Limits...
Inherent signals in sequencing-based Chromatin-ImmunoPrecipitation control libraries.

Vega VB, Cheung E, Palanisamy N, Sung WK - PLoS ONE (2009)

Bottom Line: The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives.We found that copy number plays a major influence in both ChIP-enriched as well as control libraries.Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks.

View Article: PubMed Central - PubMed

Affiliation: Computational and Mathematical Biology Group, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT

Background: The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.

Methodology: First, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.

Conclusions: We found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights.

Show MeSH

Related in: MedlinePlus

Mapping bias was apparent within repeat regions.Tag overabundance and paucity in the three mouse WCEseq libraries across various repeat classes, illustrating the biases due to mapping problems. Statistically significant deviations from random expectation (p-value<1e-3) were marked with stars.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2666154&req=5

pone-0005241-g003: Mapping bias was apparent within repeat regions.Tag overabundance and paucity in the three mouse WCEseq libraries across various repeat classes, illustrating the biases due to mapping problems. Statistically significant deviations from random expectation (p-value<1e-3) were marked with stars.

Mentions: Another likely source for systematic bias lies in the mapping procedures. For the purpose of assessing this bias, we used the repeat regions as a surrogate for heavily biased regions. We found that a number of repeat classes were significantly enriched (p<1e-3) for WCEseq tags, while some were unexpectedly depleted of tags (Fig. 3). The depleted region could be ascribed to mapping ambiguities in these repeats which resulted in the removal of these multiply mapped tags, as typically only uniquely mapped tags are retained. Satellite regions were found to be enriched in all the three WCE libraries. This was not unexpected as satellites have been previously reported to be unduly enriched in tags from ChIP-enriched libraries as well [10], marked by conspicuous spikes in otherwise flat genomic segments.


Inherent signals in sequencing-based Chromatin-ImmunoPrecipitation control libraries.

Vega VB, Cheung E, Palanisamy N, Sung WK - PLoS ONE (2009)

Mapping bias was apparent within repeat regions.Tag overabundance and paucity in the three mouse WCEseq libraries across various repeat classes, illustrating the biases due to mapping problems. Statistically significant deviations from random expectation (p-value<1e-3) were marked with stars.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666154&req=5

pone-0005241-g003: Mapping bias was apparent within repeat regions.Tag overabundance and paucity in the three mouse WCEseq libraries across various repeat classes, illustrating the biases due to mapping problems. Statistically significant deviations from random expectation (p-value<1e-3) were marked with stars.
Mentions: Another likely source for systematic bias lies in the mapping procedures. For the purpose of assessing this bias, we used the repeat regions as a surrogate for heavily biased regions. We found that a number of repeat classes were significantly enriched (p<1e-3) for WCEseq tags, while some were unexpectedly depleted of tags (Fig. 3). The depleted region could be ascribed to mapping ambiguities in these repeats which resulted in the removal of these multiply mapped tags, as typically only uniquely mapped tags are retained. Satellite regions were found to be enriched in all the three WCE libraries. This was not unexpected as satellites have been previously reported to be unduly enriched in tags from ChIP-enriched libraries as well [10], marked by conspicuous spikes in otherwise flat genomic segments.

Bottom Line: The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives.We found that copy number plays a major influence in both ChIP-enriched as well as control libraries.Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks.

View Article: PubMed Central - PubMed

Affiliation: Computational and Mathematical Biology Group, Genome Institute of Singapore, Singapore, Singapore.

ABSTRACT

Background: The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.

Methodology: First, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.

Conclusions: We found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights.

Show MeSH
Related in: MedlinePlus