diff --git a/.gitignore b/.gitignore new file mode 100644 index 0000000..269d309 --- /dev/null +++ b/.gitignore @@ -0,0 +1,14 @@ +#compile python modules +*.pyc + +#Setuptools distribution folder +/dist/ + +#Python egg metadata, regenerated from source files by setuptools +/*.egg-info +/*.egg + +# +.DS_Store + + diff --git a/LICENSE b/LICENSE new file mode 100644 index 0000000..95fbd0a --- /dev/null +++ b/LICENSE @@ -0,0 +1,22 @@ +The MIT License (MIT) + +Copyright (c) 2015 Stanford University + +Permission is hereby granted, free of charge, to any person obtaining a copy +of this software and associated documentation files (the "Software"), to deal +in the Software without restriction, including without limitation the rights +to use, copy, modify, merge, publish, distribute, sublicense, and/or sell +copies of the Software, and to permit persons to whom the Software is +furnished to do so, subject to the following conditions: + +The above copyright notice and this permission notice shall be included in all +copies or substantial portions of the Software. + +THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR +IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, +FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE +AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER +LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, +OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE +SOFTWARE. + diff --git a/MANIFEST.in b/MANIFEST.in new file mode 100644 index 0000000..cecafe0 --- /dev/null +++ b/MANIFEST.in @@ -0,0 +1,5 @@ +include README.md +include LICENSE +include nucleoatac/standard_vplot.VMat +include example/* + diff --git a/README.md b/README.md new file mode 100644 index 0000000..cfc9f74 --- /dev/null +++ b/README.md @@ -0,0 +1,57 @@ +# NucleoATAC +Python package for calling nucleosomes using ATAC-Seq data. + +Non-Python Dependencies: + +* bedGraphToBigWig (from USCS Genome Browser: http://hgdownload.cse.ucsc.edu/admin/exe/) +(must be on PATH) + +Python Dependencies: + +* python 2.7 +* bx-python +* scipy +* numpy +* pysam +* cython +* matplotlib + +Tested only on Ubuntu OS. + +Installation: + +1. Clone repository. +2. Run `python setup.py install` within main directory +(use --user flag for user specific install. See https://docs.python.org/2/install/ for more details) + +Necessary files prior to calling nucleosomes: +* BAM file with aligned data. Generally this will be filtered for high mapping quality, etc. +* Bed file with open chromatin regions. This file must be sorted, and must not contain any overlapping regions! +* Tn5 bias track in bigWig format (or alternatively, gDNA insertions). This must span all regions in bed file (with several hundred additional flanking bases)! + + +How to call nucleosomes with mostly default parameters: + +``` +nucleoatac run --bed <bed_file> --bam <bam_file> --bias <Tn5_bias_track> --out <output_basename> --cores <num_cores_to_use> +``` + +This command is actually running 3 different commands. For greater versatility, run each separately. To see options, use help: + +``` +nucleoatac occ --help +``` + +``` +nucleoatac vprocess --help +``` +``` +nucleoatac nuc --help +``` + + +There is an additional nucleoatac call that can be made (to make a V-plot for specific sites): +``` +nucleoatac vplot --help +``` + diff --git a/bias_model/Human.PWM.txt b/bias_model/Human.PWM.txt new file mode 100644 index 0000000..c672a61 --- /dev/null +++ b/bias_model/Human.PWM.txt @@ -0,0 +1,4 @@ +0.840670406462 0.9666090742495 0.929684509606 1.071886557995 0.7997846167775 1.185153593475 0.822059024821 0.7357916346195 0.806541836888 0.643302166961 1.24334576292 1.28021185724 1.097136548525 1.27563558011 0.6086877368615 1.107767288192 0.8366320562835 0.924266259849 0.783703051031 0.8228817857685 1.076371510055 +1.05083196339 0.953504938524 1.29824892591 0.9388735168795 1.61692228526 0.7012930269375 1.122145787175 1.146965873215 1.2830066904 1.43811004046 0.665358216826 0.672207685113 0.8559204963725 0.8365523557765 1.69890306358 0.876213491886 0.9074774433175 1.066666539765 1.1151256444 1.350103850425 1.068801376385 +1.068801376385 1.350103850425 1.1151256444 1.066666539765 0.9074774433175 0.876213491886 1.69890306358 0.8365523557765 0.8559204963725 0.672207685113 0.66710672673 1.43811004046 1.2830066904 1.146965873215 1.122145787175 0.7012930269375 1.61692228526 0.9388735168795 1.29824892591 0.953504938524 1.05083196339 +1.076371510055 0.8228817857685 0.783703051031 0.924266259849 0.8366320562835 1.107767288192 0.6086877368615 1.27563558011 1.097136548525 1.28021185724 1.21947260924 0.643302166961 0.806541836888 0.7357916346195 0.822059024821 1.185153593475 0.7997846167775 1.071886557995 0.929684509606 0.9666090742495 0.840670406462 diff --git a/bias_model/README.txt b/bias_model/README.txt new file mode 100644 index 0000000..bbf4d66 --- /dev/null +++ b/bias_model/README.txt @@ -0,0 +1,6 @@ +Human.PWM.txt and Yeast.PWM.txt were derived from mapped read starts from genomic samples from Human and Yeast nextera sequences, respectively. + +ScoreSequence.py was used to generate scores for individual chromosomes using the PWM files. + + + diff --git a/bias_model/ScoreSequence.py b/bias_model/ScoreSequence.py new file mode 100644 index 0000000..aba5fcc --- /dev/null +++ b/bias_model/ScoreSequence.py @@ -0,0 +1,139 @@ +#!/usr/bin/env python + +##### IMPORT MODULES ##### +# import necessary for python +from __future__ import division +import os +import sys +import string +import datetime +from optparse import OptionParser +import numpy as np +import gzip +from Bio import SeqIO +from Bio.Seq import Seq, reverse_complement + +#### OPTIONS #### +# read options from command line +opts = OptionParser() +opts.add_option("-i", "--input", default="", +help="<inputFile> Accepts FASTA file with single Sequence") +opts.add_option("-p", "--pwm", default="", +help="<pwmFile> File with normalized Tn5 transposon preference. Insertion point should be 11th position") +opts.add_option("-c", "--chrN", default="", +help="<chrN> name of chromosome (i.e. chr1)") +opts.add_option("-o", "--out", default="", +help="<outFile> Base name for Output file") +options, arguments = opts.parse_args() + +##### INPUTS AND OUTPUTS ##### +in_file = options.input +pwm_file = options.pwm +chrN=options.chrN +a=10#bases around transposition center included in motif + +if options.input == "": + sys.exit("Requires an input file, use --input to designate. Accepts FASTA files.") + +if options.out=="": + out = os.path.splitext(os.path.basename(in_file))[0] +else: + out=options.out + + +#open PWM file +pwm=np.loadtxt(pwm_file) +w=len(pwm[0]) +pwm=np.log(pwm) + +##write function to determine the pwm score... +def scorer_mono(seq,m): + #m should be array of size 4 * len(seq) + S=0 + for i in range(len(seq)): + if seq[i]=='A': + S=S+m[0][i] + elif seq[i]=='C': + S=S+m[1][i] + elif seq[i]=='G': + S=S+m[2][i] + elif seq[i]=='T': + S=S+m[3][i] + #logS=np.log(S) + return S + +def scorer_di(seq,m): + #m should be array of size 16 * len(seq) + S=0 + for i in range((len(seq)-1)): + if seq[i:(i+2)]=='AA': + S=S+m[0][i] + elif seq[i:(i+2)]=='AC': + S=S+m[1][i] + elif seq[i:(i+2)]=='AG': + S=S+m[2][i] + elif seq[i:(i+2)]=='AT': + S=S+m[3][i] + elif seq[i:(i+2)]=='CA': + S=S+m[4][i] + elif seq[i:(i+2)]=='CC': + S=S+m[5][i] + elif seq[i:(i+2)]=='CG': + S=S+m[6][i] + elif seq[i:(i+2)]=='CT': + S=S+m[7][i] + elif seq[i:(i+2)]=='GA': + S=S+m[8][i] + elif seq[i:(i+2)]=='GC': + S=S+m[9][i] + elif seq[i:(i+2)]=='GG': + S=S+m[10][i] + elif seq[i:(i+2)]=='GT': + S=S+m[11][i] + elif seq[i:(i+2)]=='TA': + S=S+m[12][i] + elif seq[i:(i+2)]=='TC': + S=S+m[13][i] + elif seq[i:(i+2)]=='TG': + S=S+m[14][i] + elif seq[i:(i+2)]=='TT': + S=S+m[15][i] + #logS=np.log(S) + return S + +def Scorer(seq,m): + if len(m)==4: + return scorer_mono(seq,m) + elif len(m)==16: + return scorer_di(seq,m) + +#Get sequence to score + +#if no chromosome is given, just use first sequence in fasta input +if chrN=='': + rec=SeqIO.parse(in_file,'fasta').next() + source=rec.seq + chrN=rec.id +else: + for rec in SeqIO.parse(in_file,'fasta'): + if rec.id==chrN: + source=rec.seq + +Seq_length=len(source) + +#open output file for writing scores +if options.chrN!='': + output=open(out+'.'+chrN+'.Scores.bed','w') +else: + output=open(out+'.Scores.bed','w') + + +for i in range(a,Seq_length-w+a): + forwardseq=str(source[i-a:i+w-a]).upper() + f=Scorer(forwardseq,pwm) + output.write('\t'.join([chrN,str(i),str(i+1),str(f)])+'\n') + + +output.close() + + diff --git a/bias_model/Yeast.PWM.txt b/bias_model/Yeast.PWM.txt new file mode 100644 index 0000000..9c26385 --- /dev/null +++ b/bias_model/Yeast.PWM.txt @@ -0,0 +1,4 @@ +0.8249902267275 0.963678911394 0.977394731096 1.094331796415 0.838984693388 1.140060661135 0.8634411405355 0.7351558672495 0.8570707149595 0.657202162157 1.24925773037 1.325312968185 1.11984130425 1.322947131275 0.617562709625 1.052434771076 0.8703150237785 0.9013347604265 0.784602391176 0.79790349428 1.073410201865 +1.080695856195 0.957660337654 1.292056428965 0.8915020935565 1.6430442671 0.718367258387 1.14008567413 1.09189268792 1.22147931095 1.34084133077 0.614913565933 0.687329339637 0.815718787164 0.814494923033 1.696092676885 0.971494577519 0.8253162076685 1.11547971422 1.09140085467 1.426465114795 1.082995843845 +1.082995843845 1.426465114795 1.09140085467 1.11547971422 0.8253162076685 0.971494577519 1.696092676885 0.814494923033 0.815718787164 0.687329339637 0.614126251469 1.34084133077 1.22147931095 1.09189268792 1.14008567413 0.718367258387 1.6430442671 0.8915020935565 1.292056428965 0.957660337654 1.080695856195 +1.073410201865 0.79790349428 0.784602391176 0.9013347604265 0.8703150237785 1.052434771076 0.617562709625 1.322947131275 1.11984130425 1.325312968185 1.22925904295 0.657202162157 0.8570707149595 0.7351558672495 0.8634411405355 1.140060661135 0.838984693388 1.094331796415 0.977394731096 0.963678911394 0.8249902267275 diff --git a/bin/nucleoatac b/bin/nucleoatac new file mode 100644 index 0000000..3458a5c --- /dev/null +++ b/bin/nucleoatac @@ -0,0 +1,264 @@ +#!/usr/bin/env python +""" +Script to run nucleoatac. +Various functions for calling nucleosomes using ATAC-Seq data. + +Copyright (c) 2015, Alicia Schep <aschep@stanford.edu> + +This code is free software; you can redistribute it and/or modify it under the terms +of the MIT License (see the file COPYING included with the distribution). + +@author: Alicia Schep +@version: 0 +@contact: aschep@stanford.edu +""" + +###-----------Import modules---------------#### + +import sys +import argparse +from nucleoatac.Magic import * +import time + +#from nucleoatac import occ, vprocess, nuc + +def main(): + """The Main function for calling nucleoatac + + """ + #Parse options... + parser = nucleoatac_parser() + args = parser.parse_args() + call = args.call + if call == "occ": + from nucleoatac.run_occ import run_occ + print('---------Computing Occupancy and Nucleosomal Insert Distribution----------------') + run_occ(args) + elif call == "vprocess": + from nucleoatac.run_vprocess import run_vprocess + print('---------Processing VPlot-------------------------------------------------------') + run_vprocess(args) + elif call == "nuc": + from nucleoatac.run_nuc import run_nuc + print('---------Obtaining nucleosome signal and calling positions----------------------') + run_nuc(args) + elif call == "run": + occ_args = parser.parse_args(map(str,['occ','--bed',args.bed,'--bam',args.bam,'--out',args.out,'--cores',args.cores])) + vprocess_args = parser.parse_args(['vprocess','--isizes',args.out+'.nuc_dist.txt','--out',args.out]) + nuc_args_list = ['nuc','--bed',args.bed,'--bam',args.bam,'--out',args.out,'--cores', str(args.cores), + '--occ_track', args.out + '.occ.bw','--vmat', args.out + '.VMat'] + if args.bias is not None: + nuc_args_list.extend(['--bias', args.bias]) + elif args.gdna is not None: + nuc_args_list.extend(['--gdna', args.gdna]) + if args.write_all: + nuc_args_list.extend(['--write_all']) + nuc_args = parser.parse_args(nuc_args_list) + from nucleoatac.run_occ import run_occ + from nucleoatac.run_vprocess import run_vprocess + from nucleoatac.run_nuc import run_nuc + print('---------Step1: Computing Occupancy and Nucleosomal Insert Distribution---------') + run_occ(occ_args) + print('---------Step2: Processing Vplot------------------------------------------------') + run_vprocess(vprocess_args) + print('---------Step3: Obtaining nucleosome signal and calling positions---------------') + run_nuc(nuc_args) + elif call == "vplot": + from nucleoatac.make_vplot import make_vplot + print('---------Making VPlot-----------------------------------------------------------') + make_vplot(args) + + +def nucleoatac_parser(): + """Prepares argparse object + + """ + argparser = argparse.ArgumentParser(description = "%(prog)s -- Nucleosome Calling for ATAC-Seq", + epilog = "For command line options for each command, type %(prog)s COMMAND -h") + argparser.add_argument("--version", action="version", version="%(prog)s "+NUCLEOATAC_VERSION) + subparsers = argparser.add_subparsers(dest = 'call' ) + + add_run_parser( subparsers) + + add_occ_parser( subparsers) + + add_vprocess_parser( subparsers) + + add_nuc_parser( subparsers) + + add_vplot_parser( subparsers) + + + return argparser + +def add_occ_parser( subparsers): + """Add argument parsers for the occ utility + + """ + parser = subparsers.add_parser("occ", help = "nucleoatac function: Call nucleosome occupancy") + group1 = parser.add_argument_group('Required', 'Necessary arguments') + group1.add_argument('--bed', metavar='bed_file' , help = 'Peaks in bed format', required=True) + group1.add_argument('--bam', metavar='bam_file', + help = 'Sorted (and indexed) BAM file', required=True) + group1.add_argument('--out', metavar='basename', + help="give output basename", required=True) + group2 = parser.add_argument_group('General Options', '') + group2.add_argument('--write_peaks', action="store_true", default = False, + help ="write bed file of occupancy peaks") + group2.add_argument('--cores', metavar = 'int',default=1, + help='Number of cores to use',type=int) + group3 = parser.add_argument_group('Occupancy parameter', 'Change with caution') + group3.add_argument('--lower',metavar="int",default=105, + help="lower limit in insert size. default is 105",type=int) + group3.add_argument('--upper',metavar="int",default=251, + help="upper limit in insert size. default is 251",type=int) + group3.add_argument('--flank',metavar="int",default=60, + help="Distance on each side of dyad to include",type=int) + group3.add_argument('--min_occ', metavar = "float", default=0.1,type=float, + help="Occupancy cutoff for determining nucleosome distribution. Default is 0.1") + group3.add_argument('--nuc_sep',metavar='int',default=120,type=int, + help = "minimum separation between occupany peaks") + group3.add_argument('--reads', metavar = "int", default=1, type= int, + help="Reads cutoff for determining nucleosome distribution. Default is 1.") + return + +def add_vprocess_parser( subparsers): + """Add argument parsers for the vprocess utility + + """ + parser = subparsers.add_parser("vprocess", help = "nucleoatac function: Make processed vplot to use for nucleosome calling") + group1 = parser.add_argument_group('Required', 'Necessary arguments') + group1.add_argument('--out', metavar='output_basename',required=True) + group2 = parser.add_argument_group('VPlot and Insert Size Options', 'Optional') + group2.add_argument('--isizes', metavar='file' , help = 'Insert distribution file') + group2.add_argument('--vplot', metavar='vmat_file', + help = 'Accepts VMat file. Default is Vplot from S. Cer.') + group3 = parser.add_argument_group('Size parameers', 'Use sensible values') + group3.add_argument('--lower',metavar="int",default=105, + help="lower limit (inclusive) in insert size. default is 105",type=int) + group3.add_argument('--upper',metavar="int",default=251, + help="upper limit (exclusive) in insert size. default 251",type=int) + group3.add_argument('--flank',metavar="int",default=60, + help="distance on each side of dyad to include",type=int) + group4 = parser.add_argument_group('Options', '') + group4.add_argument('--smooth', metavar = "float", default = 0.75, type = float, + help="SD to use for gaussian smoothing. Use 0 for no smoothing.") + group4.add_argument('--plot_extra', action='store_true',default=False, + help="Make some additional plots") + return + +def add_nuc_parser( subparsers): + """Add argument parsers for the nuc utility + + """ + parser = subparsers.add_parser("nuc", help = "nucleoatac function: Call nucleosome positions and make signal tracks") + group1 = parser.add_argument_group('Required', 'Necessary arguments') + group1.add_argument('--bed', metavar='bed_file' , help = 'Regions for which \ + to do stuff.', required=True) + group1.add_argument('--vmat', metavar='vdensity_file', help = "VMat object", required=True) + group1.add_argument('--bam', metavar='bam_file', + help = 'Accepts sorted BAM file', required=True) + group1.add_argument('--out', metavar='basename', + help="give output basename", required=True) + group2 = parser.add_argument_group("Encouraged","Use only one of these options") + group2.add_argument('--bias', metavar = 'bias_track', + help = 'Bigwig format') + group2.add_argument('--gdna', metavar = 'gdna_track', + help = 'Bigwig format') + group3 = parser.add_argument_group('General options', '') + group3.add_argument('--cores', metavar = 'num_cores',default=1, + help='Number of cores to use',type=int) + group3.add_argument('--write_all', action="store_true", default = False, + help="write all tracks") + group3.add_argument('--occ_track', metavar='bw_track', + help = 'occupancy track. should be in bw file format. prevent re-calculation of occ if already calculated') + group5 = parser.add_argument_group('Nucleosome/NFR calling parameters','Change with caution') + group5.add_argument('--min_z', metavar='float', default = 3, + help = 'Z-score threshold for nucleosome calls. Default is 3', type = float) + group5.add_argument('--min_lr', metavar='float', default = 0, + help = 'Log likelihood ratio threshold for nucleosome calls. Default is 0', type = float) + group5.add_argument('--nuc_sep',metavar='int',default=120,type=int, + help = "Minimum separation between non-redundant nucleosomes. Default is 120") + group5.add_argument('--redundant_sep',metavar='int',default=25,type=int, + help = "Minimum separation between redundant nucleosomes. Not recommended to be below 15. Default is 25") + group5.add_argument('--sd',metavar='int', type=int, + default=10, help = "Standard deviation for smoothing. Affect the \ + resolution at which nucleosomes can be positioned. Not recommended to \ + exceed 25 or to be smaller than 10. Default is 10" ) + group5.add_argument('--max_nfr_occ', metavar= 'float', default = 0.25, + help = 'Maximum occupancy for NFR. Default is 0.25', type = float) + group6 = parser.add_argument_group('Down-sampling option','Use only if downsampling desired') + group6.add_argument('--seed', metavar = 'random_seed',type=int, default=500, + help='Random seed; only used if downsampling. Default is 500.') + group6.add_argument('--downsample', metavar = 'downsample_ratio', + type = float, default=1, + help='Fraction of reads to use. Default is to use all reads (1).') + return + + +def add_run_parser( subparsers): + """Add argument parsers for the run utility + + """ + parser = subparsers.add_parser("run", help = "Main nucleoatac utility-- runs through occupancy determination & calling nuc positions") + group1 = parser.add_argument_group('Required', 'Necessary arguments') + group1.add_argument('--bed', metavar='bed_file' , help = 'Regions for which \ + to do stuff.', required=True) + group1.add_argument('--bam', metavar='bam_file', + help = 'Accepts sorted BAM file', required=True) + group1.add_argument('--out', metavar='output_basename', + help="give output basename", required=True) + group2 = parser.add_argument_group("Encouraged","Use only one of these options") + group2.add_argument('--bias', metavar = 'bias_track', + help = 'Bigwig format') + group2.add_argument('--gdna', metavar = 'gdna_track', + help = 'Bigwig format') + group3 = parser.add_argument_group('General options', '') + group3.add_argument('--cores', metavar = 'num_cores',default=1, + help='Number of cores to use',type=int) + group3.add_argument('--write_all', action="store_true", default = False, + help="write all tracks") + return + +def add_vplot_parser( subparsers): + """Add argument parsers for the vplot utility + + """ + parser = subparsers.add_parser("vplot", help = "nucleoatac funcion-- make vplot") + group1 = parser.add_argument_group('Required', 'Necessary arguments') + group1.add_argument('--bed', metavar='bed_file' , help = 'Positions around which \ + to generate VPlot', required = True) + group1.add_argument('--bam', metavar='bam_file', help = 'Accepts sorted BAM file',required=True) + group1.add_argument('--out', metavar='basename', required=True) + group2 = parser.add_argument_group('General options', '') + group2.add_argument('--cores', metavar = 'int',default=1,help = "number of cores to use") + group3 = parser.add_argument_group('VMat option', 'Size, scaling of VPlot') + group3.add_argument('--lower',metavar="int", help = 'lower limit on insert \ + size',default=0,type=int) + group3.add_argument('--upper', metavar="int",help="upper limit on insert \ + size",default=250,type=int) + group3.add_argument('--flank', metavar="int", + help="how many bases on each side of site (or center of site) to include", + type=int,default=250) + group3.add_argument('--scale', action="store_true", default = False) + group3.add_argument('--weight',metavar="int", + type=int,help="column in which weight information is included") + group3.add_argument('--strand',metavar="int", + type=int,help="column in which strand information is included") + group3.add_argument('--not_atac', action="store_false", default = True, help = "Don't use atac offsets") + return + + + +if __name__ == '__main__': + print "Command run: "+ ' '.join(map(str,sys.argv)) + print "nucleoatac version "+NUCLEOATAC_VERSION + print "start run at: " + time.strftime("%Y-%m-%d %H:%M") + try: + main() + print "end run at: " + time.strftime("%Y-%m-%d %H:%M") + except KeyboardInterrupt: + sys.stderr.write("User interrupted nucleoatac.") + sys.exit(0) + + diff --git a/example/README.txt b/example/README.txt new file mode 100644 index 0000000..5a5efdb --- /dev/null +++ b/example/README.txt @@ -0,0 +1,8 @@ + +Example files to test out functions + +example.bam created by intersecting a bam file with example.slopped.bed (which has 1000bp flank on each side of example.bed) + +example.Scores.bw has scores for ranges in example.slopped.bed + + diff --git a/example/example.Scores.bw b/example/example.Scores.bw new file mode 100644 index 0000000..3301944 Binary files /dev/null and b/example/example.Scores.bw differ diff --git a/example/example.VMat b/example/example.VMat new file mode 100644 index 0000000..b30d90b --- /dev/null +++ b/example/example.VMat @@ -0,0 +1,137 @@ +#VMat Descriptor File +#Contains VMat and pertinent information +#i_lower +115 +#i_upper +245 +#mat +0.145162894422 0.117797606792 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0.0863422569016 0.0908862508297 0.0909912457854 0.0926448200329 0.101581421151 0.110723869167 0.102326600539 0.0971752419833 0.102890147184 0.0947878258096 0.0881336898629 0.0876279610229 0.0886429371894 0.0912604543401 0.0967122855392 0.108258950919 0.114365473658 0.107367738984 0.0945909494703 0.0879436854072 0.0832625124926 0.0789510002027 0.0777627727013 0.0855600238552 0.0978514457769 0.108502539265 0.106091985356 0.0993368263853 0.0917522461155 0.0801748481728 0.0704445219058 0.0627491889576 0.0650675269609 0.0746862338577 0.0899715595302 0.0977167879292 0.0907373010392 0.0820265008721 0.0745135844822 0.0659159243007 0.0577723641074 0.0507681036632 0.0466543493863 0.0505759596769 0.057366406584 0.0607254055914 diff --git a/example/example.bam b/example/example.bam new file mode 100644 index 0000000..4755bfe Binary files /dev/null and b/example/example.bam differ diff --git a/example/example.bam.bai b/example/example.bam.bai new file mode 100644 index 0000000..2da130e Binary files /dev/null and b/example/example.bam.bai differ diff --git a/example/example.bed b/example/example.bed new file mode 100644 index 0000000..10e3e05 --- /dev/null +++ b/example/example.bed @@ -0,0 +1,19 @@ +chrII 706612 707705 +chrII 729940 730718 +chrIV 311778 313289 +chrIV 392222 394373 +chrIV 1269060 1271604 +chrIV 1475365 1478960 +chrIX 92428 94830 +chrV 97436 98293 +chrV 191321 197328 +chrV 326413 327608 +chrVI 64431 66036 +chrVII 199495 200698 +chrXI 33449 34725 +chrXII 512984 516410 +chrXII 727436 731427 +chrXIII 586195 586950 +chrXIII 745807 747061 +chrXIV 369889 370746 +chrXIV 762635 764437 diff --git a/example/example.slopped.bed b/example/example.slopped.bed new file mode 100644 index 0000000..8460338 --- /dev/null +++ b/example/example.slopped.bed @@ -0,0 +1,19 @@ +chrII 705612 708705 +chrII 728940 731718 +chrIV 310778 314289 +chrIV 391222 395373 +chrIV 1268060 1272604 +chrIV 1474365 1479960 +chrIX 91428 95830 +chrV 96436 99293 +chrV 190321 198328 +chrV 325413 328608 +chrVI 63431 67036 +chrVII 198495 201698 +chrXI 32449 35725 +chrXII 511984 517410 +chrXII 726436 732427 +chrXIII 585195 587950 +chrXIII 744807 748061 +chrXIV 368889 371746 +chrXIV 761635 765437 diff --git a/example/single_read.bam b/example/single_read.bam new file mode 100644 index 0000000..8b7afdf Binary files /dev/null and b/example/single_read.bam differ diff --git a/example/single_read.bam.bai b/example/single_read.bam.bai new file mode 100644 index 0000000..1404639 Binary files /dev/null and b/example/single_read.bam.bai differ diff --git a/nucleoatac/Magic.py b/nucleoatac/Magic.py new file mode 100644 index 0000000..b4c4cb4 --- /dev/null +++ b/nucleoatac/Magic.py @@ -0,0 +1,3 @@ +#Any constants desired for program + +NUCLEOATAC_VERSION = "0"#version diff --git a/nucleoatac/NucleosomeCalling.py b/nucleoatac/NucleosomeCalling.py new file mode 100644 index 0000000..c77c28b --- /dev/null +++ b/nucleoatac/NucleosomeCalling.py @@ -0,0 +1,496 @@ +#!/usr/bin/env python +""" +Script with classes and functions for nucleosome calling. + +@author: Alicia Schep, Greenleaf Lab, Stanford University +""" + +import numpy as np +from scipy import optimize, signal +import PyATAC as PA +import Occupancy as Occ +from copy import copy +from bisect import bisect_left +import pysam +import bx.bbi.bigwig_file +import pyximport; pyximport.install(setup_args={"include_dirs":np.get_include()}) +import multinomial_cov + + +import warnings +warnings.filterwarnings('error') + + +class SignalTrack(PA.Track): + """Class for getting V-plot signal""" + def __init__(self, chrom, start, end): + PA.Track.