title: Impact of library preparation on downstream analysis and interpretation of RNA-Seq data: comparison between Illumina PolyA and NuGEN Ovation protocol.
abstract: OBJECTIVE
  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
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America.
authors: Zhifu
authors: Z
authors: Sun
authors: Yan W
authors: YW
authors: Asmann
authors: Asha
authors: A
authors: Nair
authors: Yuji
authors: Y
authors: Zhang
authors: Liguo
authors: L
authors: Wang
authors: Krishna R
authors: KR
authors: Kalari
authors: Aditya V
authors: AV
authors: Bhagwate
authors: Tiffany R
authors: TR
authors: Baker
authors: Jennifer M
authors: JM
authors: Carr
authors: Jean-Pierre A
authors: JP
authors: Kocher
authors: Edith A
authors: EA
authors: Perez
authors: E Aubrey
authors: EA
authors: Thompson
mesh_terms: D002460:Cell Line; D003627:Data Interpretation, Statistical; D004847:Epithelial Cells / Q000378:metabolism; D005091:Exons / Q000235:genetics; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D011061:Poly A / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D062085:RNA, Long Noncoding / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*; D013223:Statistics as Topic
chemical_list: D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D011061:Poly A
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