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      Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota

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          Abstract

          Background

          Recent studies have suggested that bacteria associated with the placenta—a “placental microbiome”—may be important in reproductive health and disease. However, a challenge in working with specimens with low bacterial biomass, such as placental samples, is that some or all of the bacterial DNA may derive from contamination in dust or commercial reagents. To investigate this, we compared placental samples from healthy deliveries to a matched set of contamination controls, as well as to oral and vaginal samples from the same women.

          Results

          We quantified total 16S rRNA gene copies using quantitative PCR and found that placental samples and negative controls contained low and indistinguishable copy numbers. Oral and vaginal swab samples, in contrast, showed higher copy numbers. We carried out 16S rRNA gene sequencing and community analysis and found no separation between communities from placental samples and contamination controls, though oral and vaginal samples showed characteristic, distinctive composition. Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed. Authentically present microbiota should yield mostly similar results regardless of the purification method used—this was seen for oral samples, but no placental bacterial lineages were (1) shared between extraction methods, (2) present at >1 % of the total, and (3) present at greater abundance in placental samples than contamination controls.

          Conclusions

          We conclude that for this sample set, using the methods described, we could not distinguish between placental samples and contamination introduced during DNA purification.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s40168-016-0172-3) contains supplementary material, which is available to authorized users.

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          Most cited references21

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          Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.

          The ongoing revolution in high-throughput sequencing continues to democratize the ability of small groups of investigators to map the microbial component of the biosphere. In particular, the coevolution of new sequencing platforms and new software tools allows data acquisition and analysis on an unprecedented scale. Here we report the next stage in this coevolutionary arms race, using the Illumina GAIIx platform to sequence a diverse array of 25 environmental samples and three known "mock communities" at a depth averaging 3.1 million reads per sample. We demonstrate excellent consistency in taxonomic recovery and recapture diversity patterns that were previously reported on the basis of metaanalysis of many studies from the literature (notably, the saline/nonsaline split in environmental samples and the split between host-associated and free-living communities). We also demonstrate that 2,000 Illumina single-end reads are sufficient to recapture the same relationships among samples that we observe with the full dataset. The results thus open up the possibility of conducting large-scale studies analyzing thousands of samples simultaneously to survey microbial communities at an unprecedented spatial and temporal resolution.
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            R: A Language and environmental for statistical computing

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              Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.

              We constructed error-correcting DNA barcodes that allow one run of a massively parallel pyrosequencer to process up to 1,544 samples simultaneously. Using these barcodes we processed bacterial 16S rRNA gene sequences representing microbial communities in 286 environmental samples, corrected 92% of sample assignment errors, and thus characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.
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                Author and article information

                Contributors
                parry@mail.med.upenn.edu
                bushman@mail.med.upenn.edu
                Journal
                Microbiome
                Microbiome
                Microbiome
                BioMed Central (London )
                2049-2618
                23 June 2016
                23 June 2016
                2016
                : 4
                : 29
                Affiliations
                [ ]Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, 3610 Hamilton Walk, Philadelphia, PA 19104-6076 USA
                [ ]Department of Obstetrics and Gynecology, Maternal and Child Health Research Program, Perelman School of Medicine at the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104 USA
                Article
                172
                10.1186/s40168-016-0172-3
                4917942
                27338728
                d78264a2-122d-4f3f-897d-45f7d99d1d31
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.

                History
                : 22 April 2016
                : 30 May 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: T32 AI007632
                Award ID: P30 AI045008
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/http://dx.doi.org/10.13039/100000912, March of Dimes Foundation;
                Award ID: March of Dimes Prematurity Research Center at the University of Pennsylvania
                Categories
                Research
                Custom metadata
                © The Author(s) 2016

                placenta,microbiome,16s rrna gene,low biomass samples,reagent contamination

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