Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

By Marcus J. Claesson, Qiong Wang, Orla O'Sullivan, Rachel Greene-Diniz, James R. Cole, R. Ross, P. O’Toole. 2010

941 citations recorded by the source corpus.

High-throughput molecular technologies can profile microbial communities at high resolution even in complex environments like the intestinal microbiota. Recent improvements in next-generation sequencing technologies allow for even finer resolution. We compared phylogenetic profiling of both longer (454 Titanium) sequence reads with shorter, but more numerous, paired-end reads (Illumina). For both approaches, we targeted six tandem combinations of 16S rRNA gene variable regions, in microbial DNA extracted from a human faecal sample, in order to investigate their limitations and potentials. In silico evaluations predicted that the V3/V4 and V4/V5 regions would provide the highest classification accuracies for both technologies. However, experimental sequencing of the V3/V4 region revealed significant amplification bias compared to the other regions, emphasising the necessity for experimental validation of primer pairs. The latest developments of 454 and Illumina technologies offered higher resolution compared to their previous versions, and showed relative consistency with each other. However, the majority of the Illumina reads could not be classified down to genus level due to their shorter length and higher error rates beyond 60 nt. Nonetheless, with improved quality and longer reads, the far greater coverage of Illumina promises unparalleled insights into highly diverse and complex environments such as the human gut.

Subfields: Food Science, Molecular Biology

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Citations in the corpus

Sensitivity and correlation of hypervariable regions in 16S rRNA genes in phylogenetic analysis (2016, methodology): “Recently, meta-genomic methods provided by next-generation sequencing technology such as Roche 454 [6, 7] and Illumina [8] have facilitated a remarkable expansion of our knowledge regarding uncultured bacteria [7].”
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The Intestinal Microbiome in Early Life: Health and Disease (2014, background): “Although there is still no consensus on this, comparative analysis with full length Sanger sequencing of the 16S rRNA gene supports the use of primers that amplify the V4 region (26), as they yield the most taxonomically informative sequences (27).”
Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing (2015, neutral): “The use of universal PCR primers is based on the hypothesis that the primers used are complementary to conserved regions of genes in the environment, resulting in amplification; in turn, heterogeneity is found inside of the fragments flanked by the primers, in hypervariable regions [15] [16].”
Next‐generation sequencing technologies for environmental DNA research (2012, background): “Another drawback for 454 is its relatively high cost for reagents per megabase sequencing output (Claesson et al. 2010).”
Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics (2014, background): “The latter biases can produce differences in estimated diversity levels depending on the sequencing platform, the discriminatory power of the region of the 16S rRNA molecule targeted [53], and the fact that some taxa such as Planctomycetes, which were seen in higher abundance in the metagenomes (Fig”
Ecology and exploration of the rare biosphere (2015, neutral): “Illumina reads show a decrease in 3 ʹ read quality 175 .”