NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes

Abstract

Background: Massive high-throughput sequencing of short, hypervariable segments of the 16S ribosomal RNA (rRNA) gene has transformed the methodological landscape describing microbial diversity within and across complex biomes. However, several studies have shown that the methodology rather than the biological variation is responsible for the observed sample composition and distribution. This compromises meta-analyses, although this fact is often disregarded. Results: To facilitate true meta-analysis of microbiome studies, we developed NG-Tax, a pipeline for 16S rRNA gene amplicon sequence analysis that was validated with different mock communities and benchmarked against QIIME as a frequently used pipeline. The microbial composition of 49 independently amplified mock samples was characterized by sequencing two variable 16S rRNA gene regions, V4 and V5-V6, in three separate sequencing runs on Illumina’s HiSeq2000 platform. This allowed for the evaluation of important causes of technical bias in taxonomic classification: 1) run-to-run sequencing variation, 2) PCR–error, and 3) region/primer specific amplification bias. Despite the short read length (~140 nt) and all technical biases, the average specificity of the taxonomic assignment for the phylotypes included in the mock communities was 97.78%. On average 99.95% and 88.43% of the reads could be assigned to at least family or genus level, respectively, while assignment to ‘spurious genera’ represented on average only 0.21% of the reads per sample. Analysis of α- and β-diversity confirmed conclusions guided by biology rather than the aforementioned methodological aspects, which was not achieved with QIIME. Conclusions: Different biological outcomes are commonly observed due to 16S rRNA region-specific performance. NG-Tax demonstrated high robustness against choice of region and other technical biases associated with 16S rRNA gene amplicon sequencing studies, diminishing their impact and providing accurate qualitative and quantitative representation of the true sample composition. This will improve comparability between studies and facilitate efforts towards standardization.

Exploring groundwater microbial communities for natural attenuation potential of micropollutants

“…OTU-classification was performed using the NG-Tax pipeline (30). A large portion (7-55%) of OTUs could not be classified even at phylum level (Figure 3a).…”

“…Sequence analysis of the raw data was performed in NG-Tax using default settings (30). In short: paired-end libraries were demultiplexed using read pairs with perfectly matching barcodes.…”

Organohalide-respiringDesulfolunaspecies isolated from marine environments

“…A 2-step PCR strategy was applied to generate barcoded amplicons from the V1—V2 region of bacterial 16S rRNA genes as described previously [36]. Sequence analysis was performed using NG-Tax [37]. Operational taxonomic units (OTUs) were assigned taxonomy using uclust [38] in an open reference approach against the SILVA 16S rRNA gene reference database (LTPs128_SSU) [39].…”

Fifty Percent Human – how art brings us in touch with our microbial cohabitants

“…', a biomolecular analysis of microbial community composition was performed. PCR-amplified fragments of microbial 16S ribosomal RNA (rRNA) genes of all three samples were analysed using MiSeq Illumina sequencing to explore the archaeal and bacterial content of the samples (Ramiro-Garcia et al, 2016). Similarity of the observed microbial communities was plotted using principal component analysis (PCA) (Fig.…”