__init__(self, chrom, start, end, "signal") + def calculateSignal(self, mat, vmat): + offset=self.start-mat.start-vmat.w + if offset<0: + raise Exception("Insufficient flanking region on \ + mat to calculate signal") + self.vals = signal.correlate(mat.get(vmat.i_lower,vmat.i_upper, + mat.start + offset, mat.end - offset), + vmat.mat,mode = 'valid')[0] + +class NormSignalTrack(PA.Track): + """Class for storing normalized signal track""" + def __init__(self, chrom, start, end): + PA.Track.__init__(self, chrom, start, end, "normalized signal") + def calculateNormSignal(self, raw, bias): + self.vals = raw.get(self.start, self.end) - bias.get(self.start,self.end) + +class BiasTrack(PA.Track): + """Class for getting Bias Signal Track-- Background model""" + def __init__(self, chrom, start, end): + PA.Track.__init__(self, chrom, start, end, "bias") + def calculateBackgroundSignal(self, mat, vmat, nuc_cov): + offset=self.start-mat.start-vmat.w + if offset<0: + raise Exception("Insufficient flanking region on \ + mat to calculate signal") + self.vmat = vmat + self.bias_mat = mat + self.cov = PA.CoverageTrack(self.chrom, self.start, self.end) + self.cov.calculateCoverage(self.bias_mat, vmat.i_lower, + vmat.i_upper, vmat.w*2+1) + self.nuc_cov = nuc_cov.vals + self.vals = signal.correlate(self.bias_mat.get(vmat.i_lower,vmat.i_upper, + self.bias_mat.start + offset, + self.bias_mat.end - offset), + vmat.mat,mode = 'valid')[0] + self.vals = self.vals * self.nuc_cov/ self.cov.vals + + + + + +class SignalDistribution: + """Class for determining distribution of signal""" + def __init__(self, position, vmat, bias_mat, reads): + self.position = position + self.reads = reads + self.vmat = vmat + bias_mat = bias_mat.get(vmat.i_lower,vmat.i_upper,position - vmat.w,position + vmat.w + 1) + self.prob_mat = bias_mat / np.sum(bias_mat) + self.probs = self.prob_mat.flatten() + def simulateReads(self): + sim_vect = np.random.multinomial(self.reads,self.probs) + sim_mat = np.reshape(sim_vect, self.vmat.mat.shape) + return sim_mat + def simulateDist(self, numiters = 1000): + self.scores = map(lambda x: np.sum(self.simulateReads() * self.vmat.mat),range(numiters)) + def analStd(self): + flatv = np.ravel(self.vmat.mat) + var = multinomial_cov.makeCov(self.probs, flatv, self.reads) + return np.sqrt(var) + def analMean(self): + return np.sum(self.prob_mat * self.vmat.mat * self.reads) + + + +def norm(x, v, w, mean): + """compute values of normal pdf with given mean and sd at values in x""" + norm = (1.0/(np.sqrt(2*np.pi*v)) * + np.exp(-(x - mean)**2/(2*v))) + norm = norm * (w/max(norm)) + return norm + +class Nucleosome: + """Class for storing information about a single nucleosome""" + def __init__(self, pos,nuctrack): + self.chrom = nuctrack.chrom + self.pos = pos + self.nfr_cov = nuctrack.nfr_cov.get(pos = pos) + self.nuc_cov = nuctrack.nuc_cov.get(pos = pos) + self.nuc_signal = nuctrack.nuc_signal.get(pos = pos) + self.norm_signal = nuctrack.norm_signal.get(pos = pos) + self.smoothed = nuctrack.smoothed.get(pos= pos) + def getLR(self,nuctrack): + mat = nuctrack.mat.get(nuctrack.params.lower,nuctrack.params.upper, + self.pos - nuctrack.params.vmat.w, self.pos + nuctrack.params.vmat.w +1) + null_mat = nuctrack.bias_mat.get(nuctrack.params.lower,nuctrack.params.upper, + self.pos - nuctrack.params.vmat.w, self.pos + nuctrack.params.vmat.w +1) + bias_mat =nuctrack.bias_mat_prenorm.get(nuctrack.params.lower,nuctrack.params.upper, + self.pos - nuctrack.params.vmat.w, self.pos + nuctrack.params.vmat.w +1) + nuc_model = nuctrack.params.vmat.mat * bias_mat + nuc_model = nuc_model / np.sum(nuc_model) + null_model = null_mat / np.sum(null_mat) + nuc_lik = np.sum(np.log(nuc_model) * mat) + null_lik = np.sum(np.log(null_model) * mat) + self.lr = nuc_lik - null_lik + def getZScore(self, nuctrack): + s = SignalDistribution(self.pos, nuctrack.params.vmat, nuctrack.bias_mat, + self.nuc_cov) + std = s.analStd() + self.z = self.norm_signal / std + def getOcc(self, nuctrack): + self.occ = nuctrack.occ.get(pos = self.pos) + def getFuzz(self, nuctrack): + def addNorms(x,params): + """Add several normal distributions together""" + l = len(x) + fit = np.zeros(l) + i = len(params)/3 + for j in range(i): + fit += norm(x,params[j*3],params[3*j+1],params[3*j+2]) + return fit + def err_func(pars,y): + """error function for normal fit; to be used for fitNorm""" + x = np.linspace(0,len(y)-1,len(y)) + return sum((addNorms(x, pars) - y)**2) + def fitNorm(guess, bound, sig): + """Fit a normal to the signal with lower and upperbounds to sd""" + a = (sig,) + res = optimize.minimize(err_func,guess,args = a, bounds=bound,method="L-BFGS-B") + return res + index = self.pos - nuctrack.start + allnucs = nuctrack.sorted_nuc_keys + x = bisect_left(allnucs,index) + if x == 0: + left = index - nuctrack.params.nonredundant_sep/3 + means = (nuctrack.params.nonredundant_sep/3,) + elif index - allnucs[x-1] < nuctrack.params.nonredundant_sep: + left = allnucs[x-1] + means = (index - allnucs[x-1],0) + else: + left = index - nuctrack.params.nonredundant_sep/3 + means = (nuctrack.params.nonredundant_sep/3,) + if x == len(allnucs)-1: + right = index + nuctrack.params.nonredundant_sep/3 + 1 + elif allnucs[x+1] - index < nuctrack.params.nonredundant_sep: + right = allnucs[x+1] + means += (allnucs[x+1] - left,) + else: + right = index + nuctrack.params.nonredundant_sep/3 +1 + sig = nuctrack.smoothed.vals[left:right] + sig[sig<0] = 0 + if len(means)==1: + bounds = ((2**2,50**2),(0.001,max(sig)*1.1),(means[0]-10,means[0]+10)) + guesses = (nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[0]) + elif len(means)==2: + bounds = ((2**2,50**2),(0.001,max(sig)*1.1),(means[0]-10,means[0]+10), + (2**2,50**2),(0.001,max(sig)*1.1),(means[1]-10,means[1]+10)) + guesses = (nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[0], + nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[1]) + elif len(means)==3: + bounds = ((2**2,50**2),(0.001,max(sig)*1.1),(means[0]-10,means[0]+10), + (2**2,50**2),(0.001,max(sig)*1.1),(means[1]-10,means[1]+10), + (2**2,50**2),(0.001,max(sig)*1.1),(means[2]-10,means[2]+10)) + guesses = (nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[0], + nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[1], + nuctrack.params.smooth_sd ** 2,max(sig)*0.9,means[2]) + res= fitNorm(guesses, bounds, sig) + self.fuzz= np.sqrt(res['x'][0]) + self.weight = res['x'][1] + self.fit_pos = res['x'][2]+left + def writeNucPos(self, handle): + out = [self.chrom, self.pos, self.pos + 1, self.z, self.occ, self.lr, + self.norm_signal, self.smoothed, self.nuc_signal, self.nuc_cov, self.nfr_cov, + self.fuzz] + handle.write("\t".join(map(str, out)) + "\n") + +class NFR: + """Class for storing information about a single NFR region""" + def __init__(self, left, right, nuctrack): + self.chrom = nuctrack.chrom + self.leftpos = left + self.rightpos = right + self.nuc = np.max(nuctrack.nuc_signal.get(left,right)) + self.nfr = np.mean(nuctrack.nfr_cov.get(left,right)) + self.occ = np.mean(nuctrack.occ.get(left,right)) + self.max_occ = np.max(nuctrack.occ.get(left,right)) + def writeNFRPos(self, handle): + out = [self.chrom, self.leftpos, self.rightpos, self.occ, self.nfr, self.nuc, self.max_occ] + handle.write("\t".join(map(str, out)) + "\n") + + +class NucParameters: + """Class for storing parameters related to nucleosome calling""" + def __init__(self, vmat, insertsizes, sd = 25, nonredundant_sep = 120, redundant_sep = 25, + min_z = 3, min_lr = 0, downsample = None, min_reads = 1, + seed = 500, min_nfr_len = 1, max_nfr_len = 1000, max_nfr_occ = 0.2, min_nfr_ins = 0.05, + out = None, bias = None, gdna = None, write_all = False, occ_track = None, + bam = None, occ_params = None): + self.vmat = vmat + self.lower = vmat.i_lower + self.upper= vmat.i_upper + self.window = vmat.mat.shape[1] + self.insertsizes= insertsizes + self.min_reads = min_reads + self.min_z = min_z + self.min_lr = min_lr + self.smooth_sd = sd + self.redundant_sep = redundant_sep + self.nonredundant_sep = nonredundant_sep + self.downsample = downsample + self.seed = seed + self.min_nfr_len = min_nfr_len + self.max_nfr_len = max_nfr_len + self.max_nfr_occ = max_nfr_occ + self.min_nfr_ins = min_nfr_ins + self.out = out + self.bias = bias + self.gdna = gdna + self.write_all = write_all + self.occ_track = occ_track + self.bam = bam + self.occ_params = occ_params + +class NucChunk(PA.ChromChunk): + """Class for storing and determining collection of nucleosome positions + """ + def __init__(self, chrom, start, end): + PA.ChromChunk.__init__(self, chrom, start, end) + def initialize(self, parameters): + self.params = parameters + def extract_reads(self, bamfile): + self.reads = PA.ReadList(self.chrom, self.start - self.params.window, + self.end + self.params.window) + self.reads.extract_reads(bamfile,i_upper=self.params.upper, downsample = self.params.downsample, + seed = self.params.seed) + def getReadMat(self): + self.mat = PA.ReadMat2D(self.chrom, self.start - self.params.window, + self.end + self.params.window, 0, self.params.upper) + self.mat.makeReadMat(self.reads) + def makeBiasMat(self, biasfile = None, gdna = False): + self.bias_mat = PA.BiasMat2D(self.chrom, self.start - self.params.window, + self.end + self.params.window, 0, self.params.upper) + if biasfile and gdna: + bias_track = PA.InsertionBiasTrack(self.chrom, self.start - self.params.window - self.params.upper/2, + self.end + self.params.window + self.params.upper/2 + 1, log = False) + bias_track.read_track(biasfile) + self.bias_mat.makeBiasMat(bias_track) + elif biasfile: + bias_track = PA.InsertionBiasTrack(self.chrom, self.start - self.params.window - self.params.upper/2, + self.end + self.params.window + self.params.upper/2 + 1, log = True) + bias_track.read_track(biasfile) + self.bias_mat.makeBiasMat(bias_track) + self.bias_mat_prenorm = PA.BiasMat2D(self.chrom, self.start - self.params.window, + self.end + self.params.window, 0, self.params.upper) + self.bias_mat_prenorm.mat = copy(self.bias_mat.mat) + self.bias_mat.normByInsertDist(self.params.insertsizes) + def getNucSignal(self): + """Gets Nucleosome Signal Track""" + self.nuc_cov = PA.CoverageTrack(self.chrom, self.start, + self.end) + self.nuc_cov.calculateCoverage(self.mat, self.params.lower, self.params.upper, + self.params.window) + self.bias = BiasTrack(self.chrom, self.start, + self.end) + self.bias.calculateBackgroundSignal(self.bias_mat, self.params.vmat, self.nuc_cov) + self.nuc_signal = SignalTrack(self.chrom, self.start, + self.end) + self.nuc_signal.calculateSignal(self.mat, self.params.vmat) + self.norm_signal = NormSignalTrack(self.chrom, self.start, self.end) + self.norm_signal.calculateNormSignal(self.nuc_signal,self.bias) + def getNFR(self): + """get number of reads of sub-nucleosomal length""" + self.nfr_cov = PA.CoverageTrack(self.chrom, self.start, self.end) + self.nfr_cov.calculateCoverage(self.mat, 0, self.params.lower, + self.params.window) + def smoothSignal(self): + """Smooth thenormalized signal track""" + window_len = 6 * self.params.smooth_sd + 1 + self.smoothed = PA.Track(self.chrom,self.start,self.end, "Smooth Signal") + tmp = copy(self.norm_signal.vals) + self.smoothed.assign_track(tmp) + self.smoothed.vals[ self.smoothed.vals < 0] = 0 + self.smoothed.smooth_track(window_len, window = "gaussian", + sd = self.params.smooth_sd, mode = 'same', + norm = True) + def getOcc(self, occ_bwh = None): + """gets occupancy track-- either reads in from bw handle given, or makes new""" + if occ_bwh is None and self.params.occ_params is None: + raise Exception("Error with getOcc functoin for NucChunk. Need either bw handle or OccCalcParams") + elif occ_bwh is not None and self.params.occ_params is not None: + raise Warning("both occupancy track and parameters given-- will only use track") + if occ_bwh is not None: + self.occ = PA.Track(self.chrom,self.start,self.end,"Occupancy") + self.occ.read_track(occ_bwh) + else: + self.occ = Occ.OccupancyTrack(self.chrom, self.start, self.end) + self.occ.calculateOccupancyMLE_smooth(self.mat, self.params.occ_params) + def findAllNucs(self): + """Find peaks in data""" + self.nuc_collection = {} + combined = self.norm_signal.vals + self.smoothed.vals + #find peaks in normalized sigal + cands1 = PA.call_peaks(combined, min_signal = 0, + sep = self.params.redundant_sep, + boundary = self.params.nonredundant_sep/2, order = self.params.redundant_sep/2) + for i in cands1: + nuc = Nucleosome(i + self.start, self) + if nuc.nuc_cov > self.params.min_reads: + nuc.getLR(self) + if nuc.lr > self.params.min_lr: + nuc.getZScore(self) + if nuc.z >= self.params.min_z: + nuc.getOcc(self) + self.nuc_collection[i] = nuc + self.sorted_nuc_keys = np.array(sorted(self.nuc_collection.keys())) + self.nonredundant = PA.reduce_peaks( self.sorted_nuc_keys, + map(lambda x: self.nuc_collection[x].z, self.sorted_nuc_keys), + self.params.nonredundant_sep) + self.redundant = np.setdiff1d(self.sorted_nuc_keys, self.nonredundant) + def fit(self): + x = np.linspace(0,self.length -1,self.length) + fit = np.zeros(self.length) + for nuc in self.sorted_nuc_keys: + self.nuc_collection[nuc].getFuzz(self) + fit += norm(x,self.nuc_collection[nuc].fuzz**2, self.nuc_collection[nuc].weight, self.nuc_collection[nuc].fit_pos) + self.fitted = PA.Track(self.chrom, self.start, self.end, + "Fitted Nucleosome Signal") + self.fitted.assign_track(fit) + def findNFRs(self): + """Find NFR regions based on nfr reads, nucleosomes""" + self.nfrs = {} + nucpos = [i for i in self.sorted_nuc_keys if self.nuc_collection[i].occ > self.params.max_nfr_occ] + gaps =[nucpos[i] - nucpos[i-1] for i in range(1,len(nucpos))] + for i in range(len(gaps)): + if gaps[i]<self.params.min_nfr_len+147 or gaps[i]>self.params.max_nfr_len+147: + continue + leftnucbound = nucpos[i] + 73 + rightnucbound = nucpos[i+1] - 73 + if max(self.occ.vals[leftnucbound:rightnucbound]) < self.params.max_nfr_occ and np.mean(self.ins.vals[leftnucbound:rightnucbound]) > self.params.min_nfr_ins: + self.nfrs[leftnucbound] = NFR(leftnucbound + self.start, rightnucbound + self.start + 1,self) + def makeInsertionTrack(self): + """make insertion track for chunk""" + self.ins = PA.InsertionTrack(self.chrom, self.start,self.end) + self.ins.calculateInsertions(self.reads) + def process(self, params, bamfile, occfile, biasfile = None, gdna = False): + """wrapper to carry out all methods needed to call nucleosomes and nfrs""" + self.initialize(params) + self.extract_reads(bamfile) + self.getReadMat() + if biasfile is not None: + self.makeBiasMat(biasfile) + elif gdna: + self.makeBiasMat(biasfile, gdna = True) + else: + self.makeBiasMat() + self.getNucSignal() + self.getNFR() + self.smoothSignal() + self.getOcc(occ_bwh = occfile) + self.findAllNucs() + self.fit() + self.makeInsertionTrack() + self.findNFRs() + def removeData(self): + """remove data from chunk-- deletes all attributes""" + names = self.