The nucleoatac command line function contains several subcommands used for calling nucleosomes. The "run" subcommand is a wrapper for the other three using standard options-- for more flexibility, run "occ" then "vprocess" then "nuc". For each subcommand, options can be viewed as `nucleoatac subcommand --help`. ###Necessary Files Prior to using nucleoatac to call nucleosomes, you will need at least three types of files: 1) Bam file with aligned reads. These generally will be filtered for reads not mapping to mitochondria & reads with high mapping quality. 2) Fasta file with genome used in alignment. This file must be indexed (using [samtools](http://www.htslib.org/) faidx) 3) Sorted bed file with regions for which nucleosome analysis is to be performed. These regions will generally be broad open-chromatin regions (i.e. regions called by MACS2 with the --broad flag). It is potentially advisable to extend these regions a bit (e.g. using bedtools slop). Regions should not overlap so it is advisable to use bedtools merge on these regions. ###run For calling nucleosomes with mostly default parameters, use ``` nucleoatac run --bed --bam --fasta --out ``` Outputs: See outputs for occ, vprocess, and nuc ###occ ``` nucleoatac occ --bed --bam --out ``` Outputs: * *output_basename.occ.bedgraph.gz*: Bedgraph track with nucleosome occupancy score * *output_basename.occ.lower_bound.bedgraph.gz*: Bedgraph track with lower bound estimate for nucleosome occupancy score * *output_basename.occ.upper_bound.bedgraph.gz*: Bedgraph track with upper bound estimate nucleosome occupancy score * *output_basename.nuc_dist.txt* and *output_basename.nuc_dist.eps*: text file and EPS plot with estimate of fragment size at nucleosomes * *output_basename.fragmentsizes.txt*: Text file with fragment size distribution within input peaks * *output_basename.occ_fit.txt* and *output_basename.occ_fit.eps*: text file and EPS plot of model for NFR and nucleosomal distributions * *output_basename.occpeaks.bed.gz*: Peaks from nucleosome occupancy track -- low resolution nucleosome calls. Columns are (1) chrom (2) dyad position (0-based) (3) dyad position (1-based) (4) occupancy (5) occupancy lower bound (6) occupancy upper bound (7) # of reads in 121 bp window ###vprocess ``` nucleoatac vprocess --sizes --out ``` The will generally be the output_basename.nuc_dist.txt output from `occ`. Outputs: * *output_basename.VMat*: text file with normalized V-plot for cross-correlation ###nuc ``` nucleotac nuc ``` * *output_basename.nucpos.bed.gz*: Nucleosome dyad calls text file. Columns are: (1) chrom, (2) dyad position (0-based), (3) dyad position(1-based), (4) z-score, (5) nucleosome occupancy estimate (6) lower bound for nucleosome occupancy estimate, (7) upper bound for nucleosome occupancy estimate (8) log likelihood ratio, (9) normalied nucleoatac signal value, (10) cross-correlation signal value before normalization, (11) number of potentially nucleosome-sized fragments, (12) number of fragments smaller than nucleosome sized, (13) "fuzziness" (measure of how wide signal peak is) * *output_basename.nucpos.redundant.bed.gz*: Includes nucleosome position calls that were within the minimum separation for non-redundant calls. * *output_basename.nucleoatac_signal.bedgraph.gz*: Bedgraph track with normalized cross-correlation signal * *output_basename.nucleoatac_signal.smooth.bedgraph.gz*: Smoothed version of output_basename.nucleoatac_signal.bedgraph.gz Also includes only positive signal * *output_basename.occ.bedgraph.gz*: Bedgraph track with nucleosome occupancy score ###merge ``` nucleoatac merge ``` * *output_basename.nucmap_combined.bed.gz*: Combines low resolution nucleosome calls from occ function and higher resolution calls from nuc to create more comprehensive map ###nfr ``` nucleoatac nfr ``` * *output_basename.nfrpos.bed.gz*: NFR positions. Columns are (1) chrom, (2) left boundary (0-based), (3) right boundary (1-based), (4) mean occupancy, (5) minimum upper bound occupancy, (6) insertion density, (7) bias density