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

In a cross-site study we evaluated 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 found that all of the kits were capable of performing significant ribosomal depletion, though there were 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 among kits suggested that transcript length may be a key factor in library production efficiency. These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them. Overall design: The Universal Human Reference RNA (Agilent) was diluted to 500 ng/ul in 200ul of RNase-free water and 3.94ul of the Spike-in RNA Variant Control E2 Mix (Lexogen) were added. The sample was split into two aliquots, one of which was then heated at 94° C for 1 hour and 27 minutes. 1ul of ERCC RNA Spike-In Mix 1 was added to both the intact and degraded samples. The final intact and degraded RNA samples were then diluted to 25 ng/uL and were distributed to each of the ten genomics core facilities (members of ABRF) for ribo-depletion and library preparation following vendor protocol. Each site prepared between one and four library types. Indices were assigned by the group to prevent overlapping among libraries. Libraries were pooled at an equimolar concentration from each kit using site-specific quantification and pooling SOPs and return each pool along with individual un-pooled libraries to the designated sequencing site. The sequencing site quantified each pool, multiplexed and sequenced over three high output paired-end 75bp runs on the Illumina NextSeq 500. contributor: The Association of Biomolecular Resource Facilities (ABRF) DNA Sequencing Research Group (DSRG) members

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Herbert ZT, Kershner JP, Butty VL, Thimmapuram J, Choudhari S, Alekseyev YO, Fan J, Podnar JW, Wilcox E, Gipson J, Gillaspy A, Jepsen K, BonDurant SS, Morris K, Berkeley M, LeClerc A, Simpson SD, Sommerville G, Grimmett L, Adams M, Levine SS