__dict__.keys() + for name in names: + delattr(self,name) + +class NucChunkSet(PA.ChunkSet): + """Class for storing sets of NucChunks + """ + def __init__(self, name, chunks): + PA.ChunkSet.__init__(self, name, chunks) + def process(self, params): + """process chunkset-- for all chunks, perform all methods needed to + obtain and write nucleosome signal and positions + """ + if params.bias is not None and params.gdna is not None: + raise Exception("NucChunkSet.process shoud only have either a bias or gdna input, not both") + bamfile = pysam.Samfile(params.bam, "rb") + if params.occ_track: + occfile = bx.bbi.bigwig_file.BigWigFile(open(params.occ_track,'rb')) + else: + occfile = None + if params.bias is not None: + biasfile = bx.bbi.bigwig_file.BigWigFile(open(params.bias,'rb')) + elif params.gdna is not None: + biasfile = bx.bbi.bigwig_file.BigWigFile(open(params.bias,'rb')) + else: + biasfile = None + handles = {} + if params.write_all: + outputs = ['nucpos','nucpos.redundant','nfrpos','nucsig','smoothsig','ins','nuc_cov', + 'nfr_cov','background','rawsig','fitted'] + else: + outputs = ['nucpos','nucpos.redundant','nfrpos','nucsig','smoothsig','ins'] + if params.occ_track is None: + outputs.append('occ') + for i in outputs: + handles[i] = open('tmp_nucleoatac/' + self.name + '.' + params.out + '.' + + i + '.tmp.bed','w') + for chunk in self.chunks: + if params.bias: + chunk.process(params, bamfile, occfile, biasfile, gdna = False) + elif params.gdna: + chunk.process(params, bamfile, occfile, biasfile, gdna = True) + else: + chunk.process(params, bamfile, occfile) + for key in chunk.nonredundant: + chunk.nuc_collection[key].writeNucPos(handles['nucpos']) + for key in chunk.redundant: + chunk.nuc_collection[key].writeNucPos(handles['nucpos.redundant']) + for key in chunk.nfrs.keys(): + chunk.nfrs[key].writeNFRPos(handles['nfrpos']) + chunk.ins.write_track(handles['ins']) + chunk.norm_signal.write_track(handles['nucsig']) + chunk.smoothed.write_track(handles['smoothsig']) + if params.write_all: + chunk.nuc_signal.write_track(handles['rawsig']) + chunk.nuc_cov.vals = chunk.nuc_cov.vals / params.window * 10.0 + chunk.nuc_cov.write_track(handles['nuc_cov']) + chunk.bias.write_track(handles['background']) + chunk.nfr_cov.vals = chunk.nfr_cov.vals / params.window * 10.0 + chunk.nfr_cov.write_track(handles['nfr_cov']) + chunk.fitted.write_track(handles['fitted']) + if params.occ_track is None: + chunk.occ.write_track(handles['occ']) + chunk.removeData() + for key in handles.keys(): + handles[key].close() + bamfile.close() + +def read_bed(bedfile, genome_dict, max_offset = 0): + """Make a dictionary of Chromosome insances from a bedfile""" + infile = open(bedfile,"r") + out = [] + while 1: + in_line = infile.readline().rstrip('\n').split("\t") + if not in_line[0]: + break + chrom = in_line[0] + start = int(in_line[1]) + if start < max_offset: + start = max_offset + end = int(in_line[2]) + if end > (genome_dict[chrom] - max_offset): + end = genome_dict[chrom] - max_offset + out.extend([NucChunk(chrom, start, end)]) + infile.close() + return out + + +def makeChunkSetList(bed_list, number): + """turn list of ChromChunks into a list of ChunkSet (sets of ChromChunks)""" + l = len(bed_list) + bases = 0.0 + for i in range(l): + bases += bed_list[i].end - bed_list[i].start + if number >= l: + sets = [] + z = int(np.log10(l)+1) + for j in range(l): + sets.append(NucChunkSet(str(j).zfill(z),bed_list[j:(j+1)])) + else: + x = min(number*3,l) + n = bases/x + sets = [] + z = int(np.log10(x)+1) + included = 0 + i = 0 + for j in range(x): + a = i + while included < (j+1)*n and i < l: + included += bed_list[i].end - bed_list[i].start + i += 1 + b = i + sets.append(NucChunkSet(str(j).zfill(z),bed_list[a:b])) + return sets diff --git a/nucleoatac/Occupancy.py b/nucleoatac/Occupancy.py new file mode 100644 index 0000000..fbd64bb --- /dev/null +++ b/nucleoatac/Occupancy.py @@ -0,0 +1,277 @@ +#!/usr/bin/env python +""" +Classes for computing nucleosome occupancy + +@author: Alicia Schep, Greenleaf Lab, Stanford University +""" + + +from scipy import signal, optimize, stats +import numpy as np +import pysam +import matplotlib.pyplot as plt +import PyATAC as PA + +class InsertDistribution: + """Class for modelling insert size distribution""" + def __init__(self, i_lower = 0, i_upper =2000): + self.i_lower = i_lower + self.i_upper = i_upper + def getInsertSizes(self,bam, bed_list, maxreads = 500000): + """Use bamfile to get insertsize distribution + + Uses maxreads (or total reads if less) reads to determine + """ + bamfile=pysam.Samfile(bam, "rb") + i=0#counter + sizes = np.zeros(self.i_upper - self.i_lower) + # loop through reads + for chunk in bed_list: + for p2_rds in bamfile.fetch(chunk.chrom,chunk.start,chunk.end): + #check mapping quality + if p2_rds.mapq>=30 and p2_rds.is_proper_pair and not p2_rds.is_reverse: + # get insert size + ilen = abs(p2_rds.tlen)-8 + if ilen<self.i_upper and ilen>=self.i_lower: + sizes[ilen - self.i_lower]+=1 + #increase counter by 1 + i+=1 + if i>maxreads: + break + bamfile.close() + self.insertsizes = PA.InsertSizes(sizes / sum(sizes), self.i_lower, self.i_upper) + def modelNFR(self, boundary = 115): + """Model NFR distribution with exponential distribution""" + b = np.where(self.insertsizes.get(self.i_lower,boundary) == max(self.insertsizes.get()))[0][0]+10 + self.i_lower + def exp_pdf(x,*p): #defines the PDF + k=p[0] + a=p[1] + x=x-b + return a*k*np.exp(-k*x) + x = np.array(range(b,boundary)) + p0 = (.1,1) + coeff, var_matrix = optimize.curve_fit(exp_pdf,x, self.insertsizes.get(b,boundary), + p0=p0) + nfr = np.concatenate((self.insertsizes.get(self.i_lower,boundary), exp_pdf(np.array(range(boundary,self.i_upper)),*coeff))) + nfr[nfr==0] = min(nfr[nfr!=0])*0.01 + self.nfr_fit = PA.InsertSizes(nfr,self.i_lower,self.i_upper) + nuc = np.concatenate((np.zeros(boundary-self.i_lower), + self.insertsizes.get(boundary,self.i_upper) - + self.nfr_fit.get(boundary,self.i_upper))) + nuc[nuc<=0]=min(min(nfr)*0.1,min(nuc[nuc>0])*0.001) + self.nuc_fit = PA.InsertSizes(nuc, self.i_lower, self.i_upper) + def plotFits(self,filename=None): + """plot the Fits""" + fig = plt.figure() + plt.plot(range(self.i_lower,self.i_upper),self.insertsizes.get(), + label = "Observed") + #plt.plot(range(self.i_lower,self.i_upper),self.smoothed.get(), label = "Smoothed") + plt.plot(range(self.i_lower,self.i_upper),self.nuc_fit.get(), label = "Nucleosome Fit") + plt.plot(range(self.i_lower,self.i_upper),self.nfr_fit.get(), label = "NFR Fit") + plt.legend() + plt.xlabel("Distance between Insertions (inclusive)") + plt.ylabel("Relative Frequency") + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + out = np.vstack((self.insertsizes.get(), #self.smoothed.get(), + self.nuc_fit.get(), self.nfr_fit.get())) + np.savetxt(filename2,out,delimiter="\t") + else: + fig.show() + +class OccupancyCalcParams: + """Class with parameters for occupancy determination""" + def __init__(self, i_lower, i_upper , insert_dist): + self.i_lower = i_lower + self.i_upper = i_upper + self.smooth_mat = np.tile(signal.gaussian(151,25),(i_upper-i_lower,1)) + nuc_probs = insert_dist.nuc_fit.get(i_lower,i_upper) + self.nuc_probs = nuc_probs /np.sum(nuc_probs) + nfr_probs = insert_dist.nfr_fit.get(i_lower,i_upper) + self.nfr_probs = nfr_probs /np.sum(nfr_probs) + self.alphas = np.linspace(0, 1, 101) + self.x = map(lambda alpha: np.log(alpha * self.nuc_probs + (1 - alpha) * self.nfr_probs), self.alphas) + self.w = 75 + self.l = len(self.x) + + +def calculateOccupancy(inserts, params): + """function to calculate occupancy based on insert distribution + also takes OccupancyCalcParams as input + """ + logliks = np.array(map(lambda j: np.sum(params.x[j]*inserts),range(params.l))) + logliks[np.isnan(logliks)] = -float('inf') + occ = params.alphas[np.argmax(logliks)] + return occ + + + +class OccupancyTrack(PA.Track): + """Class for computing nucleosome occupancy""" + def __init__(self, chrom, start, end): + PA.Track.__init__(self, chrom, start, end, "occupancy") + def calculateOccupancyMLE_smooth(self, mat, params): + """Calculate Occupancy track""" + offset=self.start - mat.start + if offset<params.w: + raise Exception("For calculateOccupancyMLE_smooth, mat does not have sufficient flanking regions"),offset + self.vals=np.ones(self.end - self.start)*float('nan') + for i in range(len(self.vals)): + new_inserts = np.sum(mat.get(i_lower = params.i_lower,i_upper = params.i_upper, + start = self.start+i-params.w, end = self.start+i+params.w+1)*params.smooth_mat, + axis = 1) + if sum(new_inserts)>0: + self.vals[i] = calculateOccupancy(new_inserts,params) + +class OccPeak: + def __init__(self, pos, chunk): + """Class for storing occupancy peaks""" + self.chrom = chunk.chrom + self.pos = pos + self.occ = chunk.occ.get(pos = pos) + self.reads = chunk.cov.get(pos = pos) + def writePeak(self, handle): + """write bed line for peak""" + handle.write("\t".join(map(str,[self.chrom,self.pos,self.pos+1,self.occ,self.reads]))+"\n") + +class OccupancyParams: + """Class for storing parmeers related to Occupancy determination""" + def __init__(self, occ_calc_params, sep = 120, min_occ = 0.25, min_reads = 1, flank = 60, + write_peaks = True, out = None, bam = None): + self.occ_calc_params = occ_calc_params + self.sep = sep + self.min_occ = min_occ + self.min_reads = min_reads + self.flank = flank + self.write_peaks = write_peaks + self.out = out + self.bam = bam + +class OccChunk(PA.ChromChunk): + """Class for calculating occupancy and occupancy peaks + """ + def __init__(self, chrom, start, end): + PA.ChromChunk.__init__(self, chrom, start, end) + self.peaks = {} + def calculateOcc(self, bamfile, params): + """calculate occupancy for chunk""" + self.reads = PA.ReadList(self.chrom, self.start - 75, self.end + 75) + self.reads.extract_reads(bamfile, i_upper = params.i_upper) + self.mat = PA.ReadMat2D(self.chrom, self.start - 75, self.end + 75 , 0, params.i_upper) + self.mat.makeReadMat(self.reads) + self.occ = OccupancyTrack(self.chrom,self.start,self.end) + self.occ.calculateOccupancyMLE_smooth(self.mat, params) + def getCov(self, i_upper): + """Get read coveraged (smoothed in same way as occupancy) for regions""" + self.cov = PA.CoverageTrack(self.chrom, self.start, self.end) + self.cov.calculateCoverageSmooth(self.mat, 0, i_upper, 151, sd = 25) + def callPeaks(self, sep, min_occ, min_reads): + """Call peaks of occupancy profile""" + peaks = PA.call_peaks(self.occ.vals, sep = sep, min_signal = min_occ) + for peak in peaks: + tmp = OccPeak(peak + self.start, self) + if tmp.reads > min_reads: + self.peaks[peak] = tmp + def getNucDist(self, flank, i_upper): + """Get nucleosomal insert distribution""" + nuc_dist = np.zeros(i_upper) + for peak in self.peaks.keys(): + sub = self.mat.get(start = self.peaks[peak].pos-flank,end = self.peaks[peak].pos+1+flank) + sub_sum = np.sum(sub,axis=1) + sub_sum = sub_sum / float(sum(sub_sum)) + nuc_dist += sub_sum + return(nuc_dist) + def process(self, bamfile, params): + """proces chunk -- calculat occupancy, get coverage, call peaks""" + self.calculateOcc(bamfile, params.occ_calc_params) + self.getCov(params.occ_calc_params.i_upper) + self.callPeaks(params.sep, params.min_occ, params.min_reads) + def removeData(self): + """remove data from chunk-- deletes all attributes""" + names = self.__dict__.keys() + for name in names: + delattr(self, name) + + + +class OccChunkSet(PA.ChunkSet): + """Class for storing sets of OccChunks + """ + def __init__(self, name, chunks): + PA.ChunkSet.