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Analysis of stranded information using an automated procedure for strand specific RNA sequencing
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-8879-9245
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0003-4313-1601
2014 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, no 1, article id 631Article in journal (Refereed) Published
Abstract [en]

Background: Strand specific RNA sequencing is rapidly replacing conventional cDNA sequencing as an approach for assessing information about the transcriptome. Alongside improved laboratory protocols the development of bioinformatical tools is steadily progressing. In the current procedure the Illumina TruSeq library preparation kit is used, along with additional reagents, to make stranded libraries in an automated fashion which are then sequenced on Illumina HiSeq 2000. By the use of freely available bioinformatical tools we show, through quality metrics, that the protocol is robust and reproducible. We further highlight the practicality of strand specific libraries by comparing expression of strand specific libraries to non-stranded libraries, by looking at known antisense transcription of pseudogenes and by identifying novel transcription. Furthermore, two ribosomal depletion kits, RiboMinus and RiboZero, are compared and two sequence aligners, Tophat2 and STAR, are also compared. Results: The, non-stranded, Illumina TruSeq kit can be adapted to generate strand specific libraries and can be used to access detailed information on the transcriptome. The RiboZero kit is very effective in removing ribosomal RNA from total RNA and the STAR aligner produces high mapping yield in a short time. Strand specific data gives more detailed and correct results than does non-stranded data as we show when estimating expression values and in assembling transcripts. Even well annotated genomes need improvements and corrections which can be achieved using strand specific data. Conclusions: Researchers in the field should strive to use strand specific data; it allows for more confidence in the data analysis and is less likely to lead to false conclusions. If faced with analysing non-stranded data, researchers should be well aware of the caveats of that approach.

Place, publisher, year, edition, pages
2014. Vol. 15, no 1, article id 631
Keywords [en]
Antisense RNA, Bioinformatics, Ribosomal depletion, RNA sequencing, Strand specificity
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-161768DOI: 10.1186/1471-2164-15-631Scopus ID: 2-s2.0-84904776692OAI: oai:DiVA.org:kth-161768DiVA, id: diva2:795690
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council
Note

QC 20150317

Available from: 2015-03-17 Created: 2015-03-17 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Analysis of RNA and DNA sequencing data: Improved bioinformatics applications
Open this publication in new window or tab >>Analysis of RNA and DNA sequencing data: Improved bioinformatics applications
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Massively parallel sequencing has rapidly revolutionized DNA and RNA research. Sample preparations are steadfastly advancing, sequencing costs have plummeted and throughput is ever growing. This progress has resulted in exponential growth in data generation with a corresponding demand for bioinformatic solutions. This thesis addresses methodological aspects of this sequencing revolution and applies it to selected biological topics.

Papers I and II are technical in nature and concern sample preparation and data anal- ysis of RNA sequencing data. Paper I is focused on RNA degradation and paper II on generating strand specific RNA-seq libraries.

Paper III and IV deal with current biological issues. In paper III, whole exomes of cancer patients undergoing chemotherapy are sequenced and their genetic variants associ- ated to their toxicity induced adverse drug reactions. In paper IV a comprehensive view of the gene expression of the endometrium is assessed from two time points of the menstrual cycle.

Together these papers show relevant aspects of contemporary sequencing technologies and how it can be applied to diverse biological topics. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. p. 135
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:2
Keywords
RNA sequencing, exome sequencing, bioinformatics, gene expression, differential expression, variant calling
National Category
Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-184158 (URN)978-91-7595-894-1 (ISBN)
Public defence
2016-04-22, Inghesalen, Tomtebodavägen 18A, Solna, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20160329

Available from: 2016-03-29 Created: 2016-03-29 Last updated: 2016-03-29Bibliographically approved

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Emanuelsson, Olof

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