dc.contributor.author | Jervis-Bardy, Jake | |
dc.contributor.author | Leong, Lex EX | |
dc.contributor.author | Marri, Shashikanth | |
dc.contributor.author | Smith, Renee J | |
dc.contributor.author | Choo, Jocelyn M | |
dc.contributor.author | Smith-Vaughan, Heidi C | |
dc.contributor.author | Nosworthy, Elizabeth | |
dc.contributor.author | Morris, Peter S | |
dc.contributor.author | O'Leary, Stephen | |
dc.contributor.author | Rogers, Geraint B | |
dc.contributor.author | Marsh, Robyn L | |
dc.date.accessioned | 2018-01-19T04:25:10Z | |
dc.date.available | 2018-01-19T04:25:10Z | |
dc.date.issued | 2015 | |
dc.identifier.issn | 2049-2618 | |
dc.identifier.doi | 10.1186/s40168-015-0083-8 | |
dc.identifier.uri | http://hdl.handle.net/10072/172148 | |
dc.description.abstract | Background: The rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a
growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal
that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a
workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data
quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric
upper-respiratory samples from several anatomical sites.
Results: A workflow for processing sequence data was developed based on commonly available tools. Data generated
from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial
reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic
units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and
closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as
potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their
objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically
containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and
spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of
taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious
signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between
the sample types.
Conclusions: The application of the presented workflow to a set of challenging clinical samples demonstrates its
utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would
otherwise be highly misleading output. While other approaches could potentially achieve similar improvements,
the methodology employed here represents an accessible means to exclude the signal from contamination and
other artefacts. | |
dc.description.peerreviewed | Yes | |
dc.language | English | |
dc.language.iso | eng | |
dc.publisher | BioMed Central | |
dc.relation.ispartofpagefrom | 19-1 | |
dc.relation.ispartofpageto | 19-11 | |
dc.relation.ispartofjournal | Microbiome | |
dc.relation.ispartofvolume | 3 | |
dc.subject.fieldofresearch | Microbiology not elsewhere classified | |
dc.subject.fieldofresearch | Ecology | |
dc.subject.fieldofresearch | Microbiology | |
dc.subject.fieldofresearch | Medical Microbiology | |
dc.subject.fieldofresearchcode | 060599 | |
dc.subject.fieldofresearchcode | 0602 | |
dc.subject.fieldofresearchcode | 0605 | |
dc.subject.fieldofresearchcode | 1108 | |
dc.title | Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data | |
dc.type | Journal article | |
dc.type.description | C1 - Articles | |
dc.type.code | C - Journal Articles | |
dcterms.license | http://creativecommons.org/licenses/by/4.0 | |
dc.description.version | Published | |
gro.rights.copyright | © 2015 Jervis-Bardy et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. | |
gro.hasfulltext | Full Text | |
gro.griffith.author | Smith-Vaughan, Heidi | |