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

By Marcus J. Claesson, Orla O'Sullivan, Qiong Wang, J. Nikkilä, Julian R. Marchesi, H. Smidt, Willem M. de Vos, R. P. Ross, P. O’Toole. PLoS ONE, 2009

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Background Variations 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 Findings Over 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. Conclusions The 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.

Subfields: Food Science, Infectious Diseases, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2619e2fbc5b818677d55a0f35f6229d54c8dfbc2

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Citations in the corpus

Microbes inside—from diversity to function: the case of Akkermansia (2012, background): “In recent years, next generation sequencing has been heavily implemented in the studies of the vertebrate intestinal microbial diversity (Dethlefsen et al., 2008; Claesson et al., 2009; Turnbaugh et al., 2009; Caporaso et al., 2010).”
Soil microbial community under a nurse-plant species changes in composition, biomass and activity as the nurse grows (2013, methodology): “Short reads in V1eV3 regions have been shown to be sufficient to characterize microbial community accurately, especially when using a confidence threshold of 50% in taxonomic assignation (Liu et al., 2007; Claesson et al., 2009; Jeraldo et al., 2011).”
454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants (2011, methodology): “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 se”
Gut microbiome of the Hadza hunter-gatherers (2014, methodology): “Thermal cycling consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 50 s, annealing at 40 °C for 30 s, and extension at 72 °C for 60 s, with a final extension step at 72 °C for 5 min (Claesson et al., 2009).”
Fat, Fiber and Cancer Risk in African Americans and Rural Africans (2015, neutral): “59 and at considerably lower cost.”
Continuous application of different organic additives can suppress tomato disease by inducing the healthy rhizospheric microbiota through alterations to the bulk soil microflora (2018, methodology): “2009) and 802R (5′-TACNVGGGTATCTAATCC3′) (Ahmed et al. 2009), and ITS1F (5′-CTTG GTCATTTAGAGGAAGTAA-3 ′) (Gardes and Bruns 1993) and ITS2 (5′-GCTGCGTTCTTCAT CGATGC-3′) (White et al.”
454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. (2013, methodology): “The RDP Classifier was used to assign 16S rRNA gene sequences to a taxonomical hierarchy with a confidence threshold of 50%, because the DNA sequences were < 250 bp (Claesson et al., 2009).”
Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs (2013, methodology): “However, the same bootstrap value obtained by the naı̈ve Bayesian classifier may result in different precision and coverage for different taxonomic ranks, as well as for different regions [16,19].”
Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions (2010, neutral): “these amplicons and HITChip hybridisations of full-length 16S rRNA from our previous study (5).”
Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects (2010, methodology): “Quantification of bacterial taxa in human fecal samples by pyrosequencing of 16S rRNA tags has been validated and it showed a high correlation with other molecular methods such as qRT-PCR and phylogenetic microarray [30,31].”