ATAC2GRN: optimized ATAC-seq and DNase1-seq pipelines for rapid and accurate genome regulatory network inference
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Chromatin accessibility profiling assays such as ATAC-seq and DNase1-seq offer the opportunity to rapidly characterize the regulatory state of the genome at a single nucleotide resolution. Optimization of molecular protocols has enabled the molecular biologist to produce next-generation sequencing libraries in several hours, leaving the analysis of sequencing data as the primary obstacle to wide-scale deployment of accessibility profiling assays. To address this obstacle we have developed an optimized and efficient pipeline for the analysis of ATAC-seq and DNase1-seq data.
We executed a multi-dimensional grid-search on the NIH Biowulf supercomputing cluster to assess the impact of parameter selection on biological reproducibility and ChIP-seq recovery by analyzing 4560 pipeline configurations. Our analysis improved ChIP-seq recovery by 15% for ATAC-seq and 3% for DNase1-seq and determined that PCR duplicate removal improves biological reproducibility by 36% without significant costs in footprinting transcription factors. Our analyses of down sampled reads identified a point of diminishing returns for increased library sequencing depth, with 95% of the ChIP-seq data of a 200 million read footprinting library recovered by 160 million reads.
We present optimized ATAC-seq and DNase-seq pipelines in both Snakemake and bash formats as well as optimal sequencing depths for ATAC-seq and DNase-seq projects. The optimized ATAC-seq and DNase1-seq analysis pipelines, parameters, and ground-truth ChIP-seq datasets have been made available for deployment and future algorithmic profiling.
KeywordsDNA footprinting Pipeline ATAC-seq DNase1-seq Regulation Optimization
Assay for Transposase-Accessible Chromatin using sequencing
Area under the curve
DNase1 hypersenstivity profiling
Gene regulatory network
Open chromatin region
Polymerase chain reaction
Receiver operating characteristic (curve)
Regulatory control of RNA expression is a key component of the central dogma of molecular biology. Under the central dogma, protein and RNA products regulate RNA expression. Modules of expression compose expression states associated with specialized biological activities. The information contained within a genome is expressed in a temporally and spatially coordinated program of ever increasing differentiation. This program starts with the embryonic stem cell and grows progressively more specialized. Control over RNA expression is predominantly achieved through the cooperative behavior of multiple proteins that bind DNA called transcription factors (TF) which modify chromatin and recruit RNA polymerase II (RNAP) prior to transcriptional elongation and mRNA expression .
Higher order control over RNA expression has been postulated to exist in terms of an ‘epigenetic landscape’  in which expression of transcription factors at specific developmental states and lineages restricts the possible set of expression modules that can be activated. Thus, cells are limited to a stable expression state vector that is associated with a given lineage and cell type. Multiple RNA profiling technologies have been developed to rapidly and comprehensively identify stable transcriptional states including DNA microarrays and, recently, RNA-seq via Next-Generation Sequencing (NGS) . While RNA profiling technologies may comprehensively identify the RNA stable state associated with a given cell type (or cell in the case of single cell RNA-seq), they do not provide information on the chromatin accessibility patterns and transcription factor binding events that produce this stable state.
Chromatin accessibility profiling was developed to interrogate the genomic circumstances that drives a given expression state . These techniques profile the accessibility of the DNA to enzymatic digestion or transposition, that is regions of the genome occupied by protein complexes (e.g., nucleosomes, transcription factor complexes). Commonly used techniques for chromatin accessibility profiling include DNase1 hypersensitivity profiling (DNase1-seq)  and the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) . Briefly, nuclei are extracted from a sample and treated with concentrations of enzyme that result in fewer cuts/insertions in regions of high nucleosome occupancy. At lower read depths (e.g., 40–60 million reads), the nucleosome occupancy of the genome can be effectively identified. At higher read depths (e.g., > 200 million reads), subtly lower rates of enzyme activity reveal genomic intervals of transcription factor bound DNA, commonly described as ‘footprints’ . These footprints describe a subset of the protein bound genomic DNA and enable genome-wide reconstruction of the cellular gene regulatory network from a single experiment. Importantly, transcription factor binding kinetics and DNA-binding domain types impact the presence and detectability of this footprint, with some TFs leaving well-defined footprints and others leaving poorly-defined footprints . Despite these limitations, DNase1 and ATAC-seq-based genomic footprinting represent a rapid and cost-effective means to build a model of cellular gene regulation that can later be supplemented with traditional gene regulatory network inference methods [9, 10, 11, 12].
