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BMC Microbiology

, 17:23

First Online: 21 January 2017Received: 02 July 2016Accepted: 09 January 2017DOI: 10.1186-s12866-017-0927-4

Cite this article as: Castelino, M., Eyre, S., Moat, J. et al. BMC Microbiol 2017 17: 23. doi:10.1186-s12866-017-0927-4

Abstract

BackgroundThe composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA ribosomal ribonucleic acid gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols



MethodsDNA was obtained from two low density mock communities of 11 diverse bacterial strains with and without human DNA supplementation and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R V1-V3; and 347F and 803R V3-V4. The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME.

ResultsThere was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms.

ConclusionsThe results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.

KeywordsSkin microbiome Bacterial microbiome Low biomass 16s rRNA gene Miseq platform Next generation sequencing Methods Optimisation Primer selection Abbreviations16S rRNA gene16S ribosomal ribonucleic acid – A component of the 30S small subunit of prokaryotic ribosomes

bpBase pair

DNADeoxyribonucleic acid

GbGigabyte

HVHealthy volunteer

MbMegabyte

NCTCNational collection of type cultures

QQuality score eg: Q30 is Phred Score 30, 99.9% base call accuracy. I in 1000 probability of incorrect base call

V1-V3Hypervariable region 1 through to 3 on the 16S rRNA gene

V3-V4Hypervariable region 3 through to 4 on the 16S rRNA gene

V4-V5Hypervariable region 4 through to 5 on the 16S rRNA gene

Electronic supplementary materialThe online version of this article doi:10.1186-s12866-017-0927-4 contains supplementary material, which is available to authorized users.

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Autor: Madhura Castelino - Stephen Eyre - John Moat - Graeme Fox - Paul Martin - Pauline Ho - Mathew Upton - Anne Barton

Fuente: https://link.springer.com/







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