Cross-Site Comparison of Ribosomal Depletion Kits for Illumina RNAseq Library Construction

Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel RNA sequencing technologies requires either... [ view full abstract ]

Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel RNA sequencing technologies requires either enrichment of mature poly-adenylated transcripts or targeted depletion of the rRNA fraction. The latter method is of particular interest because it is compatible with degraded samples such as those extracted from FFPE, and it also captures transcripts that are not poly-adenylated such as some non-coding RNAs. Here we provide a cross-site study that evaluates the performance of ribosomal RNA removal kits from Illumina, Takara/Clontech, Kapa Biosystems, Lexogen, New England Biolabs and Qiagen on intact and degraded RNA samples. We find that all of the kits are capable of performing significant ribosomal depletion, though there are large differences in their ease of use. Most kits perform well on both intact and degraded samples and all identify ~14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM, though the fraction of reads that are protein coding or in annotated lncRNAs varies between the different methodologies. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.