Abstract
BackgroundThe 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 FindingsIn 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.ConclusionsResults 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.
Highlights
High-throughput sequencing has significantly accelerated the characterization of human bacteria in several phenotypes and diseases by metagenomic [1] and 16S ribosomal RNA (rRNA)-based techniques [2]
(2) Difference of extracted bacteria was observed between the two DNA extraction methods
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
Summary
High-throughput sequencing has significantly accelerated the characterization of human bacteria in several phenotypes and diseases by metagenomic [1] and 16S rRNA-based techniques [2]. Obesity was associated with reducing bacterial diversity and altering representation of bacterial genes and metabolic pathways [4]. While these reports provided new insights, a wide range of approaches will be needed to reveal the role of specific bacteria in relation to phenotypes and diseases [2]. One approach is a systematic analysis using high-throughput methods to examine links between bacteria and host genetic factors, such as DNA polymorphisms, DNA methylation, and gene expression. 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
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