Non-specific amplification of human DNA is a major challenge for 16S rRNA gene sequence analysis.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
01 10 2020
01 10 2020
Historique:
received:
21
04
2020
accepted:
16
09
2020
entrez:
2
10
2020
pubmed:
3
10
2020
medline:
2
1
2021
Statut:
epublish
Résumé
The targeted sequencing of the 16S rRNA gene is one of the most frequently employed techniques in the field of microbial ecology, with the bacterial communities of a wide variety of niches in the human body have been characterised in this way. This is performed by targeting one or more hypervariable (V) regions within the 16S rRNA gene in order to produce an amplicon suitable in size for next generation sequencing. To date, all technical research has focused on the ability of different V regions to accurately resolve the composition of bacterial communities. We present here an underreported artefact associated with 16S rRNA gene sequencing, namely the off-target amplification of human DNA. By analysing 16S rRNA gene sequencing data from a selection of human sites we highlighted samples susceptible to this off-target amplification when using the popular primer pair targeting the V3-V4 region of the gene. The most severely affected sample type identified (breast tumour samples) were then re-analysed using the V1-V2 primer set, showing considerable reduction in off target amplification. Our data indicate that human biopsy samples should preferably be amplified using primers targeting the V1-V2 region. It is shown here that these primers result in on average 80% less human genome aligning reads, allowing for more statistically significant analysis of the bacterial communities residing in these samples.
Identifiants
pubmed: 33004967
doi: 10.1038/s41598-020-73403-7
pii: 10.1038/s41598-020-73403-7
pmc: PMC7529756
doi:
Substances chimiques
DNA, Bacterial
0
RNA, Ribosomal, 16S
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
16356Subventions
Organisme : Science Foundation Ireland
ID : 12/RC/2273
Pays : Ireland
Organisme : Science Foundation Ireland
ID : 15/CDA/3630
Pays : Ireland
Organisme : Health Research Board
ID : MRCG2016-25
Pays : Ireland
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