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 39 and 59 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 39-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 59-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/39-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 59-terminal 5-nt barcode extensions for a set of 20 barcoded 39 adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

“…We reviewed the sequence alignments of unannotated reads to the genome and found no evidence of novel highly expressed miRNA genes. We also analyzed miRNA sequence variation as described previously (25)(26)(27) and found no novel SNPs in positions known to affect miRNA biogenesis or their target mRNA regulation (Dataset S1).…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…Note the position of the HEL2i domain between HEL1 and HEL2 domains as a result of blastp search, which is in contrast with the structural data from RIG-I, which place the HEL2i domain inside the HEL2 domain (Kowalinski et al, 2011;Luo et al, 2011)}. (B) An electron microscopy reconstruction of mammalian Dicer with marked regions (left panel) and several mapped domains (right panel).…”

“…The most studied model for viral infections in birds is Marek's disease, which is a consequence of a Herpesvirus infection in poultry. Publications linked to Marek's disease addressed virus encoded miRNAs (Morgan et al, 2008;Yao et al, 2008;Zhao et al, 2009;Muylkens et al, 2010;Luo et al, 2011;Xu et al, 2011;Zhao et al, 2011;Coupeau et al, 2012;Strassheim et al, 2012), changes in host miRNA expression during infection (Yao et al, 2008;Lambeth et al, 2009a;Xu et al, 2010;Tian et al, 2012;Stik et al, 2013;Dinh et al, 2014;Li et al, 2014b;Li et al, 2014c;Lian et al, 2015a;Lian et al, 2015b;Han et al, 2016), or attempts to block the virus with RNAi Chen et al, 2009b;Lambeth et al, 2009b). A similar set of articles has been found for other studied viruses infecting birds -e.g.…”

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

“…These biases can be present and even can be amplified in the final relative concentration analysis by the three main techniques, microarrays, qRT-PCR, and sequencing. [77][78][79] C. Detection of point mutations and polymorphisms…”