Assay

ATAC-seq: sanity checks and pitfalls

Tn5 transposase inserts sequencing adapters into open chromatin, producing paired-end fragments with a nucleosome-periodic size distribution. The pipeline is alignment, mitochondrial and duplicate removal, Tn5 offset shifting (+4/-5), peak calling and a consensus peak set for counting. Quality lives in the fragment-size histogram, the TSS enrichment score and the fraction of reads in peaks; libraries can look fine by read count and be unusable. Blacklisted regions and chrM reads are the two contaminants that most often masquerade as biology.

Who this is for: Epigenomics groups and immunologists profiling accessibility in sorted cell populations, typically 2-4 replicates per condition. They need peaks they can trust, a differential accessibility test, and motif enrichment, and they routinely skip the fragment-size and TSS-enrichment checks that reveal a failed library.