__init__(self, name, chunks) + def process(self, params): + """Process occupancy chunk set -- get occupancy track, call peaks, get nuc_dist""" + bamfile = pysam.Samfile(params.bam, "rb") + nuc_dist = np.zeros(params.occ_calc_params.i_upper) + handles = {} + outputs = ['occ'] + if params.write_peaks: + outputs.extend(['occ_peaks']) + for i in outputs: + handles[i] = open('tmp_nucleoatac/' + self.name + '.' + params.out + '.' + + i + '.tmp.bed','w') + for chunk in self.chunks: + chunk.process(bamfile, params) + chunk.occ.write_track(handles['occ']) + if params.write_peaks: + for key in sorted(chunk.peaks.keys()): + chunk.peaks[key].writePeak(handles['occ_peaks']) + nuc_dist += chunk.getNucDist(params.flank, params.occ_calc_params.i_upper) + chunk.removeData() + for key in handles.keys(): + handles[key].close() + bamfile.close() + return nuc_dist + +def makeChunkSetList(bed_list, number): + """turn list of ChromChunks into a list of ChunkSet (sets of ChromChunks)""" + l = len(bed_list) + bases = 0.0 + for i in range(l): + bases += bed_list[i].end - bed_list[i].start + if number >= l: + sets = [] + z = int(np.log10(l)+1) + for j in range(l): + sets.append(OccChunkSet(str(j).zfill(z),bed_list[j:(j+1)])) + else: + x = min(number*3,l) + n = bases/x + sets = [] + z = int(np.log10(x)+1) + included = 0 + i = 0 + for j in range(x): + a = i + while included < (j+1)*n and i < l: + included += bed_list[i].end - bed_list[i].start + i += 1 + b = i + sets.append(OccChunkSet(str(j).zfill(z),bed_list[a:b])) + return(sets) + + +def read_bed(bedfile, genome_dict, max_offset = 0): + """Make a dictionary of Chromosome insances from a bedfile""" + infile = open(bedfile,"r") + out = [] + while 1: + in_line = infile.readline().rstrip('\n').split("\t") + if not in_line[0]: + break + chrom = in_line[0] + start = int(in_line[1]) + if start < max_offset: + start = max_offset + end = int(in_line[2]) + if end > (genome_dict[chrom] - max_offset): + end = genome_dict[chrom] - max_offset + out.extend([OccChunk(chrom, start, end)]) + infile.close() + return out + + + diff --git a/nucleoatac/PyATAC.py b/nucleoatac/PyATAC.py new file mode 100644 index 0000000..37801bf --- /dev/null +++ b/nucleoatac/PyATAC.py @@ -0,0 +1,679 @@ +#!/usr/bin/env python +""" +General tools for dealing with ATAC-Seq data using Python. + +@author: Alicia Schep, Greenleaf Lab, Stanford University +""" + +import os +from scipy import signal +import numpy as np +import warnings +import matplotlib.pyplot as plt +import matplotlib.cm as cm +import pysam + +def shell_command(cmd): + """Conduct shell command. If error code, raise error""" + code = os.system(cmd) + if code!=0: + raise Exception("error with shell command: "+cmd) + + + +#Smoothing function +def smooth(sig, window_len, window='flat', sd = None, mode = 'valid', + norm = True): + """smoothes input signal using either flat or gaussian window + + options for window are "flat" and "gaussian" + if window is "gaussian", sd should be provided. + for guassian default sd is (window_len-1)/6 + norm means whether window should integrate to 1 + """ + if window not in ['flat','gaussian']: + raise Exception("Incorrect window input for smooth. Options are flat, gaussian") + if window_len%2 != 1: + warnings.warn("Window length is even number. Needs to be odd so adding 1.") + window_len += 1 + if window=='gaussian' and sd is None: + sd = (window_len-1)/6.0 + if window=="gaussian": + w = signal.gaussian(window_len,sd) + if window=="flat": + w = np.ones(window_len) + if norm: + w = w/sum(w) + smoothed = np.convolve(w, sig, mode = mode) + return smoothed + + +#Basic classes for storing ATAC-seq reads at different levels +class Read: + """Class that stores read position and length""" + def __init__(self, chrom, left, insert): + self.chrom = chrom + self.left = left#0-based + self.right = left + insert#1-based + self.insert = insert #Physical length between insertion (inclusive) + +class ReadList: + """Class that stores list of reads""" + def __init__(self, chrom, start, end): + self.chrom = chrom + self.start = start + self.end = end + self.reads = [] + def addRead(self, read): + """Add one read to thecollection""" + self.reads.extend([read]) + def extract_reads(self, bamfile,i_upper = 2000, downsample = None, seed =500, atac = True): + """Get reads that overlap with chunk + + bamfile: pysam object + flank: region on each side of chunk boundary to also include + downsample: a float between 0 and 1, None for no downsampling + """ + if downsample is not None and downsample!=1: + random = np.random.RandomState(seed) + for p2_rds in bamfile.fetch(self.chrom, max(0, self.start - i_upper), + self.end + i_upper): + if p2_rds.mapq>=30 and p2_rds.is_proper_pair and not p2_rds.is_reverse: + if atac: + #get left position + l_pos = p2_rds.pos + 4 + #get insert size + #correct by 8 base pairs to be inserion to insertion + if p2_rds.tlen<0 and p2_rds.tlen==-(p2_rds.rlen): + ilen = abs(p2_rds.tlen) - 8 + elif p2_rds.tlen<0: + continue + else: + ilen=p2_rds.tlen-8 + else: + l_pos = p2_rds.pos + if p2_rds.tlen<0 and p2_rds.tlen==-(p2_rds.rlen): + ilen = abs(p2_rds.tlen) + elif p2_rds.tlen<0: + continue + else: + ilen=p2_rds.tlen + if downsample is None or downsample == 1: + self.addRead(Read(self.chrom, l_pos, ilen)) + else: + x = random.uniform(0,1) + if x <= downsample: + self.addRead(Read(self.chrom, l_pos, ilen)) + +class ChunkMat2D: + """Class that stores reads in 2D matrix according to + center position and length""" + def __init__(self, chrom, start, end, i_lower, i_upper, mode = "centers"): + modes = ["centers","ends"] + if mode not in modes: + raise Exception("Invalid mode. Expected one of: %s" % modes) + self.mode = mode + self.chrom = chrom + self.i_lower = i_lower + self.i_upper = i_upper + self.start = start + self.end = end + self.ncol = end - start + self.nrow = i_upper - i_lower + self.mat = np.zeros((self.nrow, self.ncol)) + def get(self, i_lower = None, i_upper = None, start = None, end = None, flip = False): + """get a subset (or all) of the matrix stored by ChunkMat2D object based on + chromosal position and/or insert size""" + if i_lower is None: + i_lower = self.i_lower + if i_upper is None: + i_upper = self.i_upper + if start is None: + start = self.start + if end is None: + end = self.end + y1 = i_lower - self.i_lower + y2 = i_upper - self.i_lower + x1 = start - self.start + x2 = end - self.start + if not flip: + try: + return self.mat[y1:y2,x1:x2] + except: + raise Exception("Looks like dimensions from get probaby don't match ReadMat") + else: + if x1 < 1 or x2 > self.mat.shape[1] or y1 < 0 or y2 > self.mat.shape[0]: + raise Exception("Looks like dimensions from get probaby don't match ReadMat") + ncol = x2-x1 + if ncol%2==0: + raise Exception("Can only flip mat if the width is odd!") + else: + new = np.zeros((y2-y1,ncol)) + for j in range(y1,y2): + if (j+self.i_lower)%2==1: + new[j,:] = self.mat[j,x1:x2][::-1] + else: + new[j,:] = self.mat[j,(x1-1):(x2)][::-1][1:] + return new + def assign(self, mat): + """assign a matrix to object""" + if mat.shape != self.mat.shape: + raise Exception("Dimensions of input mat are wrong. Uh oh!") + self.mat = mat + def save(self, filename): + """Save object in a text file""" + head = ",".join(map(str,[self.chrom,self.start,self.end,self.i_lower,self.i_upper])) + np.savetxt(filename,self.mat,delimiter="\t", header = head) + @staticmethod + def open(filename): + f = open(filename,'r') + header = f.readline() + f.close() + elements = header.rstrip('\n').lstrip("#").split(',') + mat = np.loadtxt(filename, skiprows=1) + new= ChunkMat2D(elements[0],elements[1],elements[2],elements[3]) + new.assign(mat) + return new + def getIns(self): + """Collape matrix into insertions. Will reduce span on chromosome + if mode is centers""" + if self.mode == "centers": + pattern = np.zeros((self.i_upper-self.i_lower,self.i_upper + (self.i_upper-1)%2)) + mid = self.i_upper/2 + for i in range(self.i_lower,self.i_upper): + pattern[i-self.i_lower,mid+(i-1)/2]=1 + pattern[i-self.i_lower,mid-(i/2)]=1 + ins = signal.correlate2d(self.mat,pattern,mode="valid")[0] + insertion_track = InsertionTrack(self.chrom,self.start + pattern.shape[1]/2, self.end - (pattern.shape[1]/2)) + insertion_track.assign_track(ins) + return insertion_track + elif self.mode == "ends": + insertion_track = InsertionTrack(self.chrom,self.start, self.end) + insertion_track.assign_track(np.sum(self.mat),axis=0) + return insertion_track + def plot(self, filename = None, title = None, i_lower = None, + i_upper = None): + """Plot 2d ReadMat""" + if i_upper is None: + i_upper = self.i_upper + if i_lower is None: + i_lower = self.i_lower + fig = plt.figure() + plt.imshow(self.get(i_lower= i_lower, i_upper = i_upper), + origin="lower",interpolation='nearest', + extent=[self.start,self.end-1,i_lower,i_upper-1],cmap=cm.get_cmap('Greys')) + plt.xlabel(self.chrom) + plt.ylabel("Insert size") + if title: + plt.title(title) + #plt.colorbar(shrink=0.8) + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + np.savetxt(filename2,self.mat,delimiter="\t") + else: + fig.show() + + + + +class ReadMat2D(ChunkMat2D): + """Class that stores read information in 2D matrix according""" + def __init__(self, chrom, start, end, i_lower, i_upper, mode = "centers"): + ChunkMat2D.__init__(self,chrom, start, end, i_lower, i_upper ,mode) + def updateMat(self, read): + row = read.insert - self.i_lower + if self.mode == "centers": + col = (read.insert-1)/2 + read.left - self.start + if col>=0 and col<self.ncol and row<self.nrow and row>=0: + self.mat[row, col] += 1 + else: + col1 = read.left + col2 = read.right - 1 + if col1>=0 and col1<self.ncol and row<self.nrow and row>=0: + self.mat[row, col1] += 1 + if col2>=0 and col2<self.ncol and row<self.nrow and row>=0: + self.mat[row, col2] += 1 + def makeReadMat(self, readlist): + """Make 2D matrix using reads list""" + for read in readlist.reads: + self.updateMat(read) + + +class BiasMat2D(ChunkMat2D): + """Class that stores read information in 2D matrix according""" + def __init__(self, chrom, start, end, i_lower, i_upper, mode = "centers"): + ChunkMat2D.__init__(self,chrom, start, end, i_lower, i_upper ,mode) + self.mat = np.ones(self.mat.shape) + def makeBiasMat(self, bias_track): + """Make 2D matrix representing sequence bias preferences""" + offset = self.i_upper/2 + bias = bias_track.get(self.start-offset,self.end+offset) + if not bias_track.log: + nonzero = np.where(bias !=0)[0] + bias = np.log(bias + min(bias[nonzero])) + pattern = np.zeros((self.i_upper-self.i_lower,self.i_upper + (self.i_upper-1)%2)) + mid = self.i_upper/2 + for i in range(self.i_lower,self.i_upper): + pattern[i-self.i_lower,mid+(i-1)/2]=1 + pattern[i-self.i_lower,mid-(i/2)]=1 + for i in range(self.i_upper-self.i_lower): + self.mat[i]=np.exp(np.convolve(bias,pattern[i,:],mode='valid')) + def normByInsertDist(self, insertsizes): + inserts = insertsizes.get(self.i_lower,self.i_upper) + self.mat = self.mat * np.reshape(np.tile(inserts,self.mat.shape[1]),self.mat.shape,order="F") + + + +class ChromChunk: + """Class that stores reads for a particular chunk of the genome""" + def __init__(self, chrom, start, end, weight = None, strand = None): + self.chrom = chrom + self.start = start + self.end = end + self.length = end - start + self.weight = weight + self.strand= strand + + +class ChunkSet: + """Class for storing group of chunks""" + def __init__(self, name, chunks): + self.name = name + self.chunks = chunks + + + +class Chromosomes: + """Class for storing chromosome information""" + def __init__(self): + self.chrs = {} + def get_from_bam(self, bamfile): + """Get chromosome info from bam""" + bam = pysam.Samfile(bamfile, "rb") + chr_lengths=bam.lengths + chr_names=bam.references + bam.close() + for i in range(len(chr_lengths)): + if chr_names[i]!='chrM': + self.chrs[chr_names[i]]=int(chr_lengths[i]) + def write_chrs(self, filename): + """write chromosome information to text file""" + genome=open(filename,'w') + for c in self.chrs.keys(): + genome.write('\t'.join(map(str,[c,self.chrs[c]]))+'\n') + genome.close() + + + +##Funcion for reading in bed file +def read_bed(bedfile, weight_col=None, strand_col = None): + """Make a dictionary of Chromosome insances from a bedfile""" + infile = open(bedfile,"r") + out = [] + weight = None + strand = None + while 1: + in_line = infile.readline().rstrip('\n').split("\t") + if not in_line[0]: + break + if weight_col: + weight=in_line[weight_col-1] + if strand_col: + strand = in_line[strand_col-1] + out.extend([ChromChunk(in_line[0],int(in_line[1]), int(in_line[2]), + weight, strand)]) + infile.close() + return out + + +def makeChunkSetList(bed_list, number): + """turn list of ChromChunks into a list of ChunkSet (sets of ChromChunks)""" + l = len(bed_list) + x = min(number,l) + n = l/x + sets = [] + z = int(np.log10(x)+1) + for j in range(x): + a = j*n + if j == x-1: + b=l + else: + b = (j+1)*n + sets.append(ChunkSet(str(j).zfill(z),bed_list[a:b])) + return(sets) + + +#Family of classes for storing Signal Tracks + +class Track: + """Generic class for various types of signal tracks""" + def __init__(self, chrom, start, end, name = "track", vals=None , log = False): + self.name = name + self.chrom = chrom + self.start = start + self.end = end + self.length = end - start + self.log = log + def assign_track(self, vals, start = None, end = None): + """Assign values to track""" + if start: + self.start = start + if end: + self.end = end + if len(vals)!