Abstract
BackgroundCulture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species.ResultsBased on 2,900 sequenced reference genomes, we show that 16S rRNA gene copy number (GCN) is strongly linked to microbial phylogenetic taxonomy, potentially under-representing Archaea in amplicon microbial profiles. Using this relationship, we inferred the GCN of all bacterial and archaeal lineages in the Greengenes database within a phylogenetic framework. We created CopyRighter, new software which uses these estimates to correct 16S rRNA amplicon microbial profiles and associated quantitative (q)PCR total abundance. CopyRighter parses microbial profiles and, because GCN estimates are pre-computed for all taxa in the reference taxonomy, rapidly corrects GCN bias. Software validation with in silico and in vitro mock communities indicated that GCN correction results in more accurate estimates of microbial relative abundance and improves the agreement between metagenomic and amplicon profiles. Analyses of human-associated and anaerobic digester microbiomes illustrate that correction makes tangible changes to estimates of qPCR total abundance, α and β diversity, and can significantly change biological interpretation. For example, human gut microbiomes from twins were reclassified into three rather than two enterotypes after GCN correction.ConclusionsThe CopyRighter bioinformatic tools permits rapid correction of GCN in microbial surveys, resulting in improved estimates of microbial abundance, α and β diversity.
Highlights
Culture-independent molecular surveys targeting conserved marker genes, most notably 16S ribosomal RNA (rRNA), to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species
A resolution of suspicious gene copy number (GCN) was attempted by ignoring the Integrated Microbial Genomes (IMG) record and: 1) using the GCN determined by INFERNAL or RNAmmer if it was consistent with Ribosomal RNA Database (rrnDB); 2) using the value from INFERNAL or RNAmmer if this genome was not represented in rrnDB but its scaffold length was longer than 200 kbp; and 3) using IMG's 5S or 23S rRNA GCN if it agreed with rrnDB's GCN
Gene copy number taxonomic signal Greengenes provides a phylogenetic tree based on bacterial and archaeal rRNA sequences, and a taxonomic system derived from this tree [23]
Summary
Culture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species. GCN variation spans over an order of magnitude, from 1 to 15 in Bacteria, but only up to 5 in Archaea [5] This order of magnitude range biases both amplicon microbial profiles and estimates of total microbial abundance based on amplicon quantitative PCR (qPCR) data [6]. Since related species have similar GCN [8,9], it is often possible to accurately estimate GCN of an uncultured organism if a closely related sequenced relative exists [9,10], though this means dramatically reducing the search space to the subset of species with documented GCN Another possibility is to place reads on a phylogenetic tree and calculate GCN based on that of sequenced relatives using phylogenetically independent contrasts (PIC) [9,11,12]. Correcting for GCN bias is still an open problem that no readily available software adequately addresses
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
