleotide polymorphism (SNP) markers useful for the genetic analysis of crosses or populations. Stacks can generate markers for ultra-dense genetic linkage maps, facilitate the examination of population phylogeography, and help in reference genome assembly. We report here the algorithms implemented in Stacks and demonstrate their efficacy by constructing loci from simulated RAD-tags taken from the stickleback reference genome and by recapitulating and improving a genetic map of the zebrafish, Danio rerio.

SUBMITTER: Catchen JM

PROVIDER: S-EPMC3276136 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Stacks: building and genotyping Loci de novo from short-read sequences.

Catchen Julian M JM Amores Angel A Hohenlohe Paul P Cresko William W Postlethwait John H JH

G3 (Bethesda, Md.) 20110801 3

Advances in sequencing technology provide special opportunities for genotyping individuals with speed and thrift, but the lack of software to automate the calling of tens of thousands of genotypes over hundreds of individuals has hindered progress. Stacks is a software system that uses short-read sequence data to identify and genotype loci in a set of individuals either de novo or by comparison to a reference genome. From reduced representation Illumina sequence data, such as RAD-tags, Stacks ca ...[more]

PMID: 22384329

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Project description:BACKGROUND: RNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy. RESULTS: We found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9-47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92-2.21%, and on average 1.8-3.1 transcripts per reference gene assembled. CONCLUSIONS: With rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.

Dataset Information

Stacks: building and genotyping Loci de novo from short-read sequences.

Publications

Stacks: building and genotyping Loci de novo from short-read sequences.

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