Review and Evaluate the Bioinformatics Analysis Strategies of ATAC-seq and CUT&Tag Data

Efficient and reliable profiling methods are essential to study epigenetics.Tn5,one of the first identified prokaryotic transposases with high DNA-binding and tagmentation efficiency,is widely adopted in different genomic and epigenomic protocols for high-throughputly exploring the genome and epigenome.Based on Tn5,the Assay for Transposase-Accessible Chromatin using sequencing(ATAC-seq)and the Cleavage Under Targets and Tagmentation(CUT&Tag)were developed to measure chromatin accessibility and detect DNA–protein interactions.These methodologies can be applied to large amounts of biological samples with low-input levels,such as rare tissues,embryos,and sorted single cells.However,fast and proper processing of these epigenomic data has become a bottleneck because massive data production continues to increase quickly.Furthermore,inappropriate data analysis can generate biased or misleading conclusions.Therefore,it is essential to evaluate the performance of Tn5-based ATAC-seq and CUT&Tag data processing bioinformatics tools,many of which were developed mostly for analyzing chromatin immunoprecipitation followed by sequencing(ChIP-seq)data.Here,we conducted a comprehensive benchmarking analysis to evaluate the performance of eight popular software for processing ATAC-seq and CUT&Tag data.We compared the sensitivity,specificity,and peak width distribution for both narrow-type and broad-type peak calling.We also tested the influence of the availability of control IgG input in CUT&Tag data analysis.Finally,we evaluated the differential analysis strategies commonly used for analyzing the CUT&Tag data.Our study provided comprehensive guidance for selecting bioinformatics tools and recommended analysis strategies,which were implemented into Docker/Singularity images for streamlined data analysis.

AIAP: A Quality Control and Integrative Analysis Package to Improve ATAC-seq Data Analysis

Assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis.Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data;however, key quality control(QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data,including reads under peak ratio(RUPr), background(BG), promoter enrichment(Pro En), subsampling enrichment(Sub En), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package(AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity(20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed q ATACViewer for the userfriendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.

Genome assembly and transcriptome analysis provide insights into the antischistosome mechanism of Microtus fortis

Microtus fortis is the only mammalian host that exhibits intrinsic resistance against Schistosoma japonicum infection.However,the underlying molecular mechanisms of this resistance are not yet known.Here,we perform the first de novo genome assembly of M.fortis,comprehensive gene annotation analysis,and evolution analysis.Furthermore,we compare the recovery rate of schistosomes,pathological changes,and liver transcriptomes between M.fortis and mice at different time points after infection.We observe that the time and type of immune response in M.fortis are different from those in mice.M.fortis activates immune and inflammatory responses on the 10th day post infection,such as leukocyte extravasation,antibody activation,Fc-gamma receptor-mediated phagocytosis,and the interferon signaling cascade,which play important roles in preventing the development of schistosomes.In contrast,an intense immune response occurrs in mice at the late stages of infection and could not eliminate schistosomes.Infected mice suffer severe pathological injury and continuous decreases in cell cycle,lipid metabolism,and other functions.Our findings offer new insights into the intrinsic resistance mechanism of M.fortis against schistosome infection.The genome sequence also provides the basis for future studies of other important traits in M.fortis.