The process of cellular gene regulatory network (GRN) reconstruction via TF footprinting is computationally intensive and requires multiple data transformations, each of which have multiple possible parameters that may or may not significantly impact the quality and accuracy of the network generated. To assess the impact of parameter selection on network generation, we executed a computational grid search across eight parameter arguments and five different pipeline steps, with a total of 4560 different pipelines evaluated. The process of evaluating 4560 different pipelines collectively utilized 72,960 CPUs with 3.75 GB RAM each. The mean total time that each pipeline took was 2 h, and running all pipelines on the Biowulf supercomputing system consumed more than 100,000 CPU hours (http://hpc.nih.gov) for each run. The results from each of the 4560 argument combinations was then assessed against ‘ground truth’ ENCODE chromatin immunoprecipitation (ChIP)-seq results describing TF binding within the GM12878 lymphoblastoid cell line for both DNase1 hypersensitivity and ATAC-seq chromatin accessibility profiling . We present the results of this grid search to provide investigators insight to the impact of parameters on pipeline function. Additionally, we provide optimized Snakemake and bash pipelines with pre-configured algorithmic parameters capable of maximizing biological reproducibility and network accuracy for DNase1 hypersensitivity profiling and ATAC-seq .
Footprints increase linearly with reads – ChIP-seq recovery yields diminishing returns with read depths greater than 60 million readz
PCR duplicate removal
When PCR duplication removal was introduced, the reproducibility of footprinting data alignments measured as Pearson correlation between two replicates’ genomic bins increased from 0.6 to 0.82 (Fig. 3a). Introducing PCR duplication removal did not significantly affect ChIP-seq recovery for the footprints produced from Wellington or HINT. Although the pipelines with highest AUCs for HINT incorporated PCR duplicate removal, this improvement was not statistically significant (Fig. 3b). Therefore, PCR duplicate removal did not improve the ability of Wellington TF footprinting to recapitulate ChIP-seq data. Overall, PCR duplicate removal improves biological reproducibility without significant impacts on the detection of transcription factor binding events.
Open chromatin regions
Assessment of replicate correlation scores indicates that smaller peak sizes show higher mean reproducibility scores for both DNase- and ATAC-seq but also limited the area in which footprints could be called. This smaller search space seems to explain the lower AUC seen in lower peak sizes. To confirm that open chromatin regions extended beyond where reads aligned within known regulatory regions, a subset of promoters were visually inspected. Visual inspection of BAM files in IGV indicated that the use of progressively larger peak sizes results in errors of open chromatin region calls in genomic intervals associated with large numbers of aligned reads (Additional file 4: Figure S3a). Thus, a tradeoff exists between expanding footprinting across cis-regulatory regions and containing footprinting within truly open chromatin. The pipelines with the highest recovery of ChIP-seq used peak sizes of 200–500. Altering the minimum distance between peaks had no effect on reproducibility or footprint value (Additional file 4: Figure S3C-E).
Reproducibility vs. transcription factor ChIP recovery
ChIP optimized/reproducibility optimized pipelines
Optimized Snakemake pipelines for ATAC and DNase1 footprinting are available for download at the Chiorini Lab github page (www.github.com/ChioriniLab).
Chromatin accessibility profiling and transcription factor footprinting analyses represent an exciting opportunity to glean new insights on fundamental principles of gene regulation and expression. Herein, we present an optimized and integrated pipeline for the robust analysis of chromatin accessibility data that processes all steps from raw FASTQ input to production of a final, optimized gene regulatory network. These pipelines are freely accessible, and our approaches and data are available for additional optimization as new algorithms for footprinting analysis become available.
