Lifeind ehf

Reykjavík, Iceland

Lifeind ehf

Reykjavík, Iceland

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Felsani A.,Scientific consultant of Genomnia s.r.l. | Gudmundsson B.,University of Iceland | Nanni S.,Catholic University | Brini E.,Catholic University | And 8 more authors.
Briefings in Functional Genomics | Year: 2015

Different ChIP-Seq protocolsmay have a significant impact on the final outcome in terms of quality, number and distribution of called peaks. Sample DNA undergoes a long procedure before the final sequencing step, and damaged DNA can result in excessive mismatches in the alignment with reference genome. In this letter, we present the effect of well-defined modifications (timing of formaldehyde crosslink reversal, brand of the sonicator) of standard ChIP-Seq protocol on parallel samples derived from the same cell line correlating the initial DNA quality control metrics to the final bioinformatics analysis results. © The Author 2014. Published by Oxford University Press. All rights reserved.


Thormar H.G.,University of Iceland | Thormar H.G.,Lifeind ehf | Gudmundsson B.,University of Iceland | Gudmundsson B.,Lifeind ehf | And 8 more authors.
Clinical Chemistry | Year: 2013

Background: The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. Methods: We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. Results: The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed betweensample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Conclusions: Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. Copyright © 2013 American Association for Clinical Chemistry.

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