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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:BackgroundTarget enrichment is a critical component of targeted deep next-generation sequencing for the cost-effective and sensitive detection of mutations, which is predominantly performed by either hybrid selection or PCR. Despite the advantages of efficient enrichment, PCR-based methods preclude the identification of PCR duplicates and their subsequent removal. Recently, this limitation was overcome by assigning a unique molecular identifier(UMI) to each template molecule. Currently, several commercial library construction kits based on PCR enrichment are available for UMIs, but there have been no systematic studies to compare their performances. In this study, we evaluated and compared the performances of five commercial library kits from four vendors: the Archer® Reveal ctDNA™ 28 Kit, NEBNext Direct® Cancer HotSpot Panel, Nugen Ovation® Custom Target Enrichment System, Qiagen Human Comprehensive Cancer Panel(HCCP), and Qiagen Human Actionable Solid Tumor Panel(HASTP).ResultsWe evaluated and compared the performances of the five kits using 50 ng of genomic DNA for the library construction in terms of the library complexity, coverage uniformity, and errors in the UMIs. While the duplicate rates for all kits were dramatically decreased by identifying unique molecules with UMIs, the Qiagen HASTP achieved the highest library complexity based on the depth of unique coverage indicating superb library construction efficiency. Regarding the coverage uniformity, the kits from Nugen and NEB performed the best followed by the kits from Qiagen. We also analyzed the UMIs, including errors, which allowed us to adjust the depth of unique coverage and the length required for sufficient complexity. Based on these comparisons, we selected the Qiagen HASTP for further performance evaluations. The targeted deep sequencing method based on PCR target enrichment combined with UMI tagging sensitively detected mutations present at a frequency as low as 1% using 6.25 ng of human genomic DNA as the starting material.ConclusionThis study is the first systematic evaluation of commercial library construction kits for PCR-based targeted deep sequencing utilizing UMIs. Because the kits displayed significant variability in different quality metrics, our study offers a practical guideline for researchers to choose appropriate options for PCR-based targeted sequencing and useful benchmark data for evaluating new kits.

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Dataset Information

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

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

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