Diminishing return on AUC at 60-80 M reads
Recovery of transcription factor footprints within the ATAC/DNase sequencing signal requires deep sequencing to identify the protein bound region of DNA protected from enzymatic cleavage. We identified a linear relationship between read depth and footprints quantity for both the DNase and ATAC-seq data. Our results indicate that even 200 million reads is insufficient to exhaust the biological information gathered from footprinting, an observation also noted in Barozzi et al. . Deeper sequencing and downsampling will be necessary to determine if there is a natural plateau to the number of footprints that can be detected across the genome.
We noted diminishing returns in ChIP recovery as sequencing depth increases over 60–80 million reads for both ATAC and DNase-seq. Mean AUC at the 100 million read depth was within 80% of the 200 million read depth AUCs for both data types, and mean AUC at the 160 million read depth was within 95% of the 200 million read depth mean AUC for both data types. This plateau was observed in all individual TF without obvious differences in DNA binding kinetics between TF. One explanation is that all TF share the same kinetic properties, or footprints detected at higher read ranges describe transient TF binding that is not detectable in a ChIP-seq assay, or that the footprints detected at higher read depths have diminishing accuracy. Regardless, our data supports that 100 million reads is a defensible read depth for a sequencing run and 160 million reads is an optimal read depth.
Reproducibility vs. transcription factor ChIP recovery
Utilizing Picard to remove putative PCR duplicates improves biological reproducibility scores between biological (DNase1-seq) and technical (ATAC-seq) replicates. Similarly, smaller peak sizes of open chromatin regions (OCRs) lead to reproducibility improvements. Because of these small peak sizes, there is no reproducibility-optimized Wellington pipeline, as Wellington requires the sizes of shoulder and footprint sequences to sum to a value greater than 50 base pairs. Picard PCR duplicate removal is used in some AUC-optimized pipelines, whereas small peak sizes are not used in any AUC-optimized pipelines.
PCR duplicate removal takes out identically-ended sequencing reads (reads that look like the result of PCR bias). For example, in a chromatin accessibility assay performed on a population of 50,000 cells, it is expected that the enzyme cuts the same base pair position in multiple genomes at some unknown rate. Pipelines that used PCR duplicate removal exhibited as much as a 50% reduction in the number of footprints found relative to their untreated counterpart pipelines. In our data, TF footprinting detection by Wellington was negatively impacted by PCR duplicate removal, with a mean reduction of 84% of the AUC above random. Similarly, larger peak sizes increase the genomic interval in which footprinting is detected while decreasing the degree of biological reproducibility. Lost opportunities to call footprints, in the case of peak size, or lost information to call footprints, in the case of PCR duplicate removal, may explain the apparent contradictions we observed between increased reproducibility and diminished AUC scores for certain pipelines.
We noted that pipelines optimized for reproducibility in alignment and peak calling do not score well on AUC, and vice versa. This tradeoff is explicable by the two argument differences between these pipelines: HOMER peak size and bias correction. High reproducibility was found in samples with small peak sizes, and visual inspection determined those peaks overlapped with only the highest points of alignment, leading to safe and reliable peak assertions. However, as peaks are the only regions in which footprints are searched for, fracturing of open chromatin regions limits footprinting in regulatory regions and can reduce AUC. HINT’s known DNase bias correction increases reproducibility in DNase-seq footprints but reduces mean AUC. This phenomenon is currently inexplicable. Interestingly, none of the evaluated bias correction methods improved ATAC-seq footprint reproducibility or mean AUC.
It is worth noting that overlap of footprints between replicates was higher in DNase1-seq pipelines optimized for reproducibility relative to AUC-optimized or default pipelines, and lower in ATAC-seq pipelines optimized for reproducibility relative to AUC-optimized or default pipelines. It is not clear why this is the case. Using an AUC-optimized pipeline with HINT results in the highest biological information possible gleaned from footprinting.
We also found that AUCs representing ChIP recovery for ATAC-seq data in GM12878 were universally lower than ChIP recovery AUCs from DNase-seq data. Our optimized pipelines for ATAC-seq data used very sensitive alignment but were otherwise identical to optimized pipelines for DNase-seq for each algorithm. It may be the case that algorithms have been optimized on DNase-seq data and modes for using ATAC-seq data are not mature. Neither optimized pipeline using HINT utilized either known DNase1 bias or bias estimated from the data. As footprinting algorithms continue to include ATAC-seq training data, it is very likely that performance on this kind of data will improve.
