Computational protocol: Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

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Protocol publication

[…] Hi-C data were mapped, and reads were filtered as described previously. Corrected contact probability matrices at 10kb resolution were obtained using iterative correction . Both steps were performed using the hiclib library for python, publicly available at [...] Paired-end sequencing reads were mapped independently using Bowtie 2.1.0 to the S. pombe reference genome (ASM294v2) for each Hi-C library. Mapping with iteratively increasing truncation length was used to maximize yield of valid Hi-C interactions, using the flags ‘--score-min L,-0.6,-0.2’, as described previously. Only read pairs where both reads uniquely aligned to the genome were considered for subsequent steps. Read pairs corresponding to repeat instances of the same DNA molecule were removed. Next, on the basis of their HindIII restriction fragment assignments and orientations, read pairs were classified as valid Hi-C products, non-ligation, or self-ligation products. The following fragment-level filters were then applied, as described, which remove read pairs: with one end adjacent to the restriction site (possible un-ligated molecules), from restriction fragments with very high or low counts, from very large or small restriction fragments, and separated by very few restriction fragments (as these may be strongly influenced by inefficiencies in restriction). Filters used hiclib default values, except for the last filter, which used a more stringent 4 instead of 2 fragments. For downstream analyses, only valid Hi-C read-pairs were considered. Furthermore, read pairs from biological replicates were pooled after applying fragment-level filters. The number of valid read pairs used were as follows: 61,873,904 for wild type; 12,521,720 for rad21-K1; 16,821,386 cdc10-v50 (G1 arrested cells); and 18,549,406 for clr4Δ. […]

Pipeline specifications

Software tools hiclib, Bowtie
Application Hi-C analysis
Organisms Schizosaccharomyces pombe