= self.end - self.start: + raise Exception("The values being assigned to track do not \ + span the start to end of the track") + self.vals = vals + def write_track(self, handle, start = None, end = None, vals = None): + """Write track to output file handle + + If vals are specified use those values + Othersise use self.vals + """ + if start is None: + start = self.start + if end is None: + end = self.end + if vals is None: + vals=self.vals + if len(vals)!=self.end-self.start: + print len(vals),self.end-self.start + raise Exception("Error! Inconsistency between length of \ + values and start/end values") + for k in range(start, end): + k2 = k - start + handle.write('\t'.join(map(str, [self.chrom, k, k + 1, + vals[k2]])) + '\n') + def read_track(self, bwh, start = None, end = None, flank = None): + """Read track values from BigWig file handle""" + if start: + self.start = start + if end: + self.end = end + if flank: + self.start = self.start - flank + self.end = self.end + flank + self.vals = bwh.get_as_array(self.chrom, self.start, + self.end) + if self.vals is None: + raise Exception("No values were read for desired track. \ + Perhaps the chromsome is not included in the bigwig file?") + def log(self, pseudo = 1): + if self.log: + print "Logging a track that is already log..." + adjusted = self.vals + pseudo + self.vals = np.log(adjusted) + self.log = True + def exp(self): + if not self.log: + print "taking exponent of a non-logged track..." + self.vals = np.exp(self.vals) + self.log = False + def smooth_track(self, window_len, window='flat', sd = None, + mode = 'valid', norm = True): + """ smoothing of track""" + self.smoothed=True + self.vals = smooth(self.vals, window_len, window = window, sd = sd, + mode = mode, norm = norm) + if mode == 'valid': + self.start = self.start + window_len/2 + self.end = self.end - window_len/2 + self.length = self.end - self.start + def get(self, start = None, end = None, pos = None): + if pos: + try: + return self.vals[pos-self.start] + except: + raise Exception("Looks like position given doesn't match track") + else: + if start is None: + start = self.start + if end is None: + end = self.end + x1 = start - self.start + x2 = end - self.start + try: + return self.vals[x1:x2] + except: + raise Exception("Looks like dimensions from get probaby don't match track, or there are no vals in track") + def plot(self, name = None, start = None, end = None, filename=None): + """plot the values """ + if start is None: + start = self.start + if end is None: + end = self.end + if name is None: + name = self.name + values = self.get(start,end) + fig = plt.figure() + plt.plot(range(start,end),values) + plt.xlabel(self.chrom) + plt.ylabel(name) + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + out = np.vstack((range(start,end),values)) + np.savetxt(filename2,out,delimiter="\t") + else: + fig.show() + +class InsertionBiasTrack(Track): + """Class for getting and storing insertion bias""" + def __init__(self, chrom, start, end, log = True): + Track.__init__(self, chrom, start, end, name = "insertion bias", log = log) + def get(self, start = None, end = None, pos = None, log = None): + out = Track.get(self, start, end, pos) + if log is None: + return out + elif log: + if self.log: + return out + else: + return np.log(out) + else: + if self.log: + return np.exp(out) + else: + return out + def getLocalbias(self, windowlen = 55, window = "flat"): + """Make bias a reflection of score in a given window""" + if self.log: + ebias = np.exp(self.vals) + else: + ebias = self.vals + smoothed = smooth(ebias,windowlen, window, norm = False) + flank = windowlen/2 + if self.log: + self.vals = np.log(ebias[flank:-flank]/(smoothed-ebias[flank:-flank])) + else: + self.vals = ebias[flank:-flank]/(smoothed-ebias[flank:-flank]) + self.start = self.start + flank + self.end = self.end - flank + +class InsertionTrack(Track): + """Class for getting and storing insertion positions""" + def __init__(self, chrom, start, end): + Track.__init__(self, chrom, start, end, "insertions") + def calculateInsertions(self, readlist, flank = 0, i_lower = 0, i_upper =2000): + self.start = self.start - flank + self.end = self.end + flank + self.vals = np.zeros(self.end - self.start) + for read in readlist.reads: + if read.insert >=i_lower and read.insert<i_upper: + left = read.left - self.start + right = read.right - 1 - self.start + if left>=0 and left<self.end - self.start: + self.vals[left] +=1 + if right>=0 and right<self.end - self.start: + self.vals[right] +=1 + + +class CoverageTrack(Track): + """Class for computing read center converage""" + def __init__(self, chrom, start, end): + Track.__init__(self, chrom, start, end, "coverage") + def calculateCoverage(self,mat,i_lower,i_upper,window_len): + offset=self.start-mat.start-(window_len/2) + if offset<0: + raise Exception("Insufficient flanking region on \ + mat to calculate coverage with desired window") + i_lower=i_lower-mat.i_lower + i_upper=i_upper-mat.i_lower + if offset!=0: + collapsed = np.sum(mat.mat[i_lower:i_upper,offset:-offset],axis=0) + else: + collapsed = np.sum(mat.mat[i_lower:i_upper,],axis=0) + self.vals = smooth(collapsed, window_len, window="flat", + mode='valid',norm=False) + def calculateCoverageSmooth(self,mat,i_lower,i_upper,window_len,sd): + offset=self.start-mat.start-(window_len/2) + if offset<0: + raise Exception("Insufficient flanking region on \ + mat to calculate coverage with desired window") + i_lower=i_lower-mat.i_lower + i_upper=i_upper-mat.i_lower + if offset!=0: + collapsed = np.sum(mat.mat[i_lower:i_upper,offset:-offset],axis=0) + else: + collapsed = np.sum(mat.mat[i_lower:i_upper,],axis=0) + self.vals = smooth(collapsed, window_len, sd= sd, window="gaussian", + mode='valid',norm=False) + + + + +class InsertSizes: + """Class for storing insert sze distribution""" + def __init__(self, dist, i_lower, i_upper): + self.i_lower = i_lower + self.i_upper = i_upper + self.vals = dist + def get(self, i_lower = None, i_upper = None, size = None): + if size: + try: + return self.vals[size - self.i_lower] + except: + raise Exception("Looks like size doesn't match InsertSizes") + else: + if i_lower is None: + i_lower = self.i_lower + if i_upper is None: + i_upper = self.i_upper + y1 = i_lower - self.i_lower + y2 = i_upper - self.i_lower + try: + return self.vals[y1:y2] + except: + raise Exception("Looks like dimensions from get probaby don't match InsertSizes") + def save(self, filename): + """Save Insert Distribution information""" + f = open(filename,"w") + f.write("#i_lower\n") + f.write(str(self.i_lower)+"\n") + f.write("#i_upper\n") + f.write(str(self.i_upper)+"\n") + f.write("#isizes\n") + f.write("\t".join(map(str,self.get()))+"\n") + f.close() + @staticmethod + def open(filename): + """Create InsertDistribution object from text descriptor file""" + infile = open(filename,'r') + state = '' + for line in infile: + if '#i_lower' in line: + state = 'i_lower' + elif '#i_upper' in line: + state = 'i_upper' + elif '#isizes' in line: + state = 'isizes' + elif '#' in line: + state = 'other' + elif state == 'i_lower': + i_lower = int(line.strip('\n')) + elif state == 'i_upper': + i_upper = int(line.strip('\n')) + elif state == 'isizes': + insertsizes = np.array(map(float,line.rstrip("\n").split("\t"))) + try: + new = InsertSizes(insertsizes,i_lower,i_upper) + except NameError: + raise Exception("InsertDistribution decriptor file appeas to be missing some\ +needed components") + infile.close() + return new + +def reduce_peaks(peaks,sig, sep): + """Greedy algorithm for taking peaks and turning to set with at least sep distance + between peaks. First include peak with highest sig value, then next greatest + not within sep distance from that one, and so on""" + exclude = np.zeros(peaks.size) + keep = np.zeros(peaks.size) + st = np.argsort(sig) + j=peaks.size-1 + while j >=0: + ind = st[j] + j+= -1 + if exclude[ind]==0: + keep[ind] = 1 + exclude[ind]=1 + k = ind - 1 + while k>=0 and (peaks[ind] - peaks[k]) < sep: + exclude[k]=1 + k += -1 + k = ind + 1 + while k < peaks.size and (peaks[k]-peaks[ind]) < sep: + exclude[k]=1 + k += 1 + return peaks[keep ==1] + + + +def call_peaks(sigvals, min_signal = 0, sep = 120, boundary = None, order =1): + """Greedy algorithm for peak calling-- first call all local maxima, + then call greatest maxima as a peak, then next greatest that isn't within + 'sep' distance of that peak, and so on""" + if sum(np.isnan(sigvals))>0: + if sum(np.isnan(sigvals))==len(sigvals): + return np.array([]) + else: + replace = min(sigvals[~np.isnan(sigvals)]) + sigvals[np.isnan(sigvals)]=replace + if boundary is None: + boundary = sep/2 + random = np.random.RandomState(seed = 25) + l = len(sigvals) + peaks = signal.argrelmax(sigvals *(1+ + random.uniform(0,10**-12,l)),order=order)[0] + peaks = peaks[sigvals[peaks] >= min_signal ] + peaks = peaks[ peaks >= boundary ] + peaks = peaks[ peaks < (l - boundary)] + sig = sigvals[peaks] + return reduce_peaks(peaks, sig, sep) + + +def combine_tmp_bed(basename, track, directory = 'tmp_nucleoatac'): + """Combines temporary bed files""" + tmp_names = directory + '/*.'+ basename + '.'+ track +'.tmp.bed' + bed_name = basename + '.' + track + '.bed' + shell_command('cat ' + tmp_names + ' > ' + bed_name) + shell_command('rm ' + tmp_names) + +def combine_tmp_bed_to_bw(basename, track, directory = 'tmp_nucleoatac'): + """combines temporary bed file and turns into bigwig""" + tmp_names = directory + '/*.'+ basename + '.'+ track +'.tmp.bed' + bed_name = directory + '/' + basename + '.' + track + '.bed' + bw_name = basename + '.' + track + '.bw' + genome = directory + '/genome.txt' + shell_command('cat ' + tmp_names + ' > ' + bed_name) + shell_command('rm ' + tmp_names) + shell_command('bedGraphToBigWig '+ bed_name + ' ' + genome + ' ' + bw_name) + shell_command('rm ' + bed_name) + + + diff --git a/nucleoatac/VMat.py b/nucleoatac/VMat.py new file mode 100644 index 0000000..4ff0918 --- /dev/null +++ b/nucleoatac/VMat.py @@ -0,0 +1,221 @@ +#!/usr/bin/env python +""" +VMat Class + +@author: Alicia Schep, Greenleaf Lab, Stanford University +""" + +#Import necessary python modules +from scipy import signal, ndimage +import numpy as np +from copy import copy +import matplotlib.pyplot as plt + +class VMat_Error(Exception): + """Class for errors in VMat function""" + def __init__(self, value): + self.value = value + def __str__(self): + return repr(self.value) + + +class VMat: + """Class for storing and processing V-plot matrix""" + def __init__(self, mat, i_lower, i_upper): + """ + Assumes Vplot is centered! + Inputs: + mat = matrix (as numpy array) + i_lower = lower bound of insert sizes represented by mat + i_upper = upper bound of insert sizes represented by mat + """ + if mat.shape[0]!=i_upper-i_lower: + raise VMat_Error("mat shape is not consistent with insert limits") + self.mat = mat + self.i_upper = i_upper + self.i_lower = i_lower + self.w = mat.shape[1]/2 + def trim(self,i_lower,i_upper,w): + """reduce the size of the vplot + + i_lower is new lower bound + i_upper is new upper bound + w is new flanking region around center + """ + up = i_upper-self.i_lower + dn = i_lower-self.i_lower + left = self.w - w + right = self.w + w + 1 + if up > self.mat.shape[0] or dn < 0 or left < 0 or right > self.mat.shape[1]: + raise VMat_Error("Mat is smaller than desired trim") + self.mat = self.mat[dn:up,left:right] + self.i_lower = i_lower + self.i_upper = i_upper + self.w = w + def symmetrize(self): + """Force the V-plot to be symmetric""" + for j in range(self.i_lower,self.i_upper): + i=j-self.i_lower + if j%2==1: + lefthalf = (self.mat[i,:(self.w+1)]+self.mat[i,self.w:][::-1])*0.5 + self.mat[i,:] = np.hstack((lefthalf,lefthalf[:-1][::-1])) + else: + righthalf = (self.mat[i,(self.w):-1]+self.mat[i,:self.w][::-1])*0.5 + self.mat[i,:] = np.hstack((righthalf[::-1],righthalf,self.mat[i,-1])) + def flip(self, mode = 'same'): + """Flip V-plot""" + if mode == 'same': + new = np.zeros(self.mat.shape) + for j in range(self.i_lower,self.i_upper): + i = j - self.i_lower + if j%2==1: + new[i,:] = self.mat[i,][::-1] + else: + new[i,:-1] = self.mat[i,:-1][::-1] + #for -1 postion don't actually have values + new[i,-1] = np.mean([self.mat[i,-1],self.mat[i,1]]) + self.mat = new + elif mode == 'valid': + new = np.zeros((self.mat.shape[0],self.mat.shape[1]-2)) + for j in range(self.i_lower,self.i_upper): + i = j - self.i_lower + if j%2==1: + new[i,:] = self.mat[i,1:-1][::-1] + else: + new[i,:] = self.mat[i,:-1][::-1][1:] + self.mat = new + self.w += -1 + else: + raise Exception("Mode must be one of 'same' or 'valid'") + def smooth(self, sd = 1): + """smooth v-plot using gaussian kernel""" + self.mat = ndimage.filters.gaussian_filter(self.mat,sd, + mode='constant') + def smooth1d(self, sd = 1, axis = 1): + """smooth v-plot along one axis only""" + self.mat = ndimage.filters.gaussian_filter1d(self.mat,sd,axis, + mode='nearest') + def norm(self): + """normalize v matrix so that signal minus even background will be 1 divided by base pairs in window""" + tmp1 = self.mat / np.sum(self.mat) + tmp2 = np.ones(self.mat.shape) * (1.0 / self.mat.size) + self.mat = self.mat / (np.sum(self.mat * tmp1)- np.sum(self.mat * tmp2)) + self.mat = (self.mat / self.mat.shape[1]) * 10.0 + def norm_y(self,dist): + """normalize vplot so insertsize matches supplied distribution""" + for i in range(self.mat.shape[0]): + self.mat[i] = self.mat[i] * (dist.get(size = i + self.i_lower)/ np.sum(self.mat[i])) + def converto1d(self): + """convert the 2d matrix to a 1d representation of insertions""" + self.one_d = np.zeros(self.i_upper + self.i_upper%2 +2*self.w+1) + center = self.i_upper/2 + self.w + for j in range(self.mat.shape[0]): + for i in range(self.mat.shape[1]): + ilen=j+self.i_lower + val = copy(self.mat[j,i]) + if ilen%2==0: + self.one_d[center-(self.w-i)-(ilen/2)]+= val + self.one_d[center-(self.w-i)+(ilen/2)]+= val + else: + self.one_d[center-(self.w-i)-(ilen/2)]+= val * 0.5 + self.one_d[center-(self.w-i)+(ilen/2)]+= val * 0.5 + self.one_d[center-(self.w-i)-(ilen/2+1)]+= val * 0.5 + self.one_d[center-(self.w-i)+(ilen/2+1)]+= val * 0.5 + self.one_d = self.one_d / sum(self.one_d) + def plot(self, mat=None, title=None, filename=None): + """Plot current main matrix or specified matrix (of same dimensions)""" + if mat is None: + mat=self.mat + elif mat.shape!