title: RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.
abstract: Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.
authors.affiliation: Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
authors.forename: Neil
authors.initials: N
authors.lastname: Renwick
authors.forename: Miguel
authors.initials: M
authors.lastname: Brown
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Mihailović
authors.forename: Daniel
authors.initials: D
authors.lastname: Holoch
authors.forename: Carolina
authors.initials: C
authors.lastname: Lin
authors.forename: John T G
authors.initials: JT
authors.lastname: Pena
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Nusbaum
authors.forename: Pavel
authors.initials: P
authors.lastname: Morozov
authors.forename: Janos
authors.initials: J
authors.lastname: Ludwig
authors.forename: Tolulope
authors.initials: T
authors.lastname: Ojo
authors.forename: Shujun
authors.initials: S
authors.lastname: Luo
authors.forename: Gary
authors.initials: G
authors.lastname: Schroth
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
mesh_terms: D017931:DNA Primers; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis*; D005810:Multigene Family; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011130:RNA Ligase (ATP) / Q000032:analysis / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D017931:DNA Primers; D035683:MicroRNAs; D009841:Oligonucleotides; D011130:RNA Ligase (ATP)