├── .travis.yml ├── LICENSE ├── README.md ├── cb_sniffer.py └── requirements.txt /.travis.yml: -------------------------------------------------------------------------------- 1 | language: python 2 | python: 3 | - 3.6 4 | install: 5 | - pip install -r requirements.txt 6 | 7 | script: make test 8 | -------------------------------------------------------------------------------- /LICENSE: -------------------------------------------------------------------------------- 1 | GNU GENERAL PUBLIC LICENSE 2 | Version 3, 29 June 2007 3 | 4 | Copyright (C) 2007 Free Software Foundation, Inc. 5 | Everyone is permitted to copy and distribute verbatim copies 6 | of this license document, but changing it is not allowed. 7 | 8 | Preamble 9 | 10 | The GNU General Public License is a free, copyleft license for 11 | software and other kinds of works. 12 | 13 | The licenses for most software and other practical works are designed 14 | to take away your freedom to share and change the works. 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Of course, your program's commands 662 | might be different; for a GUI interface, you would use an "about box". 663 | 664 | You should also get your employer (if you work as a programmer) or school, 665 | if any, to sign a "copyright disclaimer" for the program, if necessary. 666 | For more information on this, and how to apply and follow the GNU GPL, see 667 | . 668 | 669 | The GNU General Public License does not permit incorporating your program 670 | into proprietary programs. If your program is a subroutine library, you 671 | may consider it more useful to permit linking proprietary applications with 672 | the library. If this is what you want to do, use the GNU Lesser General 673 | Public License instead of this License. But first, please read 674 | . 675 | -------------------------------------------------------------------------------- /README.md: -------------------------------------------------------------------------------- 1 | cb_sniffer 2 | ============= 3 | 4 | Mutation barcode caller, calls mutant and ref barcodes from 10x single cell data 5 | Bams aligned using 10x cellranger pipeline 6 | 7 | Usage 8 | ---- 9 | ```{shell} 10 | python3 cb_sniffer.py -h 11 | usage: cb_sniffer.py [-h] [-f FILTER] [-mq MAPQ] [-bq BASEQ] 12 | bam_file variant_file barcodes upn 13 | 14 | Parse CB barcodes from Single cell rna seq data 15 | 16 | positional arguments: 17 | bam_file BAM file 18 | variant_file variants file with header 19 | barcodes list of good barcodes file 20 | upn upn/sample name: will be used as prefix for out_file 21 | 22 | optional arguments: 23 | -h, --help show this help message and exit 24 | -f FILTER, --filter FILTER 25 | number of reads required per barcode default: 0 26 | -mq MAPQ, --mapq MAPQ 27 | Skip read with mapq smaller than default : 0 28 | -bq BASEQ, --baseq BASEQ 29 | Skip bases with base quality less than default : 1 30 | 31 | ``` 32 | #### Variant file 33 | ```{shell} 34 | chrm start stop ref var gene_name trv_type 35 | 19 4xxx 4xxx G A gene_name silent/misense/frame_shift 36 | ``` 37 | 38 | #### barcodes 39 | ```{shell} 40 | AACCTGAGAATGTTG-1 41 | AAACCTGAGCTACCGC-1 42 | AAACCTGAGCTGCCCA-1 43 | AAACCTGAGGTCGGAT-1 44 | AAACCTGAGTACGATA-1 45 | AAACCTGAGTGCAAGC-1 46 | AAACCTGAGTGCCATT-1 47 | AAACCTGAGTGGAGTC-1 48 | AAACCTGCAAAGTGCG-1 49 | AAACCTGCATAAGACA-1 50 | ``` 51 | 52 | ### Output files 53 | 1. variant list with barcodes supporting a variant :_AllCounts.tsv 54 | 2. read counts for Chromium cellular barcode sequence(CB_tag) : _counts_CB.tsv 55 | 3. read counts for Chromium molecular barcode(UB_tag): _counts_UB.tsv 56 | 57 | 58 | * Additional info on tags 59 | [bam_tags](https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/bam) 