=(self.i_upper-self.i_lower,self.w*2+1): + raise VMat_Error("dimensions of input mat should match \ + dim of vmat") + fig = plt.figure() + plt.imshow(mat,origin="lower",interpolation='nearest', + extent=[-self.w,self.w,self.i_lower,self.i_upper-1]) + plt.xlabel("Position relative to dyad") + plt.ylabel("Insert size") + if title: + plt.title(title) + plt.colorbar(shrink=0.8) + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + np.savetxt(filename2,mat,delimiter="\t") + else: + fig.show() + def plot_1d(self,filename=None): + """plot the 1d insertion representation of the matrix""" + fig = plt.figure() + xlim = len(self.one_d)/2 + plt.plot(range(-xlim,xlim+1),self.one_d) + plt.vlines(-73,0,max(self.one_d)*1.1,linestyles='dashed') + plt.vlines(73,0,max(self.one_d)*1.1,linestyles='dashed') + plt.xlabel("Position relative to dyad") + plt.ylabel("Insertion Frequency") + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + np.savetxt(filename2,self.one_d,delimiter="\t") + else: + fig.show() + def plot_insertsize(self,filename=None): + """plot the insert size disribution in the main matrix""" + fig = plt.figure() + ins = np.sum(self.mat,axis=1) + ins = ins/sum(ins) + plt.plot(range(self.i_lower,self.i_upper),ins) + plt.xlabel("Insert Size") + plt.ylabel("Frequency") + if filename: + fig.savefig(filename) + plt.close(fig) + #Also save text output! + filename2 = ".".join(filename.split(".")[:-1]+['txt']) + np.savetxt(filename2,ins,delimiter="\t") + else: + fig.show() + def save(self,filename): + """write text output description of VMat object attributes""" + out=open(filename,'w') + out.write('#VMat Descriptor File\n') + out.write('#Contains VMat and pertinent information\n') + out.write('#i_lower\n') + out.write(str(self.i_lower)+'\n') + out.write('#i_upper\n') + out.write(str(self.i_upper)+'\n') + out.write('#mat\n') + for row in self.mat: + out.write("\t".join(map(str,row))+'\n') + out.close() + @staticmethod + def open(filename): + """Create VMat object from text descriptor file""" + infile = open(filename,'r') + state = '' + mat = [] + for line in infile: + if '#i_lower' in line: + state = 'i_lower' + elif '#i_upper' in line: + state = 'i_upper' + elif '#mat' in line: + state = 'mat' + elif '#' in line: + state = 'other' + elif state == 'i_lower': + i_lower = int(line.strip('\n')) + elif state == 'i_upper': + i_upper = int(line.strip('\n')) + elif state == 'mat': + mat.append(map(float,line.strip('\n').split('\t'))) + try: + new = VMat(np.array(mat), i_lower, i_upper) + except NameError: + raise VMat_Error("VMat decriptor file appeas to be missing some\ +needed components") + infile.close() + return new diff --git a/nucleoatac/__init__.py b/nucleoatac/__init__.py new file mode 100644 index 0000000..e69de29 diff --git a/nucleoatac/make_vplot.py b/nucleoatac/make_vplot.py new file mode 100644 index 0000000..a708f34 --- /dev/null +++ b/nucleoatac/make_vplot.py @@ -0,0 +1,100 @@ +#!/usr/bin/env python +""" +Script to make V-plot + +@author: Alicia Schep +""" + +##### IMPORT MODULES ##### +# import necessary for python +import numpy as np +import matplotlib as mpl +mpl.use('PS') +import pysam +import PyATAC as PA +import VMat as V +from multiprocessing import Pool +from itertools import repeat +import traceback + + +def vplot_helper(arg): + """function to make vplot for a particular set of bed regions + + """ + (chunkset, args) = arg + try: + bamfile = pysam.Samfile(args.bam, "rb") + mat = np.zeros((args.upper-args.lower,2*args.flank+1)) + for chunk in chunkset.chunks: + if chunk.length > 1: + mid=(chunk.start+(chunk.end-chunk.start-1)/2) + chunk.start = mid + chunk.end = mid + 1 + chunk.length = 1 + elif chunk.length == 0: + chunk.end = chunk.start + 1 + chunk.length = 1 + reads = PA.ReadList(chunk.chrom, chunk.start - args.flank-1, chunk.end + args.flank) + reads.extract_reads(bamfile, i_upper = args.upper, atac = args.not_atac) + if chunk.strand=="-": + submat = PA.ReadMat2D(chunk.chrom, chunk.start - args.flank-1, chunk.end + args.flank, args.lower, args.upper) + else: + submat = PA.ReadMat2D(chunk.chrom, chunk.start - args.flank, chunk.end + args.flank , args.lower, args.upper) + submat.makeReadMat(reads) + if np.sum(submat.mat)==0: + continue + if chunk.strand == "-": + add = submat.get(start = chunk.start- args.flank, end = chunk.end + args.flank, flip = True) + else: + add= submat.get() + if args.scale: + mat += add/np.sum(add) + else: + mat += add + bamfile.close() + except Exception as e: + print('Caught exception when processing: '+ chunkset.name) + traceback.print_exc() + print() + raise e + return mat + +def make_vplot(args): + """function to make vplot + + """ + #read in bed list + bed_list = PA.read_bed(args.bed,weight_col=args.weight,strand_col = args.strand) + #divide intervals into sets based on number of cores + sets = PA.makeChunkSetList(bed_list, args.cores) + + ##parallel processed computation + pool = Pool(processes=args.cores) + submats=pool.map(vplot_helper,zip(sets,repeat(args))) + # sum up matrices for each chunk into matrix for all + mat = np.zeros(submats[0].shape) + for i in range(len(submats)): + mat=mat+submats[i] + + ##Turn matrix into VMat object + vmat=V.VMat(mat,args.lower,args.upper) + vmat.plot(filename=args.out+".Vplot.eps") + ##get insertion profile represented by vplot + vmat.converto1d() + vmat.plot_1d(filename=args.out+'.InsertionProfile.eps') + #get insert size dstribution represented by vplot + vmat.plot_insertsize(filename= args.out + ".InsertSizes.eps") + ##save + vmat.save(args.out+".VMat") + + + + + + + + + + + diff --git a/nucleoatac/multinomial_cov.pyx b/nucleoatac/multinomial_cov.pyx new file mode 100644 index 0000000..766253c --- /dev/null +++ b/nucleoatac/multinomial_cov.pyx @@ -0,0 +1,31 @@ +#!/usr/bin/env python +""" +Created on Mon May 12 14:40:53 2014 + +@author: alicia +""" + +from __future__ import division +import numpy as np +cimport numpy as np +cimport cython + + +DTYPE = np.float +ctypedef np.float_t DTYPE_t + + + +@cython.boundscheck(False) +def makeCov(np.ndarray[DTYPE_t, ndim=1] p, np.ndarray[DTYPE_t, ndim=1] v, int r): + if p.shape[0]!= v.shape[0]: + raise ValueError("p and v must be same shape") + cdef DTYPE_t value + cdef unsigned int i, j + for i in range(p.shape[0]): + for j in range(i,p.shape[0]): + if i==j: + value += p[i]* (1-p[i]) * v[i]**2 + else: + value += p[i] * p[j] * -2 * v[i] * v[j] + return value * r diff --git a/nucleoatac/run_nuc.py b/nucleoatac/run_nuc.py new file mode 100644 index 0000000..19a163a --- /dev/null +++ b/nucleoatac/run_nuc.py @@ -0,0 +1,105 @@ +#!/usr/bin/env python +""" +Script to call nucleosome positions-- track making, nucleosome calling, and nfr calling! + +@author: Alicia Schep +""" + +##### IMPORT MODULES ##### +# import necessary python modules +import matplotlib as mpl +mpl.use('PS') +import PyATAC as PA +import VMat as V +import NucleosomeCalling as Nuc +from multiprocessing import Pool +import Occupancy as Occ +import traceback +from itertools import repeat + +def nuc_helper(arg): + """function to call nucs for particular set of bed regions + + """ + (chunkset, params) = arg + try: + chunkset.process(params) + except Exception as e: + print('Caught exception when processing: '+ chunkset.name) + traceback.print_exc() + print() + raise e + + + +def run_nuc(args): + """run nucleosome calling-- make signal tracks, call nucleosome positions + + """ + #Make tmp directory for tmp files + try: + PA.shell_command('mkdir tmp_nucleoatac') + except: + print "Deleting old tmp_nuc folder to make new one" + PA.shell_command('rm -r tmp_nucleoatac') + PA.shell_command('mkdir tmp_nucleoatac') + + print "Reading in bedfile and setting up parameters" + #Set up parameters + ##Get chromosome info from bam + chromosomes = PA.Chromosomes() + chromosomes.get_from_bam(args.bam) + chromosomes.write_chrs('tmp_nucleoatac/genome.txt') + + vmat = V.VMat.open(args.vmat) + + bed_list = Nuc.read_bed(args.bed, chromosomes.chrs, + max_offset = vmat.mat.shape[1] + vmat.i_upper/2) + sets = Nuc.makeChunkSetList(bed_list, args.cores) + + insert_dist = Occ.InsertDistribution(0,i_upper = vmat.i_upper) + insert_dist.getInsertSizes(args.bam, bed_list) + + if args.occ_track is None: + insert_dist.modelNFR(vmat.i_lower) + occ_params = Occ.OccupancyCalcParams(0, vmat.i_upper, insert_dist) + else: + occ_params = None + + params = Nuc.NucParameters(vmat, insert_dist.insertsizes, sd = args.sd, nonredundant_sep = args.nuc_sep, + redundant_sep = args.redundant_sep, min_z = args.min_z, min_lr = args.min_lr, downsample = args.downsample, + seed = args.seed, min_nfr_len = 1, max_nfr_len = 1000, max_nfr_occ = args.max_nfr_occ, + out = args.out, gdna = args.gdna, bias= args.bias, occ_track = args.occ_track, bam = args.bam, + write_all = args.write_all, occ_params = occ_params) + + print "Processing Regions" + + ##parallel processed computation + pool = Pool(processes=args.cores) + pool.map(nuc_helper,zip(sets,repeat(params))) + + print "Merging tmp files" + + if args.write_all: + track_outputs = ['nucsig','smoothsig','ins','nuc_cov','nfr_cov','background','rawsig','fitted'] + else: + track_outputs = ['nucsig','smoothsig','ins'] + if args.occ_track is None: + track_outputs.append('occ') + + for track in track_outputs: + PA.combine_tmp_bed_to_bw(args.out, track) + + for bed in ['nucpos','nucpos.redundant','nfrpos']: + PA.combine_tmp_bed(args.out, bed) + + PA.shell_command('rm -r tmp_nucleoatac') + + + + + + + + + diff --git a/nucleoatac/run_occ.py b/nucleoatac/run_occ.py new file mode 100644 index 0000000..06c62df --- /dev/null +++ b/nucleoatac/run_occ.py @@ -0,0 +1,109 @@ +#!/usr/bin/env python +""" +Script to make nucleosome occupancy track! + +@author: Alicia Schep +""" + +##### IMPORT MODULES ##### +# import necessary python modules +import matplotlib as mpl +mpl.use('PS') +import matplotlib.pyplot as plt +import PyATAC as PA +import Occupancy as Occ +from multiprocessing import Pool +import numpy as np +from itertools import repeat +import traceback + +def occ_helper(arg): + """function to get occupancy for a set of bed regions + + """ + (chunkset, params) = arg + try: + nuc_dist = chunkset.process(params) + except Exception as e: + print('Caught exception when processing: '+ chunkset.name) + traceback.print_exc() + print() + raise e + return nuc_dist + +def run_occ(args): + """run occupancy calling + + """ + #Make tmp directory for tmp files + try: + PA.shell_command('mkdir tmp_nucleoatac') + except: + print "Deleting old tmp_nuc folder to make new one" + PA.shell_command('rm -r tmp_nucleoatac') + PA.shell_command('mkdir tmp_nucleoatac') + + + ##Get chromosome info from bam + chromosomes = PA.Chromosomes() + chromosomes.get_from_bam(args.bam) + chromosomes.write_chrs('tmp_nucleoatac/genome.txt') + + #Read in bed list + bed_list = Occ.read_bed(args.bed, chromosomes.chrs, 75) + + print "Getting insert distribution" + insert_dist = Occ.InsertDistribution(0,i_upper = args.upper) + insert_dist.getInsertSizes(args.bam, bed_list) + + print "Modelling NFR distribution" + insert_dist.modelNFR(args.lower) + insert_dist.plotFits(args.out+".fits.eps") + + print "Calculating occupancies and getting nucleosomal distribution" + + params = Occ.OccupancyParams(Occ.OccupancyCalcParams(0,args.upper, insert_dist), + sep = args.nuc_sep, min_occ = args.min_occ, min_reads = args.reads, flank = args.flank, + write_peaks = args.write_peaks, out = args.out, bam = args.bam) + + #divide intervals into sets based on number of cores + sets = Occ.makeChunkSetList(bed_list, args.cores) + + ##parallel processed computation + pool = Pool(processes=args.cores) + submats=pool.map(occ_helper,zip(sets,repeat(params))) + + mat = np.zeros(submats[0].shape) + for i in range(len(submats)): + mat=mat+submats[i] + mat = mat / sum(mat) + nuc_dist = PA.InsertSizes(mat,0,args.upper) + nuc_dist.save(args.out + '.nuc_dist.txt') + + print "Making figure" + #make figure + fig = plt.figure() + plt.plot(range(0,args.upper),nuc_dist.get(0,args.upper),label = "Nucleosome Distribution") + plt.xlabel("Fragment Size") + plt.ylabel("Frequency") + fig.savefig(args.out+'.nuc_dist.eps') + plt.close(fig) + + print "Merging tmp files" + + PA.combine_tmp_bed_to_bw(args.out, 'occ') + + if args.write_peaks: + PA.combine_tmp_bed(args.out, 'occ_peaks') + + PA.shell_command('rm -r tmp_nucleoatac') + + + + + + + + + + diff --git a/nucleoatac/run_vprocess.py b/nucleoatac/run_vprocess.py new file mode 100644 index 0000000..68781e0 --- /dev/null +++ b/nucleoatac/run_vprocess.py @@ -0,0 +1,52 @@ +#!