Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

Shaomei He, Omri Wurtzel, Kanwar Singh, Jeff L. Froula, Suzan Yilmaz, Susannah G. Tringe, Zhong Wang, Feng Chen, Erika A. Lindquist, Rotem Sorek, Philip Hugenholtz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

Original languageEnglish
Pages (from-to)807-812
Number of pages6
JournalNature Methods
Issue number10
StatePublished - Oct 2010
Externally publishedYes


Dive into the research topics of 'Validation of two ribosomal RNA removal methods for microbial metatranscriptomics'. Together they form a unique fingerprint.

Cite this