Dataset Information

PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.

ABSTRACT: High-throughput parallel sequencing is a powerful tool for the quantification of microbial diversity through the amplification of nuclear ribosomal gene regions. Recent work has extended this approach to the quantification of diversity within otherwise difficult-to-study metazoan groups. However, nuclear ribosomal genes present both analytical challenges and practical limitations that are a consequence of the mutational properties of nuclear ribosomal genes. Here we exploit useful properties of protein-coding genes for cross-species amplification and denoising of 454 flowgrams. We first use experimental mixtures of species from the class Collembola to amplify and pyrosequence the 5' region of the COI barcode, and we implement a new algorithm called PyroClean for the denoising of Roche GS FLX pyrosequences. Using parameter values from the analysis of experimental mixtures, we then analyse two communities sampled from field sites on the island of Tenerife. Cross-species amplification success of target mitochondrial sequences in experimental species mixtures is high; however, there is little relationship between template DNA concentrations and pyrosequencing read abundance. Homopolymer error correction and filtering against a consensus reference sequence reduced the volume of unique sequences to approximately 5% of the original unique raw reads. Filtering of remaining non-target sequences attributed to PCR error, sequencing error, or numts further reduced unique sequence volume to 0.8% of the original raw reads. PyroClean reduces or eliminates the need for an additional, time-consuming step to cluster reads into Operational Taxonomic Units, which facilitates the detection of intraspecific DNA sequence variation. PyroCleaned sequence data from field sites in Tenerife demonstrate the utility of our approach for quantifying evolutionary diversity and its spatial structure. Comparison of our sequence data to public databases reveals that we are able to successfully recover both interspecific and intraspecific sequence diversity.

SUBMITTER: Ramirez-Gonzalez R

PROVIDER: S-EPMC3585932 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.

Ramirez-Gonzalez Ricardo R Yu Douglas W DW Bruce Catharine C Heavens Darren D Caccamo Mario M Emerson Brent C BC

PloS one 20130301 3

High-throughput parallel sequencing is a powerful tool for the quantification of microbial diversity through the amplification of nuclear ribosomal gene regions. Recent work has extended this approach to the quantification of diversity within otherwise difficult-to-study metazoan groups. However, nuclear ribosomal genes present both analytical challenges and practical limitations that are a consequence of the mutational properties of nuclear ribosomal genes. Here we exploit useful properties of ...[more]

PMID: 23469211

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Project description:The worldwide plant economic spectrum hypothesis predicts that leaf, stem, and root traits are correlated across vascular plant species because carbon gain depends on leaves being adequately supplied with water and nutrients, and because construction of each organ involves a trade-off between performance and persistence. Despite its logical and intuitive appeal, this hypothesis has received mixed empirical support. If traits within species diverge in their responses to an environmental gradient, then interspecific trait correlations could be weakened when measured in natural ecosystems. To test this prediction, we measured relative growth rates (RGR) and seven functional traits that have been shown to be related to fluxes of water, nutrients, and carbon across 56 functionally diverse tree species on (1) juveniles in a controlled environment, (2) juveniles in forest understories, and (3) mature trees in forests. Leaf, stem, and fine root traits of juveniles grown in a controlled environment were closely correlated with each other, and with RGR. Remarkably, the seven leaf, stem, and fine root tissue traits spanned a single dimension of variation when measured in the controlled environment. Forest-grown juveniles expressed lower leaf mass per area, but higher wood and fine root tissue density, than greenhouse-grown juveniles. Traits and growth rates were decoupled in forest-grown juveniles and mature trees. Our results indicate that constraints exist on the covariation, not just the variation, among vegetative plant organs; however, divergent responses of traits within species to environmental gradients can mask interspecific trait correlations in natural environments. Correlations among organs and relationships between traits and RGR were strong when plants were compared in a standardized environment. Our results may reconcile the discrepancies seen among studies, by showing that if traits and growth rates of species are compared across varied environments, then the interorgan trait correlations observed in controlled conditions can weaken or disappear.

Dataset Information

PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.

Publications

PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.

Similar Datasets

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