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Characterization of bacteria in biopsies of colon and stools by high throughput sequencing of the V2 region of bacterial 16S rRNA gene in human.
- Source :
-
PloS one [PLoS One] 2011 Feb 10; Vol. 6 (2), pp. e16952. Date of Electronic Publication: 2011 Feb 10. - Publication Year :
- 2011
-
Abstract
- Background: The characterization of the human intestinal microflora and their interactions with the host have been identified as key components in the study of intestinal disorders such as inflammatory bowel diseases. High-throughput sequencing has enabled culture-independent studies to deeply analyze bacteria in the gut. It is possible with this technology to systematically analyze links between microbes and the genetic constitution of the host, such as DNA polymorphisms and methylation, and gene expression.<br />Methods and Findings: In this study the V2 region of the bacterial 16S ribosomal RNA (rRNA) gene using 454 pyrosequencing from seven anatomic regions of human colon and two types of stool specimens were analyzed. The study examined the number of reads needed to ascertain differences between samples, the effect of DNA extraction procedures and PCR reproducibility, and differences between biopsies and stools in order to design a large scale systematic analysis of gut microbes. It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances. Distances between samples became stable after 1,000 reads. (2) Difference of extracted bacteria was observed between the two DNA extraction methods. In particular, Firmicutes Bacilli were not extracted well by one method. (3) Quantitative and qualitative difference in bacteria from ileum to rectum colon were not observed, but there was a significant positive trend between distances within colon and quantitative differences. Between sample type, biopsies or stools, quantitative and qualitative differences were observed.<br />Conclusions: Results of human colonic bacteria analyzed using high-throughput sequencing were highly dependent on the experimental design, especially the number of sequence reads, DNA extraction method, and sample type.
- Subjects :
- Adult
Bacteria classification
Biopsy
DNA, Bacterial genetics
DNA, Bacterial isolation & purification
Female
Humans
Male
Middle Aged
Organ Specificity
Polymerase Chain Reaction
Reproducibility of Results
Sequence Analysis, RNA
Bacteria genetics
Colon microbiology
Colon pathology
Feces microbiology
High-Throughput Nucleotide Sequencing methods
RNA, Bacterial genetics
RNA, Ribosomal, 16S genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 6
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 21347324
- Full Text :
- https://doi.org/10.1371/journal.pone.0016952