Abstract
Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.
Highlights
The rise of next-generation RNA sequencing (NGS or RNASeq) of transcriptome has largely accelerated genomic and epigenetic research and allowed the discovery of new RNA species which could harness a potential to be useful in the study of particular diseases and the development of new therapeutic strategies [1, 2]
Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of long non-coding RNAs (lncRNAs) via transcriptome sequencing
Our aim is to study transcripts called long non-coding RNAs, which do not code for protein and, instead, perform as fully functional regulatory elements which have been implicated in many biological processes including transcriptional regulation of neighbouring or distant genes [6] and histone modification [7], and those related to malignancies such as gliomas [8, 9]
Summary
The rise of next-generation RNA sequencing (NGS or RNASeq) of transcriptome has largely accelerated genomic and epigenetic research and allowed the discovery of new RNA species which could harness a potential to be useful in the study of particular diseases and the development of new therapeutic strategies [1, 2]. As is usually the case during implementation of new methods, there are issues that need to be tackled with in order to maximize the accuracy and the value of gathered experimental data One such issue is the selection of a proper approach to molecular library preparation and sequencing with regard to the molecular targets of interest [3,4,5]. Many lncRNAs are antisense to coding genes, intergenic or intronic [10] and, suitable library prepration protocol needs to reflect this fact and retain strand-specific information This can be achieved via incorporation of deoxyuridine triphosphates (dUTPs) during the synthesis of the second strand which is later cleaved using uracil-DNA glycosylase [11, 12] or via starter/stopper binding sites determining the insert size and modulating the activity of reverse transcriptase [13]. Otherwise, sequencing of libraries prepared with improper approach would result in the permanent loss of valuable data about lowly expressed lncRNAs
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