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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.

Momozawa Y, Deffontaine V, Louis E, Medrano JF - PLoS ONE (2011)

Bottom Line: It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances.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.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.

View Article: PubMed Central - PubMed

Affiliation: Unit of Animal Genomics, GIGA-Research and Faculty of Veterinary Medicine, University of Liège, Liège, Belgium. Yukihide.Momozawa@guest.ulg.ac.be

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.

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.

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.

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Related in: MedlinePlus

Effect of different experimental steps measured by UniFrac distances.(A) Weighted and (B) unweighted UniFrac distances. ‘Between extractions’ was used as a base measurement for comparison with other distances. Between PCRs: distances between PCRs using the same DNAs (n = 8). Between extractions: distances between extractions of DNAs from two biopsy specimens from the same anatomic region or the same stool specimens by the Stool kit (n = 18). Between extraction kits: distances between extractions of DNAs from two biopsy specimens from the same anatomic region by the Stool kit and the Mini kit (n = 12). Between individuals: distances between 2 of 9 individuals from biopsies from transverse colon. Mean ± SD, * p<0.05, ****: p<0.0001
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pone-0016952-g005: Effect of different experimental steps measured by UniFrac distances.(A) Weighted and (B) unweighted UniFrac distances. ‘Between extractions’ was used as a base measurement for comparison with other distances. Between PCRs: distances between PCRs using the same DNAs (n = 8). Between extractions: distances between extractions of DNAs from two biopsy specimens from the same anatomic region or the same stool specimens by the Stool kit (n = 18). Between extraction kits: distances between extractions of DNAs from two biopsy specimens from the same anatomic region by the Stool kit and the Mini kit (n = 12). Between individuals: distances between 2 of 9 individuals from biopsies from transverse colon. Mean ± SD, * p<0.05, ****: p<0.0001

Mentions: Figure 5 shows both UniFrac distances between different experimental procedures. We used ‘Between extractions’ as a base measurement for comparison with other procedure steps. ‘Between extractions’ was obtained from two different specimens from the same anatomic region or the same stool specimens. Comparing the distance measurement of “Between extractions” with those of “Between extraction kits” and “Between individuals” allowed us to estimate the magnitude of the effects of ‘Between extraction kits’ and ‘Between individuals’. In all of the comparisons, differences between individuals were much larger quantitatively (t52 = 8.13, p = 7.94×10−11) and qualitatively (t52 = 9.26, p = 1.39×10−12) than any other effects. Even if we eliminated an ulcerative colitis and a Crohn’s disease patient, both UniFrac distances were not changed (data not shown). We also observed other significant differences in weighted UniFrac distance at “Between PCRs” (t24 = 2.72, p = 0.012) and at “Between extraction kits” (t28 = 2.18, p = 0.038).


Characterization of bacteria in biopsies of colon and stools by high throughput sequencing of the V2 region of bacterial 16S rRNA gene in human.

Momozawa Y, Deffontaine V, Louis E, Medrano JF - PLoS ONE (2011)

Effect of different experimental steps measured by UniFrac distances.(A) Weighted and (B) unweighted UniFrac distances. ‘Between extractions’ was used as a base measurement for comparison with other distances. Between PCRs: distances between PCRs using the same DNAs (n = 8). Between extractions: distances between extractions of DNAs from two biopsy specimens from the same anatomic region or the same stool specimens by the Stool kit (n = 18). Between extraction kits: distances between extractions of DNAs from two biopsy specimens from the same anatomic region by the Stool kit and the Mini kit (n = 12). Between individuals: distances between 2 of 9 individuals from biopsies from transverse colon. Mean ± SD, * p<0.05, ****: p<0.0001
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037395&req=5

pone-0016952-g005: Effect of different experimental steps measured by UniFrac distances.(A) Weighted and (B) unweighted UniFrac distances. ‘Between extractions’ was used as a base measurement for comparison with other distances. Between PCRs: distances between PCRs using the same DNAs (n = 8). Between extractions: distances between extractions of DNAs from two biopsy specimens from the same anatomic region or the same stool specimens by the Stool kit (n = 18). Between extraction kits: distances between extractions of DNAs from two biopsy specimens from the same anatomic region by the Stool kit and the Mini kit (n = 12). Between individuals: distances between 2 of 9 individuals from biopsies from transverse colon. Mean ± SD, * p<0.05, ****: p<0.0001
Mentions: Figure 5 shows both UniFrac distances between different experimental procedures. We used ‘Between extractions’ as a base measurement for comparison with other procedure steps. ‘Between extractions’ was obtained from two different specimens from the same anatomic region or the same stool specimens. Comparing the distance measurement of “Between extractions” with those of “Between extraction kits” and “Between individuals” allowed us to estimate the magnitude of the effects of ‘Between extraction kits’ and ‘Between individuals’. In all of the comparisons, differences between individuals were much larger quantitatively (t52 = 8.13, p = 7.94×10−11) and qualitatively (t52 = 9.26, p = 1.39×10−12) than any other effects. Even if we eliminated an ulcerative colitis and a Crohn’s disease patient, both UniFrac distances were not changed (data not shown). We also observed other significant differences in weighted UniFrac distance at “Between PCRs” (t24 = 2.72, p = 0.012) and at “Between extraction kits” (t28 = 2.18, p = 0.038).

Bottom Line: It was shown (1) that sequence coverage lower than 1,000 reads influenced quantitative and qualitative differences between samples measured by UniFrac distances.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.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.

View Article: PubMed Central - PubMed

Affiliation: Unit of Animal Genomics, GIGA-Research and Faculty of Veterinary Medicine, University of Liège, Liège, Belgium. Yukihide.Momozawa@guest.ulg.ac.be

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.

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.

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.

Show MeSH
Related in: MedlinePlus