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Sequencing Degraded RNA Addressed by 3' Tag Counting
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.
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 3, e91851- p.Article in journal (Refereed) Published
Abstract [en]

RNA sequencing has become widely used in gene expression profiling experiments. Prior to any RNA sequencing experiment the quality of the RNA must be measured to assess whether or not it can be used for further downstream analysis. The RNA integrity number (RIN) is a scale used to measure the quality of RNA that runs from 1 (completely degraded) to 10 (intact). Ideally, samples with high RIN (>8) are used in RNA sequencing experiments. RNA, however, is a fragile molecule which is susceptible to degradation and obtaining high quality RNA is often hard, or even impossible when extracting RNA from certain clinical tissues. Thus, occasionally, working with low quality RNA is the only option the researcher has. Here we investigate the effects of RIN on RNA sequencing and suggest a computational method to handle data from samples with low quality RNA which also enables reanalysis of published datasets. Using RNA from a human cell line we generated and sequenced samples with varying RINs and illustrate what effect the RIN has on the basic procedure of RNA sequencing; both quality aspects and differential expression. We show that the RIN has systematic effects on gene coverage, false positives in differential expression and the quantification of duplicate reads. We introduce 3' tag counting (3TC) as a computational approach to reliably estimate differential expression for samples with low RIN. We show that using the 3TC method in differential expression analysis significantly reduces false positives when comparing samples with different RIN, while retaining reasonable sensitivity.

Place, publisher, year, edition, pages
2014. Vol. 9, no 3, e91851- p.
Keyword [en]
SEQ, Quantification, Transcription, Degradation, Integrity, Number
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-144364DOI: 10.1371/journal.pone.0091851ISI: 000332858400109ScopusID: 2-s2.0-84897972088OAI: diva2:713408
Swedish Research CouncilKnut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience

QC 20140423

Available from: 2014-04-23 Created: 2014-04-22 Last updated: 2016-05-31Bibliographically 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. 135 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:2
RNA sequencing, exome sequencing, bioinformatics, gene expression, differential expression, variant calling
National Category
Bioinformatics and Systems Biology
Research subject
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)
Swedish Research CouncilKnut and Alice Wallenberg Foundation

QC 20160329

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

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Sigurgeirsson, BenjaminEmanuelsson, OlofLundeberg, Joakim
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