rRNA_subtraction
Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics
Metatranscriptomes generated by pyrosequencing hold significant potential for describing  functional processes in complex microbial communities. Meeting this potential requires protocols  that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as  systematic comparisons to identify methodological artifacts and test for reproducibility across data  sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S)  that uses sample-specific probes and is applicable across diverse environmental samples. To test  this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX  technology) from bacterioplankton communities at two depths in the oligotrophic open ocean,  yielding 10 data sets representing B350 Mbp. Subtractive hybridization reduced bacterial rRNA  transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from  12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for  detecting sequences replicated artificially via pyrosequencing errors and identified such replicates  as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal,  statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical  replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential  transcript abundance (o0.2% of reference genes). However, gene overlap between data sets was  remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing  library template) sharing greater than 17% of unique reference genes. These results indicate that  pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of  reliable rRNA subtraction procedures to enhance sequencing coverage across the functional  transcript pool.