/usr/bin/env python +""" +Takes as input a raw vplot and processes it for use in nucleosome calling +Use insert distribution at nuclesomal regions in peaks for normalization + +@author: Alicia Schep +""" + +##### IMPORT MODULES ##### +# import necessary for python +import os +import sys +import matplotlib as mpl +mpl.use('PS') +import VMat as V +import PyATAC as PA +import numpy as np + +def run_vprocess(args): + """process vplot + + """ + #Load vplot + if args.vplot is None: + args.vplot = os.path.dirname(os.path.realpath(sys.argv[0]))+'/../nucleoatac/standard_vplot.VMat' + vmat=V.VMat.open(args.vplot) + #Trim, Symmetrize + vmat.trim(args.lower,args.upper,args.flank) + vmat.symmetrize() + #insert size norm + if args.isizes is not None: + #read in insertsizes + nuc_dist = PA.InsertSizes.open(args.isizes) + vmat.norm_y(nuc_dist) + ##Smooth + if args.smooth > 0: + vmat.smooth(sd=args.smooth) + #normalize + vmat.norm() + #Make extra plots if requeted + if args.plot_extra: + vmat.autoCorr() + vmat.plot_auto(args.out+'.vplot.Autocorr.eps') + vmat.converto1d() + vmat.plot_1d(args.out+'.vplot.InsertionProfile.eps') + vmat.plot_insertsize(args.out+'.vplot.InsertSizes.eps') + #make plot and save + vmat.save(args.out+".VMat") + vmat.plot(filename = args.out+".VMat.eps") + + + diff --git a/nucleoatac/standard_vplot.VMat b/nucleoatac/standard_vplot.VMat new file mode 100644 index 0000000..736454d --- /dev/null +++ b/nucleoatac/standard_vplot.VMat @@ -0,0 +1,307 @@ +#VMat 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34.0 34.0 43.0 38.0 54.0 64.0 54.0 43.0 34.0 41.0 43.0 33.0 43.0 36.0 39.0 45.0 43.0 diff --git a/setup.py b/setup.py new file mode 100644 index 0000000..ffa984d --- /dev/null +++ b/setup.py @@ -0,0 +1,36 @@ +from setuptools import setup +import sys + + +def readme(): + with open('README.md') as f: + return f.read() + +if float(sys.version[:3])<2.7 or float(sys.version[:3])>=2.8: + sys.stderr.write("CRITICAL: Python version must be 2.7!\n") + sys.exit(1) + +setup(name='nucleoatac', + version='0', + description='nucleosome calling for ATAC-Seq', + long_description=readme(), + classifiers=[ + 'Development Status :: 3 - Alpha', + 'License :: OSI Approved :: MIT License', + 'Programming Language :: Python :: 2.7', + 'Topic :: Scientific/Engineering :: Bio-Informatics'], + keywords='nucleosome ATAC-Seq sequencing', + url='https://github.com/GreenleafLab/NucleoATAC', + author='Alicia Schep', + author_email='aschep@stanford.edu', + license='MIT', + packages=['nucleoatac'], + install_requires=['numpy', 'scipy','pysam','matplotlib','cython','bx-python'], + scripts=['bin/nucleoatac'], + include_package_data=True, + zip_safe=False, + test_suite='nose.collector', + tests_require=['nose']) + + + diff --git a/tests/__init__.py b/tests/__init__.py new file mode 100644 index 0000000..e69de29 diff --git a/tests/test_occupancy.py b/tests/test_occupancy.py new file mode 100644 index 0000000..3adbdac --- /dev/null +++ b/tests/test_occupancy.py @@ -0,0 +1,33 @@ +from unittest import TestCase + +import nucleoatac.PyATAC as PA +import nucleoatac.Occupancy as Occ +import numpy as np + +class Test_occupancy(TestCase): + """class to test occupancy""" + def setUp(self): + """setup Test_occupancy class by establishing parameters""" + self.insert_dist = Occ.InsertDistribution(0,3) + self.insert_dist.nfr_fit = PA.InsertSizes(np.array([0.5, 0.49, 0.01 ]),0,3) + self.insert_dist.nuc_fit = PA.InsertSizes(np.array([0.01, 0.49,0.5]),0,3) + self.params = Occ.OccupancyCalcParams(0,3,self.insert_dist) + def test_occupancy_calc1(self): + """test occupancy class for a zero occupancy situation""" + occ = Occ.calculateOccupancy(np.array([1,0,0]),self.params) + self.assertTrue(occ == 0) + def test_occupancy_calc2(self): + """test occupancy for a 50% occupancy situation""" + occ = Occ.calculateOccupancy(np.array([1,1,1]),self.params) + self.assertTrue(occ == 0.5) + def test_occupancy_calc3(self): + """test average occupancy for when you have 40 random reads, + which should be 75% from nuc dist + """ + results = np.zeros(100) + for i in range(100): + a = np.random.multinomial(10,self.insert_dist.nfr_fit.get()) + b = np.random.multinomial(30,self.insert_dist.nuc_fit.get()) + results[i] = Occ.calculateOccupancy(a+b,self.params) + self.assertTrue(abs(np.mean(results)-0.75)<0.1) + diff --git a/tests/test_pyatac.py b/tests/test_pyatac.py new file mode 100644 index 0000000..1d7750b --- /dev/null +++ b/tests/test_pyatac.py @@ -0,0 +1,129 @@ +from unittest import TestCase + +import nucleoatac.PyATAC as PA +import numpy as np +import bx.bbi.bigwig_file +import pysam + +class Test_call_peaks(TestCase): + """test the call_peaks function in pyatac""" + def test_call_peaks_case1(self): + """test case1 for call_peaks""" + peaks = PA.call_peaks(np.array([1,2,3,2,1,4,1,2,1,0,0]),min_signal=1,sep=3) + self.assertTrue(np.array_equal(peaks,np.array([2,5]))) + def test_call_peaks_case2(self): + """test case2 for call_peaks""" + peaks = PA.call_peaks(np.array([1,2,3,2,1,4,1,2,1,0,0]),min_signal=1,sep=1) + self.assertTrue(np.array_equal(peaks,np.array([2,5,7]))) + def test_call_peaks_case3(self): + """test case3 for call_peaks""" + peaks = PA.call_peaks(np.array([1,2,3,2,1,4,1,2,1,0,0]),min_signal=3,sep=2) + self.assertTrue(np.array_equal(peaks,np.array([2,5]))) + +class Test_ReadMat2D(TestCase): + """test methods for ReadMat2D""" + def test_updatedMat(self): + """test updating of readmat""" + x = PA.ReadMat2D('chrI', 500, 1000, 0, 200) + read = PA.Read('chrI', 550, 100) + x.updateMat(read) + y = np.zeros((200,500)) + y[100,99]=1 + self.assertTrue(np.array_equal(x.get(),y)) + def test_get(self): + """test get function for readmat""" + x = PA.ReadMat2D('chr1',500,1000,0,200) + x.mat[100,5] = 1 + self.assertTrue(np.array_equal(x.get(start=505,end=507,i_lower=100,i_upper=102),np.array([[1,0],[0,0]]))) + +class Test_readlist(TestCase): + """tests readlist class""" + def setUp(self): + """read in readlist""" + bam = pysam.Samfile('example/single_read.bam','rb') + self.readlist = PA.ReadList("chrII",706800,707000) + self.readlist.extract_reads(bam) + def test_chrom(self): + """test chromosome of read""" + self.assertTrue(self.readlist.reads[0].chrom == "chrII") + def test_left(self): + """test left position of read""" + self.assertTrue(self.readlist.reads[0].left == 706858) + def test_right(self): + """test right position of read""" + self.assertTrue(self.readlist.reads[0].right == 706929) + def test_insert(self): + """test insert size of read""" + self.assertTrue(self.readlist.reads[0].insert == 71) + +class Test_BiasMat(TestCase): + """test construction of BiasMat""" + def setUp(self): + """setup Test_BiasMat class with construction of a biasmat""" + bed_list = PA.read_bed('example/example.bed') + self.chunk = bed_list[0] + bias_bwh=bx.bbi.bigwig_file.BigWigFile(open('example/example.Scores.bw','rb')) + self.biastrack = PA.InsertionBiasTrack(self.chunk.chrom, self.chunk.start, self.chunk.end) + self.biastrack.read_track(bias_bwh) + self.biasmat = PA.BiasMat2D(self.chunk.chrom,self.chunk.start+100,self.chunk.end-100,100,200) + self.biasmat.makeBiasMat(self.biastrack) + def test_biasmat1(self): + """test case1 for biasmat""" + a1 = self.biastrack.get(pos = self.biasmat.start - 49) + a2 = self.biastrack.get(pos = self.biasmat.start + 50) + correct = np.exp(a1+a2) + self.assertTrue(abs(correct - self.biasmat.mat[0,0])<0.01*correct) + def test_biasmat2(self): + """test case2 for biasmat""" + a1 = self.biastrack.get(pos = self.biasmat.start + 145) + a2 = self.biastrack.get(pos = self.biasmat.start + 295) + correct = np.exp(a1+a2) + self.assertTrue(abs(correct - self.biasmat.mat[51,220]) < 0.01*correct) + def test_normByInsertDist(self): + """test that normalization by insert distribution works as expected""" + isizes = PA.InsertSizes(np.array(range(100,200)),i_lower=100,i_upper=200) + self.biasmat.normByInsertDist(isizes) + a1 = self.biastrack.get(pos = self.biasmat.start -50) + a2 = self.biastrack.get(pos = self.biasmat.start + 50) + correct = np.exp(a1+a2)*isizes.get(size = 101) + self.assertTrue(abs(correct - self.biasmat.mat[1,0])<0.01*correct) + +class Test_Ins(TestCase): + """test that conversion of readmat to insertion gives same result as insertion track""" + def setUp(self): + """setup Test_Ins class by making a readlist""" + bed_list = PA.read_bed('example/example.bed') + self.chunk = bed_list[0] + self.readlist = PA.ReadList(self.chunk.chrom, self.chunk.start, self.chunk.end) + self.readlist.addRead(PA.Read(self.chunk.chrom, self.chunk.start + 200, 93)) + self.readlist.addRead(PA.Read(self.chunk.chrom, self.chunk.start + 155, 43)) + self.readlist.addRead(PA.Read(self.chunk.chrom, self.chunk.start + 138, 90)) + self.readlist.addRead(PA.Read(self.chunk.chrom, self.chunk.start + 155, 43)) + self.readlist.addRead(PA.Read(self.chunk.chrom, self.chunk.start + 147, 73)) + def test_ins_methods(self): + """test that two methods for getting insertion track give same result""" + ins1 = PA.InsertionTrack(self.chunk.chrom, self.chunk.start, self.chunk.end) + ins1.calculateInsertions(self.readlist) + mat = PA.ReadMat2D(self.chunk.chrom,self.chunk.start,self.chunk.end,0,100) + mat.makeReadMat(self.readlist) + ins2 = mat.getIns() + self.assertTrue(np.array_equal(ins1.get(self.chunk.start+100,self.chunk.start+300),ins2.get(self.chunk.start+100,self.chunk.start+300))) + +class Test_Track(TestCase): + """Test out the Track class in PyATAC""" + def setUp(self): + """setup Test_Track class""" + bed_list = PA.read_bed('example/example.bed') + self.chunk = bed_list[0] + def test_read_and_get(self): + """test the read and get functionality of track class""" + track = PA.Track(self.chunk.chrom,self.chunk.start,self.chunk.end) + bias_bwh = bx.bbi.bigwig_file.BigWigFile(open('example/example.Scores.bw','rb')) + track.read_track(bias_bwh) + val = 1.35994655714 + self.assertTrue(abs(val - track.get(pos = 706661))<0.001) + + + + + diff --git a/tests/test_var.py b/tests/test_var.py new file mode 100644 index 0000000..d850695 --- /dev/null +++ b/tests/test_var.py @@ -0,0 +1,45 @@ +from unittest import TestCase + +import nucleoatac.NucleosomeCalling as Nuc +import nucleoatac.VMat as V +import nucleoatac.PyATAC as PA +import numpy as np +import bx.bbi.bigwig_file + +class Test_variance(TestCase): + """class for testing variance calculation on background signal + + """ + def setUp(self): + """ set up class for testing variance calculation for background signal + + """ + bed_list = PA.read_bed('example/example.bed') + chunk = bed_list[0] + vmat = V.VMat.open('example/example.VMat') + bias_bwh=bx.bbi.bigwig_file.BigWigFile(open('example/example.Scores.bw','rb')) + biastrack = PA.InsertionBiasTrack(chunk.chrom, chunk.start, chunk.end) + biastrack.read_track(bias_bwh) + biasmat = PA.BiasMat2D(chunk.chrom,chunk.start+200,chunk.end-200,100,250) + biasmat.makeBiasMat(biastrack) + self.signaldist = Nuc.SignalDistribution(chunk.start+300,vmat,biasmat,35) + def test_sd1(self): + """Make sure variance calculation is close to what is obtained by simulation + + """ + self.signaldist.simulateDist(5000) + sd1 = np.std(self.signaldist.scores) + sd2 = self.signaldist.analStd() + self.assertTrue(abs(sd1-sd2)<0.05*sd1) + def test_sd2(self): + """Make sure variance calculation is same as would be obtained through alternate calculation + + """ + var_term = np.sum(self.signaldist.prob_mat*(1-self.signaldist.prob_mat)*self.signaldist.vmat.mat**2) + tmp = self.signaldist.prob_mat *self.signaldist.vmat.mat + cov_term = np.sum(np.outer(tmp,tmp))-np.sum(tmp**2) + sd1 = np.sqrt(self.signaldist.reads * (var_term - cov_term)) + sd2 = self.signaldist.analStd() + self.assertTrue(abs(sd1-sd2)<0.001*sd1) + + diff --git a/tests/test_xcor.py b/tests/test_xcor.py new file mode 100644 index 0000000..907166c --- /dev/null +++ b/tests/test_xcor.py @@ -0,0 +1,34 @@ +from unittest import TestCase + +import nucleoatac.PyATAC as PA +import nucleoatac.NucleosomeCalling as Nuc +import nucleoatac.VMat as V +import numpy as np +import pysam + +class Test_xcor(TestCase): + """class to test occupancy""" + def setUp(self): + """setup Test_occupancy class by establishing parameters""" + bed_list = PA.read_bed('example/example.bed') + self.chunk = bed_list[0] + bam = pysam.Samfile('example/example.bam','rb') + self.vmat = V.VMat.open('example/example.VMat') + readlist = PA.ReadList(self.chunk.chrom, self.chunk.start - self.vmat.w, self.chunk.end + self.vmat.w) + readlist.extract_reads(bam) + self.vmat = V.VMat.open('example/example.VMat') + self.mat = PA.ReadMat2D(self.chunk.chrom,self.chunk.start-self.vmat.w,self.chunk.end+self.vmat.w,self.vmat.i_lower,self.vmat.i_upper) + self.mat.makeReadMat(readlist) + self.signal = Nuc.SignalTrack(self.chunk.chrom,self.chunk.start,self.chunk.end) + self.signal.calculateSignal(self.mat, self.vmat) + def test_signal_calc1(self): + """test signal calc""" + a=np.sum(self.mat.get(start = self.chunk.start, end = self.chunk.start + self.vmat.w*2 + 1)*self.vmat.mat) + b=self.signal.get(pos=self.chunk.start + self.vmat.w) + self.assertTrue(abs(a-b)<0.0001) + def test_signal_calc2(self): + """test signal calc""" + a=np.sum(self.mat.get(start = self.chunk.start+100, end = self.chunk.start+ 100 + self.vmat.w*2 + 1)*self.vmat.mat) + b=self.signal.get(pos=self.chunk.start+ 100 + self.vmat.w) + self.assertTrue(abs(a-b)<0.0001) +