Using paired-end read orientations to assess technical biases in capture Hi-C.

Hi-C and capture Hi-C (CHi-C) both leverage paired-end sequencing of chimeric fragments to gauge the strength of interactions based on the total number of paired-end reads mapped to a common pair of restriction fragments. Mapped paired-end reads can have four relative orientations, depending on the genomic positions and strands of the two reads. We assigned one paired-end read orientation to each of the four possible re-ligations that can occur between two given restriction fragments. In a large hematopoietic cell dataset, we determined the read pair counts of interactions separately for each orientation. Interactions with imbalances in the counts occur much more often than expected by chance for both Hi-C and CHi-C. Based on such imbalances, we identified target restriction fragments enriched at only one instead of both ends. By matching them to the baits used for the experiments, we confirmed our assignment of paired-end read orientations and gained insights that can inform bait design. An analysis of unbaited fragments shows that, beyond bait effects, other known types of technical biases are reflected in count imbalances. Taking advantage of distance-dependent contact frequencies, we assessed the impact of such biases. Our results have the potential to improve the design and interpretation of CHi-C experiments.