Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs
| العنوان: | Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs |
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| المؤلفون: | Markéta Hermanová, Marek Vecera, Radim Lipina, Jan Oppelt, Radim Jančálek, Kopkova Alena, Boris Tichy, Ondrej Slaby, Leos Kren, Martin Smrčka, Jiri Sana |
| المصدر: | PLoS ONE, Vol 14, Iss 2, p e0211978 (2019) PLoS ONE |
| بيانات النشر: | Public Library of Science (PLoS), 2019. |
| سنة النشر: | 2019 |
| مصطلحات موضوعية: | cDNA libraries, 0301 basic medicine, Molecular biology, Normal Distribution, Biochemistry, Transcriptome, Database and Informatics Methods, Sequencing techniques, 0302 clinical medicine, Coding region, Genomic library, DNA libraries, Multidisciplinary, Brain Neoplasms, High-Throughput Nucleotide Sequencing, RNA sequencing, Genomics, Complementary DNA, 3. Good health, Nucleic acids, Gene Expression Regulation, Neoplastic, Ribosomal RNA, 030220 oncology & carcinogenesis, Physical Sciences, Medicine, RNA, Long Noncoding, Sequence Analysis, Transcriptome Analysis, Research Article, Cell biology, Cellular structures and organelles, Forms of DNA, Bioinformatics, Sequence analysis, Science, Computational biology, Research and Analysis Methods, 03 medical and health sciences, Genetics, Humans, Non-coding RNA, Gene Library, Biology and life sciences, Sequence Analysis, RNA, cDNA library, Gene Expression Profiling, Computational Biology, RNA, DNA, Genome Analysis, Genomic Libraries, Probability Theory, Probability Distribution, Molecular biology techniques, 030104 developmental biology, Long non-coding RNAs, Reagent Kits, Diagnostic, Glioblastoma, Ribosomes, Sequence Alignment, Mathematics, GC-content |
| الوصف: | 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. |
| تدمد: | 1932-6203 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::957dcb749f2d4b9147434e51b874a383Test https://doi.org/10.1371/journal.pone.0211978Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....957dcb749f2d4b9147434e51b874a383 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 19326203 |
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