60 | 61 | 62 | 63 | Dependencies 64 | ------- 65 | 66 | python3 67 | pysam 68 | 69 | Docker image 70 | ---------- 71 | * sridnona/python3:180925.v1 72 | 73 | ```{shell} 74 | LSF_DOCKER_PRESERVE_ENVIRONMENT=false bsub -Is -q research-hpc -a "docker(sridnona/python3:180925.v1)" /bin/bash 75 | ``` 76 | -------------------------------------------------------------------------------- /cb_sniffer.py: -------------------------------------------------------------------------------- 1 | from collections import defaultdict 2 | import pysam 3 | import logging as logger 4 | import sys 5 | import argparse 6 | #print(pysam.__version__) 7 | #0.14.1 8 | #python3 9 | #3.6.5 10 | 11 | 12 | class GenomicPosition: 13 | 14 | def __init__(self, line): 15 | """ 16 | :param line: 17 | for each variant holds genomics positions 18 | and other informations 19 | """ 20 | region = line.strip().split('\t') 21 | if len(region) < 7: 22 | logger.debug("Insufficient columns fields please check variant file") 23 | sys.exit(1) 24 | self.chrm = region[0] 25 | self.start = int(region[1]) 26 | self.end = int(region[2]) 27 | self.ref = region[3] 28 | self.alt = region[4] 29 | self.gene = region[5] # gene name 30 | self.event = region[6] # 31 | # print(self, self.chrm, self.start, self.ref, self.alt,self.event) 32 | 33 | def classify(self): 34 | """ 35 | for each variant classifies if its a snp or variant 36 | :return: 37 | 0 for snp 38 | int if its insertion or deletion 39 | """ 40 | v = ['A', 'T', 'G', 'C'] 41 | if self.ref and self.alt in v: 42 | return 0 43 | 44 | else: 45 | if self.alt == '-': # deletion 46 | return len(self.ref) 47 | elif self.ref == '-': # insertion 48 | return len(self.alt) 49 | elif len(self.ref) == len(self.alt) > 1: 50 | return len(self.alt) 51 | 52 | @classmethod 53 | def good_barcodes(cls,f): 54 | """ 55 | 56 | :param f: good barcodes file 57 | :return: dict with good barcodes 58 | """ 59 | barc = {} 60 | with open(f, 'r') as barcodes: 61 | for line in barcodes: 62 | lines = line.strip() 63 | barc[lines] = 1 64 | return barc 65 | 66 | def count_barcodes(self, bam_fil, bar, indel, mapq=0, baseq=0): 67 | """ 68 | 69 | :param bam_fil: bam_file 70 | :param bar: good barcodes dict 71 | :param indel: SNP = 0 indel= int(window size) insertion or deletion 72 | :param mapq: mapping quality 73 | :param baseq: base quality 74 | :return: two dicts 1) list of all barcodes at given var pos 75 | 2) dict with ref and alt barcodes 76 | """ 77 | barcodes = defaultdict(list) # CB 78 | # ====================================== 79 | barUcodes = defaultdict(list) # UB 80 | bar_count = {'ref': [], 'alt': []} # UB 81 | with pysam.AlignmentFile(bam_fil, 'rb') as pile: 82 | logger.info("processing variant: {}\t{}\t{}\t{}\t{}\t{}\t{}".format( 83 | self.chrm,self.start, self.end, self.ref, self.alt, self.gene, self.event)) 84 | 85 | for pileupcolumn in pile.pileup(reference=self.chrm, start=self.start - 1, end=self.start, 86 | truncate=True, stepper="nofilter", max_depth=100000000): 87 | for read in pileupcolumn.pileups: 88 | 89 | if read.alignment.has_tag('CB') and read.alignment.has_tag('UB'): 90 | if read.alignment.get_tag('CB') not in bar: # filter reads with only good barcodes 91 | continue 92 | 93 | # print(read.query_position) 94 | if indel > 0: 95 | # print(int(read.alignment.query_qualities[read.query_position])) 96 | 97 | # print('{}'.format(read.query_position)) 98 | # if read.query_position == None: 99 | # continue 100 | # if read.alignment.query_qualities[read.query_position + indel + 1] == None: 101 | # continue 102 | if read.is_refskip or \ 103 | int(read.alignment.mapping_quality) < int(mapq): 104 | continue 105 | else: 106 | 107 | if read.is_del or read.is_refskip or \ 108 | int(read.alignment.query_qualities[read.query_position]) < int(baseq) or \ 109 | int(read.alignment.mapping_quality) < int(mapq): 110 | continue 111 | 112 | q_name = read.alignment.query_name 113 | q_tag = read.alignment.get_tag('CB') 114 | qU_tag = read.alignment.get_tag('UB') 115 | qstring = q_tag + ':' + qU_tag 116 | barcodes['{}'.format(q_name)].append(q_tag) 117 | barUcodes['{}'.format(q_name)].append(qstring) 118 | 119 | # barcode without mutations 120 | if indel > 0: 121 | 122 | # print('{}:{}:{}:{}:{}:{}'.format(read.indel, read.alignment.get_tag('CB'),read.alignment.cigarstring, 123 | # read.query_position,self.alt,pileupcolumn.pos)) 124 | q_name = read.alignment.query_name 125 | q_tag = read.alignment.get_tag('CB') 126 | qU_tag = read.alignment.get_tag('UB') 127 | qstring = q_tag + ':' + qU_tag 128 | barcodes['{}'.format(q_name)].append(q_tag) 129 | barUcodes['{}'.format(q_name)].append(qstring) 130 | if self.alt == '-': 131 | if read.alignment.has_tag('CB') and read.query_position == None: 132 | # and read.indel == indel and \ 133 | # read.alignment.query_alignment_sequence[read.query_position + 134 | # 1:read.query_position + read.indel + 1] == self.alt: 135 | # print('indel length', read.indel) 136 | alt_tag = read.alignment.get_tag('CB') 137 | # UB 138 | altU_tag = read.alignment.get_tag('UB') 139 | ustringa = alt_tag + ':' + altU_tag 140 | bar_count['alt'].append(ustringa) 141 | else: # ref reads 142 | # CB 143 | ref_tag = read.alignment.get_tag('CB') 144 | # UB 145 | refU_tag = read.alignment.get_tag('UB') 146 | ustring = ref_tag + ':' + refU_tag 147 | bar_count['ref'].append(ustring) 148 | # print(ustring) 149 | # print('{}\t{}\t{}'.format(ref_tag, refU_tag, 'ref')) 150 | else: 151 | if read.alignment.has_tag('CB') and read.indel == indel and \ 152 | read.alignment.query_alignment_sequence[read.query_position + 153 | 1:read.query_position + read.indel + 1] == self.alt: 154 | # print('indel length', read.indel) 155 | alt_tag = read.alignment.get_tag('CB') 156 | # UB 157 | altU_tag = read.alignment.get_tag('UB') 158 | ustringa = alt_tag + ':' + altU_tag 159 | bar_count['alt'].append(ustringa) 160 | else: # ref reads 161 | # CB 162 | ref_tag = read.alignment.get_tag('CB') 163 | # UB 164 | refU_tag = read.alignment.get_tag('UB') 165 | ustring = ref_tag + ':' + refU_tag 166 | bar_count['ref'].append(ustring) 167 | # print(ustring) 168 | # print('{}\t{}\t{}'.format(ref_tag, refU_tag, 'ref')) 169 | else: 170 | #print(indel,read.query_position,read.alignment.query_sequence[read.query_position]) 171 | if read.alignment.query_sequence[read.query_position] != self.alt: 172 | ref_tag = read.alignment.get_tag('CB') 173 | # UB 174 | refU_tag = read.alignment.get_tag('UB') 175 | ustring = ref_tag + ':' + refU_tag 176 | bar_count['ref'].append(ustring) 177 | # print('{}\t{}\t{}'.format(ref_tag, refU_tag, 'ref')) 178 | elif read.alignment.query_sequence[read.query_position] == self.alt: 179 | # CB 180 | alt_tag = read.alignment.get_tag('CB') 181 | # UB 182 | altU_tag = read.alignment.get_tag('UB') 183 | ustringa = alt_tag + ':' + altU_tag 184 | bar_count['alt'].append(ustringa) 185 | return barUcodes, bar_count 186 | 187 | def consensus_calling(self,total_barcodes, ub_counts, wt, wu, wc): 188 | """ 189 | 190 | :param total_barcodes: list of all the barcodes at variant pos 191 | :param ub_counts: ref/alt barcode counts 192 | :param wt: file handle to write vaf information for each variant 193 | :param wu: file handle to write CB:UB information for each variant 194 | :param wc: file handle to write CB information for each variant 195 | :return: variant information 196 | 197 | """ 198 | uniqub_barcodes_raw = set() # uni depth 199 | ub_raw = [] # raw depth 200 | 201 | for tupru in total_barcodes.values(): 202 | for sampleru in tupru: 203 | ub_raw.append(sampleru) 204 | uniqub_barcodes_raw.add(sampleru) 205 | # UB ==================================================================================================== 206 | logger.info("Consensus calc variant: {}\t{}\t{}\t{}\t{}\t{}\t{}".format( 207 | self.chrm, self.start, self.end, self.ref, self.alt, self.gene, self.event)) 208 | d = {} 209 | UBuniq_barcodes_raw = [] 210 | for utags in uniqub_barcodes_raw: 211 | if utags in ub_counts['alt'] and utags in ub_counts['ref']: 212 | # print 'both: ', utags) 213 | unref = ub_counts['ref'].count(utags) 214 | unalt = ub_counts['alt'].count(utags) 215 | utotl = unref + unalt 216 | un1ref = ub_counts['ref'].count(utags) 217 | un1alt = ub_counts['alt'].count(utags) 218 | ut1totl = un1ref + un1alt 219 | if unref / utotl < float(0.75): 220 | if unalt / utotl < float(0.75): 221 | continue 222 | else: 223 | un1alt = 1 224 | un1ref = 0 225 | u1totl = un1ref + un1alt 226 | else: 227 | if unalt / utotl < float(0.75): 228 | un1alt = 0 229 | un1ref = 1 230 | u1totl = un1ref + un1alt 231 | else: 232 | un1alt = 1 # this is sanity check in the final file 233 | un1ref = 1 # if both are one then the code block failed 234 | u1totl = un1ref + un1alt 235 | else: 236 | # uniq the ref UB barcode for vaf 237 | # if an UB is not seen in both ref and alt bin 238 | # meaning if a UB occurs more than once consider it only once 239 | # i.e if UB has 5 [alt] and 0[ref] 240 | # we would consider it 1[alt] and 0[ref] 241 | un1ref = list(set(ub_counts['ref'])).count(utags) 242 | # same stuff for alt UB barcodes 243 | un1alt = list(set(ub_counts['alt'])).count(utags) 244 | u1totl = un1ref + un1alt 245 | # although we Uniq the UB's above we need to print out the actual number 246 | # UB has 5 [alt] and 0[ref] 247 | # print out the same number 248 | unref = ub_counts['ref'].count(utags) 249 | unalt = ub_counts['alt'].count(utags) 250 | utotl = unref + unalt 251 | 252 | if un1alt: # append only alt barcodes that have 1 253 | UBuniq_barcodes_raw.append(utags) 254 | 255 | ubtager = '{chrm}\t{st}\t{en}\t{ref}\t{alt}\t' \ 256 | '{type}\t{gene}\t{bar}\t{r}\t{v}\t{tot}\n'.format( 257 | chrm=self.chrm, 258 | st=self.start, 259 | en=self.end, 260 | ref=self.ref, 261 | alt=self.alt, 262 | type=self.event, 263 | gene=self.gene, 264 | bar=utags, 265 | r=unref, 266 | v=unalt, tot=utotl) 267 | # print(ubtager) 268 | wu.write(ubtager) 269 | 270 | # now work on CB barcodes 271 | # GGAAAGCCACCACGTG-2:GATGTCGCGC CB = GGAAAGCCACCACGTG-2 : UB = GATGTCGCGC 272 | if utags.split(':')[0] not in d: # check if this already exists in dict(d) 273 | d[utags.split(':')[0]] = {'ref': un1ref, 'alt': un1alt} 274 | else: 275 | d[utags.split(':')[0]]['ref'] += un1ref 276 | d[utags.split(':')[0]]['alt'] += un1alt 277 | uni_alt = [] 278 | tuni_alt = [] 279 | CBuniq_barcodes_raw = [] 280 | for i, v in d.items(): 281 | # print i,v['alt'] 282 | if v['alt'] >= 1: 283 | CBuniq_barcodes_raw.append(i) 284 | uni_alt.append(v['alt']) 285 | tot1 = v['alt'] + v['ref'] 286 | tuni_alt.append(tot1) 287 | cbtager = '{chrm}\t{st}\t{en}\t{ref}\t{alt}\t' \ 288 | '{type}\t{gene}\t{bar}\t{ref1}\t{alt1}\t{tot}\n'.format( 289 | chrm=self.chrm, 290 | st=self.start, 291 | en=self.end, 292 | ref=self.ref, 293 | alt=self.alt, 294 | type=self.event, 295 | gene=self.gene, 296 | bar=i, 297 | alt1=v['alt'], 298 | ref1=v['ref'], 299 | tot=tot1) 300 | # print(cbtager) 301 | wc.write(cbtager) 302 | 303 | Uuni_alt_c = sum(uni_alt) 304 | tUuni_alt_c = sum(tuni_alt) # total 305 | UBfin_umi = ','.join(UBuniq_barcodes_raw) 306 | CBfin_umi = ','.join(CBuniq_barcodes_raw) 307 | 308 | try: 309 | uvU = round(float(Uuni_alt_c) / float(tUuni_alt_c), 2) # uni vaf 310 | except ZeroDivisionError: 311 | uvU = float(0.0) 312 | 313 | snp = '{chrm}\t{st}\t{en}\t{ref}\t{alt}\t{type}\t{gene}' \ 314 | '\t{Uunidepth}\t{Uunc}\t{Uuvaf}\t{umi}\t{Uumi}\n'.format( 315 | chrm=self.chrm, 316 | st=self.start, 317 | en=self.end, 318 | ref=self.ref, 319 | alt=self.alt, 320 | type=self.event, 321 | gene=self.gene, 322 | Uunidepth=tUuni_alt_c, 323 | Uumi=UBfin_umi, 324 | Uuvaf=uvU, 325 | umi=CBfin_umi, 326 | Uunc=Uuni_alt_c) 327 | # print(snp) 328 | wt.write(snp) 329 | logger.info("completed processing variant: {}\t{}\t{}\t{}\t{}\t{}\t{}".format( 330 | self.chrm, self.start, self.end, self.ref, self.alt, self.gene, self.event)) 331 | return snp 332 | 333 | 334 | if __name__ == '__main__': 335 | parser = argparse.ArgumentParser(description='Parse CB barcodes from Single cell rna seq data') 336 | parser.add_argument('bam_file', help='BAM file') 337 | parser.add_argument('variant_file', help='variants file with header') 338 | parser.add_argument('barcodes', help='list of good barcodes file') 339 | parser.add_argument('upn', help='upn/sample name: will be used as prefix for out_file') 340 | parser.add_argument('-f', "--filter", type=int, default=0, 341 | help='number of reads required per barcode default: 0') 342 | parser.add_argument('-mq', "--mapq", type=int, default=0, 343 | help='Skip read with mapq smaller than default : 0') 344 | parser.add_argument('-bq', "--baseq", type=int, default=1, 345 | help='Skip bases with base quality less than default : 1') 346 | 347 | args = parser.parse_args() 348 | 349 | bam_file = args.bam_file 350 | variants = args.variant_file 351 | barcodes_good = args.barcodes 352 | outfile = args.upn 353 | counts = args.filter 354 | bq = args.baseq 355 | mq = args.mapq 356 | logger.basicConfig(filename=outfile + '.log', filemode='w+', 357 | level=logger.DEBUG, 358 | format='%(asctime)s %(levelname)s %(message)s') 359 | logger.info("Start process") 360 | num_lines_variants = sum(1 for line in open(variants)) - 1 # minus header 361 | logger.info("Number of variants:\t{}".format(num_lines_variants)) 362 | 363 | num_lines_barcode = sum(1 for lines in open(barcodes_good)) 364 | logger.info("Number of good barcodes:\t{}".format(num_lines_barcode)) 365 | bars = GenomicPosition.good_barcodes(barcodes_good) 366 | with open(outfile + '_AllCounts.tsv', 'w+') as var, \ 367 | open(outfile + '_counts_CB.tsv', 'w+') as CB, \ 368 | open(outfile + '_counts_UB.tsv', 'w+') as UB, \ 369 | open(variants, 'r') as regions: 370 | # write headers 371 | var_header = ['chr', 'start', 'end', 'ref', 'alt', 'gene', 'type', 372 | 'UB_DEPTH', 'UB_ALT', 'UB_VAF', 'CB_barcodes', 'CB:UB_barcodes'] 373 | var_h = '\t'.join(var_header) + '\n' 374 | var.write(var_h) 375 | 376 | CB_header = ['chrm', 'start', 'end', 'ref', 'alt', 377 | 'type', 'gene', 'barcode', 'ref_count', 'alt_count', 'total_CB'] 378 | CB_h = '\t'.join(CB_header) + '\n' 379 | CB.write(CB_h) 380 | 381 | UB_header = ['chrm', 'start', 'end', 'ref', 'alt', 'type', 382 | 'gene', 'barcode', 'ref_count', 'alt_count', 'total_UB'] 383 | UB_h = '\t'.join(UB_header) + '\n' 384 | UB.write(UB_h) 385 | 386 | next(regions) 387 | for lines in regions: 388 | x = GenomicPosition(lines) 389 | 390 | print(x.chrm, x.start, x.event, x.gene, x.classify()) 391 | barcode_counts = x.count_barcodes(bam_file, bars, x.classify(), mq, bq) 392 | # print(check_classify[0]) 393 | counters = x.consensus_calling(barcode_counts[0], barcode_counts[1], var, UB, CB) 394 | 395 | logger.info("end process") 396 | 397 | -------------------------------------------------------------------------------- /requirements.txt: -------------------------------------------------------------------------------- 1 | pysam==0.14.1 2 | --------------------------------------------------------------------------------