ATAC-seq exploits the observation that the pattern of transposition of a hyperactive Tn5 transposase in native chromatin mirrors genome-wide chromatin accessibility. It has been suggested that transposition observed around transcription factor binding motifs can be used to assess their occupancy in the form of footprints. However, we show that the vast majority of footprints observed at transcription factor motifs in ATAC-seq data spuriously arise from the intrinsic sequence-dependent transposition site bias of Tn5 and are also observed in naked DNA. We demonstrate that the Tn5 transposition bias can be corrected using existing tools for sequence bias correction and a novel estimate of global occupancy in order to produce more reliable estimates of footprints.ATAC-seq has emerged as a powerful method to assess genome-wide DNA accessibility in native chromatin 1 . This approach relies upon the use of a hyperactive variant of the Tn5 transposase that can cleave and tag DNA for next-generation sequencing in a single step dubbed tagmentation. Tn5 transposition in native chromatin is largely restricted to accessible regions, resulting in a rapid method to map genome-wide chromatin accessibility. Footprints of DNA-bound transcription factors (TFs) that occlude Tn5 transposition can be observed in ATAC-seq data 1 . Such footprints serve as a surrogate measure of transcription factor occupancy and this approach is conceptually identical to DNAse I footprinting 2 .

T follicular helper (Tfh) cells play a critical role in T-dependent humoral immune responses. While genetic programs controlling Tfh cell differentiation have been extensively studied using murine models, studies in humans have been hampered by the lack of a robust in vitro differentiation system for Tfh cells. We characterized epigenomic landscapes across stages of Tfh cell differentiation in a healthy human tonsil using ATAC-Seq and CUT&RUN for selected histone modifications. We combined these epigenomic datasets and integrated them with the reference human protein interactome using a novel network propagation approach. Our approach uncovered subnetworks integral to Tfh cell differentiation. These subnetworks captured known Tfh cell drivers to a greater extent than conventional gene-centric analyses would, and also revealed novel modules that may be required for Tfh cell differentiation. We find that human Tfh cell subnetworks are functionally associated with specific immune signaling cascades including cytokine receptor driven pathways. Analyses of transcriptomic data revealed that in addition to these immune pathways being significantly dysregulated in severe COVID-19, the corresponding Tfh cell subnetworks are also transcriptionally perturbed to a similar extent. This provides a molecular mechanistic basis for the previously observed impaired Tfh cell differentiation and loss of germinal centers in severe COVID-19.