Chromatin accessibility profiling assays such as ATAC-seq and DNase1-seq offer the opportunity to rapidly characterize the regulatory state of the genome at a single nucleotide resolution. Significant optimization of molecular protocols has enabled the generation of sequencing libraries in several hours. The remaining computational analysis of next generation sequencing data is the primary obstacle to wide-scale deployment of accessibility profiling assays (e.g., ATAC-seq). To address this obstacle, we have developed an optimized and efficient pipeline for the analysis of ATAC-seq and DNase1-seq data.
Our pipeline was designed to maximize ChIP recovery, the current ‘gold standard’ in transcription factor occupancy assessment. In addition, we performed a computationally intensive grid search across 4560 possible argument combinations to identify the impact of parameter selection on pipeline performance. Our analyses identified the optimal argument combinations for ATAC-seq and DNase1 analysis and these pre-optimized pipelines were translated into Snakemake pipelines for routine and reproducible deployment to supercomputing clusters. Each iteration of these analyses required approximately 11.5 CPU years to reach completion and are being made freely available at the Chiorini Lab Github to avoid duplication of effort within the scientific community. Additionally, the raw datasets and code for pipeline validation have been made publically available allowing investigators to easily profile, and rapidly improve, future pipelines and algorithms for transcription factor footprinting.
Genome wide chromatin accessibility mapping offers a unique window into the regulatory state of the cell by allowing an investigator to rapidly map cellular genomic states. We anticipate continued development in algorithms and molecular protocols that will improve the accuracy and efficiency of gene regulatory network inference from ATAC/DNase sequencing. As chromatin profiling is a valuable tool to characterize the networks and TF binding events that define a cell’s operation, we expect that chromatin profiling assays will continue to see use in cancer genomics, cell engineering, and monogenic diseases cases evading exomic diagnosis. The optimized analysis pipelines and quantitative profiling metrics for construction of ATAC/DNase-seq networks provided here contribute to the scientific community by providing an easily accessed platform for analysis and pipeline engineering.
Following quality control, the second step is to align reads to the genome using Bowtie2 . A series of alignment arguments are passed to the pipeline. These first two arguments specify if Bowtie2 is to align reads globally or locally as well as sensitively or very sensitively. A global alignment subtracts overhangs in the sequenced reads from the alignment score, whereas a local does not. “Sensitive” and “very sensitive” are modes of alignment used by Bowtie2. Running Bowtie2 on “very sensitive” causes the program to attempt more potential alignments than “sensitive” before discarding or aligning a read.
In the third step, aligned reads are converted to a binary format using (as the third argument) either SAMtools  or the Broad Institute’s Picard package (http://broadinstitute.github.io/picard). These tools are then used to sort and index the aligned reads. If Picard is used to convert the reads to binary format, the aligned reads are also queued for PCR duplicate removal via the Picard MarkDuplicates function. PCR duplicates are identical copies of reads which can introduce PCR bias to the TF footprinting analysis by over-representing a small number of highly duplicated genomic regions. In genomic footprinting, PCR duplicate removal may lower bias or, alternatively, discard biological information.
In the fourth step, the aligned reads are analyzed by the HOMER  findpeaks function to identify open chromatin regions (OCRs). OCRs are operationally described as intervals of the genome with higher aligned read density than background. Functionally, OCRs are therefore regions of the genome with low nucleosome occupancy that demonstrate higher enzymatic accessibility and read signal. Following OCR identification, the computationally expensive HINT  and Wellington  footprinting algorithms are targeted to these areas of the genome HOMER identified as peaks to search for TF footprints using alignment information. The “peak height” of an OCR corresponds to the number of reads that align to that region and is a metric that quantitatively describes the chromatin accessibility of a genomic interval. The two arguments passed to HOMER to process aligned reads are the OCR’s sizes, which are the size in base pairs of the regions that HOMER uses to scan across the genome (and are thus the lower limit on the length of a peak); and the minimum distance allowed between OCRs. These are the fourth and fifth arguments in the tested pipelines.
