Impact of library preparation on downstream analysis and interpretation of RNA-Seq data: comparison between Illumina PolyA and NuGEN Ovation protocol

PLoS One. 2013 Aug 19;8(8):e71745. doi: 10.1371/journal.pone.0071745. eCollection 2013.


Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing.

Methods and materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina's mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression.

Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and "novel" lincRNAs.

Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Data Interpretation, Statistical*
  • Epithelial Cells / metabolism
  • Exons / genetics
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Gene Library*
  • Humans
  • Poly A / genetics*
  • Polymorphism, Single Nucleotide / genetics
  • RNA, Long Noncoding / genetics
  • RNA, Messenger / genetics
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods*
  • Statistics as Topic*


  • RNA, Long Noncoding
  • RNA, Messenger
  • Poly A

Grants and funding

This work was supported by grants from the 26.2 with Donna Foundation (to EAP), the Florida Department of Health Bankhead/Coley grant program (1BG12-34201 to EAT), and Mayo Clinic Center for Individualized Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.