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.

Basic information

Original name

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

Authors

VEČEŘA, Marek (703 Slovakia, belonging to the institution), Jiří ŠÁNA (203 Czech Republic, belonging to the institution), Jan OPPELT (203 Czech Republic, belonging to the institution), Boris TICHÝ (203 Czech Republic, belonging to the institution), Alena KOPKOVÁ (203 Czech Republic, belonging to the institution), R. LIPINA (203 Czech Republic), Martin SMRČKA (203 Czech Republic, belonging to the institution), Radim JANČÁLEK (203 Czech Republic, belonging to the institution), Markéta HERMANOVÁ (203 Czech Republic, belonging to the institution), Leoš KŘEN (203 Czech Republic, belonging to the institution) and Ondřej SLABÝ (203 Czech Republic, guarantor, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

30204 Oncology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 2.740

RIV identification code

RIV/00216224:14740/19:00108512

Organization unit

Central European Institute of Technology

DOI

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

UT WoS

000458393400036

Keywords in English

SEQ; PROLIFERATION; CANCER

Abstract

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.

Links

NV15-33158A, research and development project

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

90091, large research infrastructures

Name: NCMG

Citovat

VEČEŘA, Marek, Jiří ŠÁNA, Jan OPPELT, Boris TICHÝ, Alena KOPKOVÁ, R. LIPINA, Martin SMRČKA, Radim JANČÁLEK, Markéta HERMANOVÁ, Leoš KŘEN and 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, vol. 14, No 2, p. 1-18. ISSN 1932-6203. Available from: 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 and 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, vol.~14, No~2, p.~1-18. ISSN~1932-6203. Available from: https://dx.doi.org/10.1371/journal.pone.0211978.

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