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High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3′ termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) – a novel RNA-seq library preparation method directed towards mRNA 3′ termini. We demonstrate the specific enrichment for 3′-terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates.

High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3' termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) - a novel RNA-seq library preparation method directed towards mRNA 3' termini. We demonstrate the specific enrichment for 3'- terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates. [ABSTRACT FROM AUTHOR]

High-throughput sequencing has emerged as the favoured method to study microRNA (miRNA) expression, but biases introduced during library preparation have been reported. We recently compared the performance (sensitivity, reliability, titration response and differential expression) of six commercially-available kits on synthetic miRNAs and human RNA, where library preparation was performed by the vendors. We hereby supplement this study with data from two further commonly used kits (NEBNext, NEXTflex) whose manufacturers initially declined to participate. NEXTflex demonstrated the highest sensitivity, which may reflect its use of partially-randomized adapter sequences, but overall performance was lower than the QIAseq and TailorMix kits. NEBNext showed intermediate performance. We reaffirm that biases are kit specific, complicating the comparison of miRNA datasets generated using different kits. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.