Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries.

Journal Article (Journal Article)

RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

Full Text

Duke Authors

Cited Authors

  • Telzrow, CL; Zwack, PJ; Esher Righi, S; Dietrich, FS; Chan, C; Owzar, K; Alspaugh, JA; Granek, JA

Published Date

  • October 19, 2021

Published In

Volume / Issue

  • 11 / 11

PubMed ID

  • 34518880

Pubmed Central ID

  • PMC8527493

Electronic International Standard Serial Number (EISSN)

  • 2160-1836

Digital Object Identifier (DOI)

  • 10.1093/g3journal/jkab301


  • eng

Conference Location

  • England