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

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, nu...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ricardo Ramirez-Gonzalez, Douglas W Yu, Catharine Bruce, Darren Heavens, Mario Caccamo, Brent C Emerson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/dedccae4fccd4142a127607f92a45452
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:dedccae4fccd4142a127607f92a45452
record_format	dspace
spelling	oai:doaj.org-article:dedccae4fccd4142a127607f92a454522021-11-18T07:55:14ZPyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.1932-620310.1371/journal.pone.0057615https://doaj.org/article/dedccae4fccd4142a127607f92a454522013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23469211/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203High-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.Ricardo Ramirez-GonzalezDouglas W YuCatharine BruceDarren HeavensMario CaccamoBrent C EmersonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 3, p e57615 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ricardo Ramirez-Gonzalez Douglas W Yu Catharine Bruce Darren Heavens Mario Caccamo Brent C Emerson PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
description	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.
format	article
author	Ricardo Ramirez-Gonzalez Douglas W Yu Catharine Bruce Darren Heavens Mario Caccamo Brent C Emerson
author_facet	Ricardo Ramirez-Gonzalez Douglas W Yu Catharine Bruce Darren Heavens Mario Caccamo Brent C Emerson
author_sort	Ricardo Ramirez-Gonzalez
title	PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
title_short	PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
title_full	PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
title_fullStr	PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
title_full_unstemmed	PyroClean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
title_sort	pyroclean: denoising pyrosequences from protein-coding amplicons for the recovery of interspecific and intraspecific genetic variation.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/dedccae4fccd4142a127607f92a45452
work_keys_str_mv	AT ricardoramirezgonzalez pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation AT douglaswyu pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation AT catharinebruce pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation AT darrenheavens pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation AT mariocaccamo pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation AT brentcemerson pyrocleandenoisingpyrosequencesfromproteincodingampliconsfortherecoveryofinterspecificandintraspecificgeneticvariation
_version_	1718422762279665664

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

Ejemplares similares