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Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

ABSTRACT: BACKGROUND: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 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. RESULTS:We 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. CONCLUSIONS:These results provide a roadmap for labs on the strengths of each of these methods and how best to utilize them.

SUBMITTER: Herbert ZT

PROVIDER: S-EPMC6389247 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

Herbert Zachary T ZT Kershner Jamie P JP Butty Vincent L VL Thimmapuram Jyothi J Choudhari Sulbha S Alekseyev Yuriy O YO Fan Jun J Podnar Jessica W JW Wilcox Edward E Gipson Jenny J Gillaspy Allison A Jepsen Kristen K BonDurant Sandra Splinter SS Morris Krystalynne K Berkeley Maura M LeClerc Ashley A Simpson Stephen D SD Sommerville Gary G Grimmett Leslie L Adams Marie M Levine Stuart S SS

BMC genomics 20180315 1

<h4>Background</h4>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 also captures transcripts that are not poly-a ...[more]

PMID: 29703133

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Project description:BackgroundMicroRNAs(miRNAs) are 18-25 nt small RNAs playing critical roles in many biological processes. The majority of known miRNAs were discovered by conventional cloning and a Sanger sequencing approach. The next-generation sequencing (NGS) technologies enable in-depth characterization of the global repertoire of miRNAs, and different protocols for miRNA library construction have been developed. However, the possible bias between the relative expression levels and sequences introduced by different protocols of library preparation have rarely been explored.ResultsWe assessed three different miRNA library preparation protocols, SOLiD, Illumina versions 1 and 1.5, using cloning or SBS sequencing of total RNA samples extracted from skeletal muscles from Hu sheep and Dorper sheep, and then validated 9 miRNAs by qRT-PCR. Our results show that SBS sequencing data highly correlate with Illumina cloning data. The SOLiD data, when compared to Illumina's, indicate more dispersed distribution of length, higher frequency variation for nucleotides near the 3'- and 5'-ends, higher frequency occurrence for reads containing end secondary structure (ESS), and higher frequency for reads that do not map to known miRNAs. qRT-PCR results showed the best correlation with SOLiD cloning data. Fold difference of Hu sheep and Dorper sheep between qRT-PCR result and SBS sequencing data correlated well (r = 0.937), and fold difference of miR-1 and miR-206 among SOLiD cloning data, qRT-PCR and SBS sequencing data was similar.ConclusionsThe sequencing depth can influence the quantitative measurement of miRNA abundance, but the discrepancy caused by it was not statistically significant as high correlation was observed between Illumina cloning and SBS sequencing data. Bias of length distribution, sequence variation, and ESS was observed between data obtained with the different protocols. SOLiD cloning data differ from Illumina cloning data mainly because of distinct methods of adapter ligation. The good correlation between qRT-PCR result and SOLiD data might be due to the similarities of the hybridization-based methods. The fold difference analysis indicated that methods based on hybridization may be superior for quantitative measurement of miRNA abundance. Because of the genome sequence of the sheep is not available, our data may not explain how the entire miRNA bias in the natural miRNAs in sheep or other mammal miRNA expression, unbiased artificially synthesized miRNA will help on evaluating the methodology of miRNA library preparation.

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Dataset Information

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

Publications

Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction.

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