Important biological information uncovered in previously unaligned reads from chromatin immunoprecipitation experiments (ChIP-Seq)

Abstract

Establishing the architecture of gene regulatory networks (GRNs) relies on chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) methods that provide genome-wide transcription factor binding sites (TFBSs). ChIP-Seq furnishes millions of short reads that, after alignment, describe the genome-wide binding sites of a particular TF. However, in all organisms investigated an average of 40% of reads fail to align to the corresponding genome, with some datasets having as much as 80% of reads failing to align. We describe here the provenance of previously unaligned reads in ChIP-Seq experiments from animals and plants. We show that a substantial portion corresponds to sequences of bacterial and metazoan origin, irrespective of the ChIP-Seq chromatin source. Unforeseen was the finding that 30%–40% of unaligned reads were actually alignable. To validate these observations, we investigated the characteristics of the previously unaligned reads corresponding to TAL1, a human TF involved in lineage specification of hemopoietic cells. We show that, while unmapped ChIP-Seq read datasets contain foreign DNA sequences, additional TFBSs can be identified from the previously unaligned ChIP-Seq reads. Our results indicate that the re-evaluation of previously unaligned reads from ChIP-Seq experiments will significantly contribute to TF target identification and determination of emerging properties of GRNs.

From trash to treasure: detecting unexpected contamination in unmapped NGS data

“…Unmapped ChIP-Seq reads from A. thaliana , Z. mays , H. sapiens , and D. Melanogaster datasets were investigated and found contaminated by foreign sequences. The authors characterised the contaminant organisms and calculated the relative abundance for each dataset by taxonomic classification [28]. Quality assessment of the working environment is crucially important, since sequence-based methods are particularly sensitive to reagents and laboratory contamination.…”