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

Abstract

BackgroundMassive 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 true meta-analyses, although this fact is often disregarded. ResultsTo 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 as the QIIME currently most 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 evaluating important factors 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 96%. On average 99.94% and 92.02% of the reads could be assigned to at least family or genus level, respectively, while assignment to 'spurious genera' represented on average only 0.02% 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 the case when samples were analysed using QIIME. 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. Conclusions

Diurnal Dynamics of Gaseous and Dissolved Metabolites and Microbiota Composition in the Bovine Rumen

“…PCR products were then purified using HighPrep™ (MagBio Europe Ltd, Kent, United Kingdom) and quantified using a Qubit in combination with the dsDNA BR Assay Kit (Invitrogen). Purified PCR products were mixed in equimolar amounts into pools together with defined synthetic mock communities which allow assessment of potential technical biases (Ramiro-Garcia et al, 2016). Pools then underwent adaptor ligation followed by sequencing on the HiSeq platform with addition of 20% PhiX (GATC-Biotech, Konstanz, Germany).…”

“…The 16S rRNA cDNA gene sequencing data was then analyzed using NG-Tax, an in-house pipeline (Ramiro-Garcia et al, 2016). Paired-end libraries were filtered to contain only read pairs with perfectly matching barcodes, and those barcodes were used to demultiplex reads by sample.…”

Effect of humic acid on anaerobic digestion of cellulose and xylan in completely stirred tank reactors: inhibitory effect, mitigation of the inhibition and the dynamics of the microbial communities.

“…16S rRNA gene sequencing data was analysed using NG-Tax, an in-house pipeline (Ramiro-Garcia et al 2016). Paired-end libraries were filtered to contain only read pairs with perfectly matching barcodes, and those barcodes were used to demultiplex reads by sample.…”

Botryococcus braunii for the production of hydrocarbons and exopolysaccharides and the role of associated bacteria

“…To process the sequence data, special pipelines are needed. Recently the NG-Tax pipeline has been developed that benchmarks against other highly used pipelines such as Qiime (Ramiro-Garcia et al 2016). In this thesis the NG-Tax pipeline ( Fig.…”

“…Illumina sequencing data were analyzed using NG-Tax pipeline (Ramiro-Garcia et al 2016) yielding a total of 9080216 sequences. Briefly, in a first step, paired-end reads of 2 x 100 nucleotides were combined and only read pairs with perfectly matching primers and barcodes were retained.…”