Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences

Abstract

Microbial community profiling using 16S rRNA gene sequences requires accurate taxonomy assignments. ‘Universal' primers target conserved sequences and amplify sequences from many taxa, but they provide variable coverage of different environments, and regions of the rRNA gene differ in taxonomic informativeness—especially when high-throughput short-read sequencing technologies (for example, 454 and Illumina) are used. We introduce a new evaluation procedure that provides an improved measure of expected taxonomic precision when classifying environmental sequence reads from a given primer. Applying this measure to thousands of combinations of primers and read lengths, simulating single-ended and paired-end sequencing, reveals that these choices greatly affect taxonomic informativeness. The most informative sequence region may differ by environment, partly due to variable coverage of different environments in reference databases. Using our Rtax method of classifying paired-end reads, we found that paired-end sequencing provides substantial benefit in some environments including human gut, but not in others. Optimal primer choice for short reads totaling 96 nt provides 82–100% of the confident genus classifications available from longer reads.

Biofloc Technology (BFT): A Tool for Water Quality Management in Aquaculture

“…However, current sequencing technologies can cover only a fraction (~600 bp) of the ribosomal genes used for taxonomic classification, which means that only 2 or 3 of the 10 hypervariable regions of the 16S gene can be used for this classification. Researchers have made efforts to elucidate whose sequences are the most information richness [73]; however, there is still a loss of information contained on the regions that cannot be covered by these sequencing platforms.…”

Atlantic Salmon (Salmo salar L.) Gastrointestinal Microbial Community Dynamics in Relation to Digesta Properties and Diet

“…Samples of each cohort and time included an even contribution of each faecal score (1 to 4), score 5 (empty gut) did not produce a sample. Tagencoded FLX amplicon pyrosequencing of the region covered by application of the 341F and 907R primers [44] was carried out by Research and Testing Laboratories (Lubbock, Texas) using a Roche 454. FLX instruments with Titanium reagents as previously detailed by Dowd et al [45].…”

CHANGES IN THE HUMAN SKIN MICROBIOME OVER ONE YEAR’S TIME

“…The first limitation was the universality of the PCR primers. The PCR primers employed to amplify the V6 hypervariable region of the rDNA may have some mismatches to the rDNAs of relevant bacteria (Soergel et al, 2012;Frank et al, 2008). Presumably, such mismatch resulted in less amplification, especially in the critical first round.…”