Abstract
BackgroundRibosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel 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 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.ResultsWe find that all of the kits are capable of performing significant ribosomal depletion, though there are differences in their ease of use. All kits were able to remove ribosomal RNA to below 20% with intact RNA and identify ~ 14,000 protein coding genes from the Universal Human Reference RNA sample at >1FPKM. Analysis of differentially detected genes between kits suggests that transcript length may be a key factor in library production efficiency.ConclusionsThese results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.
Highlights
Ribosomal RNA comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples
Ribosomal depletion is a critical method in transcriptomics that allows for efficient detection of functionally relevant coding as well as non-coding transcripts through removal of highly abundant Ribosomal RNA (rRNA) species
Use of oligo dT primer to capture the polyadenylated 3′ end of the transcripts and isolate Messenger RNA (mRNA) is routine in many RNA sequencing preparations; this method lacks the ability to handle degraded samples where most of the RNA is separated from the 3′ tail, or to isolate non-polyadenylated transcripts such as Long Non-Coding RNA (lncRNA)
Summary
Ribosomal RNA (rRNA) comprises at least 90% of total RNA extracted from mammalian tissue or cell line samples. Informative transcriptional profiling using massively parallel 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 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. RRNA is captured by complimentary oligonucleotides that are coupled to paramagnetic beads, after which the bound rRNA is precipitated and removed from the reaction Kits utilizing this method include Illumina’s RiboZero, Qiagen GeneRead rRNA depletion, and Lexogen RiboCop. The second method uses an alternative strategy, hybridizing the rRNA to DNA oligos and degrading the RNA:DNA hybrids using RNAseH. A third method that is aimed at low-input
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