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

Zachary T Herbert; Jamie P Kershner; Vincent L Butty; Jyothi Thimmapuram; Sulbha Choudhari; Yuriy O Alekseyev; Jun Fan; Jessica W Podnar; Edward Wilcox; Jenny Gipson; Allison Gillaspy; Kristen Jepsen; Sandra Splinter BonDurant; Krystalynne Morris; Maura Berkeley; Ashley LeClerc; Stephen D Simpson; Gary Sommerville; Leslie Grimmett; Marie Adams; Stuart S Levine

doi:10.1186/s12864-018-4585-1

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

BMC Genomics. 2018 Mar 15;19(1):199. doi: 10.1186/s12864-018-4585-1.

Authors

Zachary T Herbert¹, Jamie P Kershner², Vincent L Butty³, Jyothi Thimmapuram⁴, Sulbha Choudhari⁴, Yuriy O Alekseyev^{5

6}, Jun Fan⁷, Jessica W Podnar⁸, Edward Wilcox⁹, Jenny Gipson¹⁰, Allison Gillaspy¹⁰, Kristen Jepsen¹¹, Sandra Splinter BonDurant¹², Krystalynne Morris¹³, Maura Berkeley¹, Ashley LeClerc⁵, Stephen D Simpson¹³, Gary Sommerville¹, Leslie Grimmett¹, Marie Adams¹⁴, Stuart S Levine¹⁵

Affiliations

¹ Molecular Biology Core Facilities at Dana-Farber Cancer Institute, Boston, MA, USA.
² BioFrontiers Institute, Next-Gen Sequencing Facility, University of Colorado Boulder, Boulder, CO, USA.
³ MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA.
⁴ Bioinformatics Core, Purdue University, West Lafayette, IN, USA.
⁵ Microarray and Sequencing Resource Core Facility, Boston University, Boston, MA, USA.
⁶ Department of Pathology and Laboratory Medicine, Boston University, Boston, MA, USA.
⁷ Genomic Core Facility, Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA.
⁸ Genomic Sequencing and Analysis Facility, University of Texas, Austin, TX, USA.
⁹ DNA Sequencing Center, Brigham Young University, Provo, UT, USA.
¹⁰ Laboratory for Molecular Biology and Cytometry, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
¹¹ IGM Genomics Center, University of California, San Diego, La Jolla, CA, USA.
¹² UW Biotechnology Center, Gene Expression Center, University of Wisconsin-Madison, Madison, WI, USA.
¹³ Hubbard Center for Genome Studies, University of New Hampshire, Durham, NH, USA.
¹⁴ Genomics Core Facility, Van Andel Institute, Grand Rapids, MI, USA.
¹⁵ MIT BioMicro Center, Massachusetts Institute of Technology, Cambridge, MA, USA. [email protected].

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.

Keywords: ABRF; Illumina; NGS; RNAseq; Transcriptomics; rRNA depletion.

MeSH terms

Gene Expression Profiling / methods
Gene Library
High-Throughput Nucleotide Sequencing / methods*
Humans
Poly A / genetics
RNA, Ribosomal / genetics
RNA, Ribosomal / isolation & purification*
Sequence Analysis, RNA / methods*

Substances

RNA, Ribosomal
Poly A

Abstract

MeSH terms

Substances

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