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

J 2019

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

VEČEŘA, Marek, Jiří ŠÁNA, Jan OPPELT, Boris TICHÝ, Alena KOPKOVÁ et. al.

Základní údaje

Originální název

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

Autoři

VEČEŘA, Marek (703 Slovensko, domácí), Jiří ŠÁNA (203 Česká republika, domácí), Jan OPPELT (203 Česká republika, domácí), Boris TICHÝ (203 Česká republika, domácí), Alena KOPKOVÁ (203 Česká republika, domácí), R. LIPINA (203 Česká republika), Martin SMRČKA (203 Česká republika, domácí), Radim JANČÁLEK (203 Česká republika, domácí), Markéta HERMANOVÁ (203 Česká republika, domácí), Leoš KŘEN (203 Česká republika, domácí) a Ondřej SLABÝ (203 Česká republika, garant, domácí)

Vydání

Plos one, San Francisco, Public Library of Science, 2019, 1932-6203

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

30204 Oncology

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 2.740

Kód RIV

RIV/00216224:14740/19:00108512

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.1371/journal.pone.0211978

UT WoS

000458393400036

Klíčová slova anglicky

SEQ; PROLIFERATION; CANCER

Štítky

14110112, 14110131, 14110224, 14110230, 14110811, podil, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 15. 10. 2024 09:15, Ing. Martina Blahová

Anotace

V originále

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.

Návaznosti

NV15-33158A, projekt VaV

Název: Nová úroveň molekulární taxonomie glioblastomu založená na expresních profilech dlouhých nekódujících RNA: implikace pro diagnostiku a terapii

90091, velká výzkumná infrastruktura

Název: NCMG

Citovat

VEČEŘA, Marek, Jiří ŠÁNA, Jan OPPELT, Boris TICHÝ, Alena KOPKOVÁ, R. LIPINA, Martin SMRČKA, Radim JANČÁLEK, Markéta HERMANOVÁ, Leoš KŘEN a Ondřej SLABÝ. 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. Plos one. San Francisco: Public Library of Science, 2019, roč. 14, č. 2, s. 1-18. ISSN 1932-6203. Dostupné z: https://dx.doi.org/10.1371/journal.pone.0211978.

@article{1555282,
   author = {Večeřa, Marek and Šána, Jiří and Oppelt, Jan and Tichý, Boris and Kopková, Alena and Lipina, R. and Smrčka, Martin and Jančálek, Radim and Hermanová, Markéta and Křen, Leoš and Slabý, Ondřej},
   article_location = {San Francisco},
   article_number = {2},
   doi = {http://dx.doi.org/10.1371/journal.pone.0211978},
   keywords = {SEQ; PROLIFERATION; CANCER},
   language = {eng},
   issn = {1932-6203},
   journal = {Plos one},
   title = {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},
   url = {https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0211978&type=printable},
   volume = {14},
   year = {2019}
}

TY  - JOUR
ID  - 1555282
AU  - Večeřa, Marek - Šána, Jiří - Oppelt, Jan - Tichý, Boris - Kopková, Alena - Lipina, R. - Smrčka, Martin - Jančálek, Radim - Hermanová, Markéta - Křen, Leoš - Slabý, Ondřej
PY  - 2019
TI  - 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
JF  - Plos one
VL  - 14
IS  - 2
SP  - 1-18
EP  - 1-18
PB  - Public Library of Science
SN  - 19326203
KW  - SEQ
KW  - PROLIFERATION
KW  - CANCER
UR  - https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0211978&type=printable
L2  - https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0211978&type=printable
N2  - 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.
ER  -

VEČEŘA, Marek, Jiří ŠÁNA, Jan OPPELT, Boris TICHÝ, Alena KOPKOVÁ, R. LIPINA, Martin SMRČKA, Radim JANČÁLEK, Markéta HERMANOVÁ, Leoš KŘEN a Ondřej SLABÝ. 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. \textit{Plos one}. San Francisco: Public Library of Science, 2019, roč.~14, č.~2, s.~1-18. ISSN~1932-6203. Dostupné z: https://dx.doi.org/10.1371/journal.pone.0211978.

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