Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.

Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of c...

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Autores principales: Stephen J Salipante, Dhruba J Sengupta, Christopher Rosenthal, Gina Costa, Jessica Spangler, Elizabeth H Sims, Michael A Jacobs, Samuel I Miller, Daniel R Hoogestraat, Brad T Cookson, Connor McCoy, Frederick A Matsen, Jay Shendure, Clarence C Lee, Timothy T Harkins, Noah G Hoffman
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Publicado: Public Library of Science (PLoS) 2013
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spelling oai:doaj.org-article:295dd7c1af2b470e95163724ac3d7e512021-11-18T07:43:53ZRapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.1932-620310.1371/journal.pone.0065226https://doaj.org/article/295dd7c1af2b470e95163724ac3d7e512013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23734239/?tool=EBIhttps://doaj.org/toc/1932-6203Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.Stephen J SalipanteDhruba J SenguptaChristopher RosenthalGina CostaJessica SpanglerElizabeth H SimsMichael A JacobsSamuel I MillerDaniel R HoogestraatBrad T CooksonConnor McCoyFrederick A MatsenJay ShendureClarence C LeeTimothy T HarkinsNoah G HoffmanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e65226 (2013)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Stephen J Salipante
Dhruba J Sengupta
Christopher Rosenthal
Gina Costa
Jessica Spangler
Elizabeth H Sims
Michael A Jacobs
Samuel I Miller
Daniel R Hoogestraat
Brad T Cookson
Connor McCoy
Frederick A Matsen
Jay Shendure
Clarence C Lee
Timothy T Harkins
Noah G Hoffman
Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
description Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use.
format article
author Stephen J Salipante
Dhruba J Sengupta
Christopher Rosenthal
Gina Costa
Jessica Spangler
Elizabeth H Sims
Michael A Jacobs
Samuel I Miller
Daniel R Hoogestraat
Brad T Cookson
Connor McCoy
Frederick A Matsen
Jay Shendure
Clarence C Lee
Timothy T Harkins
Noah G Hoffman
author_facet Stephen J Salipante
Dhruba J Sengupta
Christopher Rosenthal
Gina Costa
Jessica Spangler
Elizabeth H Sims
Michael A Jacobs
Samuel I Miller
Daniel R Hoogestraat
Brad T Cookson
Connor McCoy
Frederick A Matsen
Jay Shendure
Clarence C Lee
Timothy T Harkins
Noah G Hoffman
author_sort Stephen J Salipante
title Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
title_short Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
title_full Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
title_fullStr Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
title_full_unstemmed Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
title_sort rapid 16s rrna next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.
publisher Public Library of Science (PLoS)
publishDate 2013
url https://doaj.org/article/295dd7c1af2b470e95163724ac3d7e51
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