Systematic assessment of commercially available low-input miRNA library preparation kits

Abstract

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

Insufficiently complex unique-molecular identifiers (UMIs) distort small RNA sequencing

“…2). In agreement with other recent studies 3,25,26 , we found a strong correlation between UMI counts and raw read counts, suggesting relatively little PCR bias exists within our samples. This suggests one might dispense with UMIs altogether in smRNA sequencing, though for low concentration samples that require more extensive amplification, we have found UMIs are helpful to maintain information of relative miRNA expression (data not shown).…”

An extension to: Systematic assessment of commercially available low-input miRNA library preparation kits

“…1A ). To aid comparison, we present here the results of all eight kits, with our previous results [ 8 ] displayed in faded colours in the figures. Following library preparation, the NEBNext and NEXTflex libraries were sequenced together (i.e.…”

“…Following library preparation, the NEBNext and NEXTflex libraries were sequenced together (i.e. on the same sequencing flow cell) with the libraries from the other six library preparation kits [ 8 ]. Previously-published samples were not re-sequenced: the new data presented here was generated at the same time as those presented earlier, thus partial figures reproduced from the earlier study to aid comparison did not entail re-analysis.…”

“…Several commercially-available kits are available to prepare miRNA libraries for sequencing, which entails addition of adapter sequences to the miRNAs followed by reverse transcription and cDNA synthesis. In a recent study, we compared the performance of six such kits (CATS, CleanTag, QIAseq, TailorMix, SMARTer-beta and srLp) with respect to detection rate sensitivity, reliability and ability to detect differentially expressed miRNAs [ 8 ]. However, two commonly used kits (NEBNext and NEXTflex) were not included.…”

“…This manuscript gives an overview on the results for all eight kits, with a focus on the NEBNext and NEXTflex kits. For more details on the other six kits we refer to Heinicke, et al [ 8 ].…”