A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities

Abstract

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVSION ranged from 65%−91% versus 44%−61% for single region QIIME analyses (p<0.01, p<0.001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively. MVRSION represents a generally applicable tool for taxonomic classification that is superior to singleregion 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

“…Comparison of DNA sequences from these PCR products is widely used in taxonomy and phylogenetic studies and clinical microbiology . Besides the conserved regions, 16S rRNA contains hypervariable regions that are highly specific for biological species or genera . Therefore, we designed primers specific for Helicobacter to elaborate reliable nested PCR.…”

“…Primers were designed to anneal in hypervariable regions specific for a particular species or genus . To identify the appropriate sector, the H pylori strain P12 16S rDNA sequence was compared to its counterparts from Escherichia coli (typical stool bacteria) and Campylobacter jejuni , which represents a species from the most‐related genus.…”

“…34 Besides the conserved regions, 16S rRNA contains hypervariable regions that are highly specific for biological species or genera. [35][36][37] Therefore, we designed primers specific for Helicobacter to elaborate reliable nested PCR.…”

Gut metabolites influence susceptibility of neonatal mice to cryptosporidiosis

“…MiSeq Sequencer, as previously described (56). Sequencing data analysis either used the V1-V9 regions and the MVRSION pipeline (57) or the V4 region and QIIME pipeline version 1.9.0(58), as previously described (56). The OTU table resulting from QIIME analysis was used as input for linear discriminant analysis (LDA) effect size (LEfSe)(59) (http://huttenhower.sph.harvard.edu/lefse/) to identify statistically significant, differentially abundant taxa between the 1-week and 6-week old mice.…”

Metagenomics-Based Phylogeny and Phylogenomic

“…Extracting phylogenetic information from massive sequencing reads is not a trivial task; however, it can be achieved with reasonable success by using several profiling tools adapted both to the analysis of amplicons of ribosomal genes and to the conserved genes between different domains [6,7]. The microbial community structure has been approached mostly using the 16S SSU-rRNA gene as phylogenetic marker, mainly due to lower sequencing costs and an acceptable relation of specificity-resolution in taxonomic assignments [8], while methods that use single-copy markers obtained from shotgun sequencing reads or assembled samples are gaining relevance because they have demonstrated strain-level resolution [9,10], a really hard issue when analyzing complex microbiomes.…”