Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine

Abstract

BackgroundVariations in the composition of the human intestinal microbiota are linked to diverse health conditions. High-throughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions.Methods and FindingsOver 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings.ConclusionsThe RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genus-level with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.

454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

“…Although it has been reported that the V4 region used in this study displayed the highest number of correctly classified sequences, followed by the V3 and V6 regions (Claesson et al, 2009), quite a large portion of effective bacterial sequences in this study could not be assigned to any taxa of different level at the 50% threshold, indicating the extent of novel sequences captured by this study. Supplementary Figure S1 shows that the unclassified sequence portions in the total community increased from the domain level to the genus level, and were significantly different among the samples, especially at the family and genus levels.…”

“…They have also been used in other microbial diversity studies employing the PCR-based pyrosequencing method (Claesson et al, 2009;Jesus et al, 2010;Murphy et al, 2010). The 5 0 -fused primer includes a 10 nucleotide 'barcode' inserted between the Roche 454 life Science (Branford, CT, USA) adapter primer A and the 563F primer.…”

“…All the raw reads were treated with the Pyrosequencing Pipeline Initial Process (Cole et al, 2009) of the Ribosomal Database Project (RDP), (1) to sort those exactly matching the specific barcodes into different samples, (2) to trim off the adapters, barcodes and primers using the default parameters, and (3) to remove sequences containing ambiguous 'N' or shorter than 150 bps (Claesson et al, 2009). The reads selected above were defined as 'raw reads' for each AS sample.…”

“…High-throughput sequencing is a promising method, as it provides enough sequencing depth to cover the complex microbial communities (Shendure and Ji, 2008). So far, it has been applied to analyze microbial communities in marine water (Qian et al, 2010), soil (Roesch et al, 2007;Lauber et al, 2009), human hand surface (Fierer et al, 2008), human distal intestine (Claesson et al, 2009), etc. But few studies have been conducted using this method to investigate AS, though this approach has been used to study the microbial community in the raw sewage (McLellan et al, 2010) and the residual biosolids after digestion of AS (Bibby et al, 2010).…”

The role of gut microbiota in the gut-brain axis: current challenges and perspectives

“…Microarrays represent an excellent choice for the high-throughput analysis of bacterial populations, because many different probes can be placed on one slide or chip, and samples thus can be tested for the presence of many different species simultaneously samples can be interrogated directly, circumventing any need for culturing, and thus non-culturable species can be reliably detected. It's been demonstrated that microarray has a power equal to new generation sequencing (Claesson et al, 2009). Several types of microarrays have been used to identify the gut microorganisms, like community genome array, functional genome array and phylogenetic oligonucleotide array (Loy et Wu et al, 2004;He et al, 2007).…”