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. 2011 Mar;77(6):2071-80.
doi: 10.1128/AEM.02477-10. Epub 2011 Jan 21.

Microarray analysis and barcoded pyrosequencing provide consistent microbial profiles depending on the source of human intestinal samples

Bartholomeus van den Bogert  1 , Willem M de Vos, Erwin G Zoetendal, Michiel Kleerebezem
Affiliations

Microarray analysis and barcoded pyrosequencing provide consistent microbial profiles depending on the source of human intestinal samples

Bartholomeus van den Bogert et al. Appl Environ Microbiol. 2011 Mar.

Abstract

Large-scale and in-depth characterization of the intestinal microbiota necessitates application of high-throughput 16S rRNA gene-based technologies, such as barcoded pyrosequencing and phylogenetic microarray analysis. In this study, the two techniques were compared and contrasted for analysis of the bacterial composition in three fecal and three small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, different forward primers were used for amplification to assess their impact on microbial profiling results. An average of 7,944 pyrosequences, spanning the V1 and V2 region of 16S rRNA genes, was obtained per sample. Although primer choice in barcoded pyrosequencing did not affect species richness and diversity estimates, detection of Actinobacteria strongly depended on the selected primer. Microbial profiles obtained by pyrosequencing and phylogenetic microarray analysis (HITChip) correlated strongly for fecal and ileal lumen samples but were less concordant for ileostomy effluent. Quantitative PCR was employed to investigate the deviations in profiling between pyrosequencing and HITChip analysis. Since cloning and sequencing of random 16S rRNA genes from ileostomy effluent confirmed the presence of novel intestinal phylotypes detected by pyrosequencing, especially those belonging to the Veillonella group, the divergence between pyrosequencing and the HITChip is likely due to the relatively low number of available 16S rRNA gene sequences of small intestinal origin in the DNA databases that were used for HITChip probe design. Overall, this study demonstrated that equivalent biological conclusions are obtained by high-throughput profiling of microbial communities, independent of technology or primer choice.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of the experimental setup for characterization of the microbial composition in fecal samples, ileal content, and ileostomy effluent using molecular approaches. The asterisks indicate that forward primers and the reverse primer used for pyrosequencing were appended with adaptor A and adaptor B, respectively.
FIG. 2.
FIG. 2.
Relative contributions of detected bacterial phyla with pyrosequencing using four different forward primers and HITChip analysis for community data at level 1. Pearson product-moment correlation coefficients (r) between pairs of profiles are shown above the bars. The phylum Firmicutes was subdivided into Bacilli, Clostridium clusters, uncultured Mollicutes, and uncultured Clostridiales. Pyrosequences that could not be classified above the confidence threshold of 80% are grouped to Unclassified_Human unique OTU, which is indicated in the microbial profiles with shadowing (black bars). Phylogenetic groups that contribute at least 1% to one of the profiles are indicated in the color key.
FIG. 3.
FIG. 3.
Comparison of the relative contributions as determined by means of HITChip, qPCR assays, and pyrosequencing for 4 phylogenetic groups in fecal (F) and small intestinal (S) samples. Relative contributions as assessed by qPCR assays were not determined for sample F3.
FIG. 4.
FIG. 4.
Total numbers of cloned sequences detected per phylogenetic group (black) and numbers of sequences showing <98% identity to the 16S rRNA sequences represented in the human unique OTU database (gray) for ileostomy samples S1 and S2. The ratio of number of sequences showing <98% identity to the 16S rRNA sequences represented in the human unique OTU database to the total number of cloned sequences is provided for each level 2 group. Sequences that could not be classified above the confidence threshold of 80% are grouped to Unclassified_ at the specific rank per taxon.
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