Beyond blacklists: a critical assessment of exclusion set generation strategies and alternative approaches

We examined the widely used Blacklist software and found that pre-generated exclusion sets were difficult to reproduce due to sensitivity to input data, aligner choice, and read length. We further explored the use of "sponge" sequences-unassembled genomic regions such as satellite DNA, ribosomal DNA, and mitochondrial DNA-as an alternative approach. We additionally investigated the effect of the T2T-CHM13 genome assembly on improving biological signals. Aligning reads to a genome that includes sponge sequences reduced signal correlation in ChIP-seq data comparably to Blacklist-derived exclusion sets while preserving biological signal. Sponge-based alignment also had minimal impact on RNA-seq gene counts, suggesting broader applicability beyond chromatin profiling. These results highlight the limitations of fixed exclusion sets, and recommend the use of the T2T-CHM13 assembly or, for the hg38 genome assembly, "sponge" sequences as an alignment-guided strategy for reducing artifacts and improving functional genomics analyses.