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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

UniFrac distances between sequencing methods.(A) Weighted and (B) unweighted UniFrac distances. Reads by 454 Standard chemistry were compared with technical replicates of the same DNAs sequenced by 454 Standard chemistry (Standard), with 454 Titanium chemistry (no trim), and with 454 Titanium chemistry with sequences trimmed at 270 bp (trim). ** p<0.01, **** p<0.0001
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pone-0016952-g003: UniFrac distances between sequencing methods.(A) Weighted and (B) unweighted UniFrac distances. Reads by 454 Standard chemistry were compared with technical replicates of the same DNAs sequenced by 454 Standard chemistry (Standard), with 454 Titanium chemistry (no trim), and with 454 Titanium chemistry with sequences trimmed at 270 bp (trim). ** p<0.01, **** p<0.0001

Mentions: Figure 3 shows both UniFrac distances comparing reads by 454 Standard chemistry versus those of the same DNAs by 454 Standard chemistry (technical replicates) and by 454 Titanium chemistry. Significant difference (t10 = 6.45, p = 7.33×10−5) in weighted UniFrac distances was observed between both chemistries. Since this difference was likely due to difference in length of reads (mean ± SD, Standard: 269.4±9.8 bp, Titanium: 329.2±11.8 bp), the Titanium reads were trimmed to 270 bp in order to correct for this effect. Difference in weighted UniFrac distance became not significant (t10 = 1.38, p = 0.199). Unweighted UniFrac distances were not different between the three sequence comparisons (Figure 3B).


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)

UniFrac distances between sequencing methods.(A) Weighted and (B) unweighted UniFrac distances. Reads by 454 Standard chemistry were compared with technical replicates of the same DNAs sequenced by 454 Standard chemistry (Standard), with 454 Titanium chemistry (no trim), and with 454 Titanium chemistry with sequences trimmed at 270 bp (trim). ** p<0.01, **** p<0.0001
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037395&req=5

pone-0016952-g003: UniFrac distances between sequencing methods.(A) Weighted and (B) unweighted UniFrac distances. Reads by 454 Standard chemistry were compared with technical replicates of the same DNAs sequenced by 454 Standard chemistry (Standard), with 454 Titanium chemistry (no trim), and with 454 Titanium chemistry with sequences trimmed at 270 bp (trim). ** p<0.01, **** p<0.0001
Mentions: Figure 3 shows both UniFrac distances comparing reads by 454 Standard chemistry versus those of the same DNAs by 454 Standard chemistry (technical replicates) and by 454 Titanium chemistry. Significant difference (t10 = 6.45, p = 7.33×10−5) in weighted UniFrac distances was observed between both chemistries. Since this difference was likely due to difference in length of reads (mean ± SD, Standard: 269.4±9.8 bp, Titanium: 329.2±11.8 bp), the Titanium reads were trimmed to 270 bp in order to correct for this effect. Difference in weighted UniFrac distance became not significant (t10 = 1.38, p = 0.199). Unweighted UniFrac distances were not different between the three sequence comparisons (Figure 3B).

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