The final step of the analysis pipeline is to pass the aligned reads and OCRs to a TF footprinting identification algorithm. We evaluated two footprinting algorithms, Wellington and HINT. Wellington utilizes a beta-binomial distribution to estimate footprints and leverages the observation that enzymatically accessible genomic DNA in the nucleus results in strand-specific increases in enzyme activity around transcription factor footprints. Wellington requires two arguments: a p-value cutoff and a desired false discovery rate. HINT is a hidden Markov model based footprinting algorithm that incorporates bias correction for known DNase1 digestion biases. Usefully, HINT also incorporates functions that enable de novo estimation of enzymatic bias from input data. The only parameter that can be passed to HINT is the mode of bias correction. Both Wellington and HINT return genomic intervals of predicted protein binding. These proposed protein binding intervals are then intersected on the genome with transcription factor position weight matrices to generate predicted transcription factor binding intervals and a final gene regulatory network.
Pipeline validation metrics
The data used to validate this pipeline came from GM12878, a lymphoblastoid cell line. DNase1-seq data used to validate the pipeline is ENCODE data  from the Stamatoyannopolous and Crawford labs downloadable at https://www.encodeproject.org/experiments/ENCSR000EJD/ and https://www.encodeproject.org/experiments/ENCSR000EMT/ , respectively. ATAC-seq data used to validate the pipeline was acquired from Buenrostro et al.  via the Gene Expression Omnibus repository GSE47753. Biological reproducibility was assessed by assembling at least two independently sampled datasets for each source (ATAC-seq and DNase1-seq). All the ATAC-seq data was produced within the Buenrostro Lab, whereas the DNase1-seq data is replicated across the Stamatoyannopoulos lab and Crawford labs, potentially impacting observed reproducibility due to technical and biological variance.
Three metrics of data reproducibility were assayed across the pipeline. The first metric was the correlation of sequence read alignments. Under this metric, the genome is divided into 10 kilobase bins and the number of reads in each bin is compared between replicates for each bin. A summary Pearson correlation coefficient is then calculated, with higher Pearson correlation coefficients between genome bin sets suggestive of similar patterns of aligned reads across the genome.
The second metric of reproducibility quantitatively assesses OCR reproducibility between replicates. Peaks are averaged across 10 kilobase bins and compared between replicates to produce a Pearson correlation coefficient that summarizes the reproducibility of the assay to detect OCRs across multiple replicates.
The final reproducibility metric assayed was the percentage of TF footprints detected as present and overlapping in two biological replicates. This metric assesses the ability of the assay to detect biologically reproducible TF binding events.
In addition to the three metrics of reproducibility, we assessed the ability of chromatin accessibility based data to recover transcription factor binding by comparing identified footprints to the ‘gold standard’ of ChIP-seq for each footprint. Transcription factor ChIP data for BHLHE40, BRCA1, CTCF, EBF1, IRF3, MXI1, NFE2, NFYA, NFYB, NRF1, STAT1, STAT3, USF2, ZNF143 and ZNF274 was downloaded from ENCODE. This data was compared to footprint coverage of the genome at all positions matching that transcription factor’s binding motif to assess the ability of transcription factor footprinting to recover known ChIP-seq binding information.
Software used in the pipeline includes FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), seqtk (https://github.com/lh3/seqtk), Bowtie2 v.2.2.9 , SAMtools v.1.3.1 , HOMER v.4.8.2 , BEDtools v.2.25.0 , BEDops v.2.4.26 , pyDNase v.0.2.4 , R v.3.4, deepTools v.2.2.4 , Picard v.2.9.2, UCSC tools v.344 , RGT v.0.9.9 [20, 21], and Trimmomatic v.0.36 . All code was translated to the Snakemake  pipeline management platform to streamline scientific supercomputing deployment and maximize data reproducibility. This work utilized the computational resources of the NIH HPC Biowulf cluster. (http://hpc.nih.gov).
These software packages were selected based on the original footprinting tutorial from Piper and found at https://pythonhosted.org/pyDNase/tutorial.html. The tested pipelines use software used both in that tutorial and in the Hardison ATAC-seq pipeline found at https://www.encodeproject.org/pipelines/ENCPL035XIO/.
ATAC-seq and DNase-seq data was downsampled using seqtk (https://github.com/lh3/seqtk) to 20, 40, 60, 80, 100, 120, 140, 160 and 200 million reads. Random seeds for seqtk were set to the same value as the number of reads being downsampled to and three permutations of the data were iteratively sampled. Downsampled samples were used to assess the impact of read number on the total number of transcription factor footprints detected and AUC recovery.
ATAC-seq fastq reads were trimmed using Trimmomatic  to remove Tn5 tagmentation adapters. The final length of the trimmed determined to be mappable without a high expected rate of random alignment was 20 and Trimmomatic was called with the arguments HEADCROP:20 CROP:20.
Alignment was performed with Bowtie2 . Alignments were performed using the --sensitive (default), −-sensitive-local, −-very-sensitive, and --very-sensitive-local pre-configured alignment parameters. Default alignments are global, requiring the entire read to align to the genome.
To process alignments for downstream analysis, SAMtools  was used to process reads without the removal of PCR duplicates. The impact of PCR duplicate removal was assessed via the use of Picard to process reads and remove PCR duplicates. The SAMtools processes used were view, sort and index, while the Picard processes used were SortSam, MarkDuplicates and BuildBamIndex.
Open chromatin regions
Open chromatin regions were identified from alignment files using HOMER  makeTagDirectory, findPeaks and pos2BED functions, as well as BEDtools  sort and merge functions. These bedfiles representing open chromatin regions were analyzed by the footprint detection algorithms, HINT  and Wellington .
To compute reproducibility in alignment space, deepTools  multiBamSummary and plotCorrelation were used to summarize the alignments in genomic bins of size 10 kb across the hg19 genome and compare these alignments between two biological replicates. High Pearson correlation coefficients describe pipelines in which alignments are mapping to the same genomic bins between two replicates.
To compute reproducibility in open chromatin region calling across the genome, deepTools’ multiBigwigSummary and plotCorrelation were used to quantify the similarity in open chromatin calling between replicates using 10 kb bins. The average score for open chromatin was calculated and compared between bins. High Pearson correlation coefficients are assigned to pipelines in which both replicates have open chromatin regions in the same genomic bins.
To produce PWM hits for individual proteins, the cisBP  database was mapped to the human hg19 genome with FIMO using a p-value cutoff of 1E-04 . Transcription factor binding was assessed via analysis of ChIP-seq data for GM12878 acquired from the ENCODE  consortium from data accessions beginning in ENCSR000D and ending in NN, ZS, ZN, ZU, ZO, ZM, ZI, NM, ZY, ZX, ZJ, ZQ, ZL, ZV and YW.
The ability of the pipeline to recapitulate known transcription factor binding events was assessed by comparison of the TF footprint predictions with known TF binding events from ChIP-seq data. Footprints overlapping known TF binding intervals via ChIP-seq were assessed as true positives, while footprints without overlapping ChIP-seq peaks were labeled as false positives. ChIP-supported protein binding sites that did not overlap a footprint were considered false negatives, and ChIP negative, footprint negative binding sites were considered to be true negatives. Predictions from footprints were ordered by score from Wellington or HINT, and binding site predictions that didn’t overlap with predictions from footprints were ordered randomly. Receiver operating characteristic (ROC) curves were produced from these predictions and the area under curve (AUC) for each transcription factor was calculated. Across all of the transcription factors tested, there were on average 10 negative TF binding sites for each positive TF binding site.
This work was supported by an NIH NIDCR intramural research grant to J.A.C. The authors thank Dr. Stephen Parker and Dr. Michael Erdos for critical review and helpful discussion.
This work was supported by a NIDCR, NIH intramural grant to JAC.
Availability of data and materials
The datasets supporting the conclusions of this article are available in the NIDCR Box repository at https://nidcr.box.com/s/g10jqfbya9dcw0y1puhij4ri594del1g. The code used is available on Github at https://github.com/ChioriniLab/ATAC2GRN.
TJFP, DGM and JAC designed research; TJFP performed research and analysis and wrote the paper; TJFP, DGM and JAC edited and approved the final manuscript.
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