From 0ee99119bc112364126abf400be46938441c9c00 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Wed, 11 Mar 2015 16:55:11 -0400 Subject: [PATCH 01/20] fix snakemake isnvs_vcf merge rule --- pipes/rules/intrahost.rules | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/pipes/rules/intrahost.rules b/pipes/rules/intrahost.rules index 6dfd14d0e..78732e547 100644 --- a/pipes/rules/intrahost.rules +++ b/pipes/rules/intrahost.rules @@ -46,7 +46,7 @@ rule isnvs_vcf: run: shell("{config[binDir]}/intrahost.py merge_to_vcf {params.refGenome} {output[0]}" + " --samples {params.samples}" - + " --isnvs " + " ".join("{config[dataDir]}/{config[subdirs][intrahost]}/vphaser2."+s+".txt" for s in params.samples) + + " --isnvs " + " ".join("{config[dataDir]}/{config[subdirs][intrahost]}/vphaser2."+s+".txt.gz" for s in params.samples) + " --assemblies " + " ".join("{config[dataDir]}/{config[subdirs][assembly]}/"+s+".fasta" for s in params.samples) ) shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]} --strip_chr_version") From b2520d3ed055b85751302d466dd1ae72d40a5732 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 10:01:03 -0400 Subject: [PATCH 02/20] isnv snakemake pipe fix --- pipes/rules/intrahost.rules | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/pipes/rules/intrahost.rules b/pipes/rules/intrahost.rules index 78732e547..7dabe6197 100644 --- a/pipes/rules/intrahost.rules +++ b/pipes/rules/intrahost.rules @@ -48,7 +48,8 @@ rule isnvs_vcf: + " --samples {params.samples}" + " --isnvs " + " ".join("{config[dataDir]}/{config[subdirs][intrahost]}/vphaser2."+s+".txt.gz" for s in params.samples) + " --assemblies " + " ".join("{config[dataDir]}/{config[subdirs][assembly]}/"+s+".fasta" for s in params.samples) + + " --strip_chr_version" ) - shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]} --strip_chr_version") + shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]}") shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") From 4da8958cbf51c4624c03d227eaa01f1851416010 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 10:24:45 -0400 Subject: [PATCH 03/20] delete unneeded stuff --- old-scripts/kga/trees.sh | 70 ---------------------------------------- 1 file changed, 70 deletions(-) delete mode 100644 old-scripts/kga/trees.sh diff --git a/old-scripts/kga/trees.sh b/old-scripts/kga/trees.sh deleted file mode 100644 index 6e5ac75a9..000000000 --- a/old-scripts/kga/trees.sh +++ /dev/null @@ -1,70 +0,0 @@ -#-------- MULTIPLE-SEQUENCE ALIGNMENT & TREE BUILDING @ BROAD --------# -# Make sure all file-names are unique when cut down to 10 characters - e.g. if analysing Lassa 'LASV-' identifier needs to be removed from the input sequence file. - -# MAKE REQUIRED SUB-DIRECTORIES -for directory in -do -bsub -o ~/log.txt -P sabeti_trees "mkdir $directory/_msa $directory/_sequences $directory/_trees $directory/_logs $directory/_temp $directory/_trees/mr-bayes" -done - -#-------- SEQUENCE ALIGNMENT --------# -# CREATE MSA USING MAFFT & TRIM USING TRIMAL -for sequences in -do -for directory in -do -bsub -R "rusage[mem=4]" -n 4 -R "span[hosts=1]" -W 4:00 -q hour -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.al1 "/idi/sabeti-scratch/kandersen/bin/mafft/core/mafft --localpair --maxiterate 1000 --reorder --ep 0.123 --preservecase --thread 4 $directory/_sequences/$sequences.fasta > $directory/_msa/$sequences.mafft.fasta && /idi/sabeti-scratch/kandersen/bin/trimal/trimal -phylip -automated1 -in $directory/_msa/$sequences.mafft.fasta -out $directory/_msa/$sequences.pruned.phy -htmlout $directory/_logs/$sequences.log.trimal.html -colnumbering" -done -done - -#-------- TREE BUILDING USING MAXIMUM LIKELIHOOD - RAXML --------# -# CREATE TREE -for sequences in -do -for directory in -do -for substitution_model in GTRGAMMA -do -for bootstraps in 500 -do -bsub -sp 100 -R "rusage[mem=2]" -n 2 -R "span[hosts=1]" -W 4:00 -q hour -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.tr1 "/idi/sabeti-scratch/kandersen/bin/raxml/raxmlHPC-PTHREADS-SSE3 -f d -T 2 -p 123421 -m $substitution_model -N 20 -n $sequences.tree1 -w $directory/_trees/ -s $directory/_msa/$sequences.pruned.phy" -bsub -sp 90 -n 4 -R "span[hosts=1]" -q week -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.tr2 "/idi/sabeti-scratch/kandersen/bin/raxml/raxmlHPC-PTHREADS-SSE3 -f d -T 4 -p 12438 -m $substitution_model -b 12438 -N $bootstraps -k -n $sequences.tree2 -w $directory/_trees/ -s $directory/_msa/$sequences.pruned.phy && /idi/sabeti-scratch/kandersen/bin/raxml/raxmlHPC-SSE3 -T 1 -m $substitution_model -n $sequences.tree3 -f b -t $directory/_trees/RAxML_bestTree.$sequences.tree1 -z $directory/_trees/RAxML_bootstrap.$sequences.tree2 -w $directory/_trees/ && mv $directory/_trees/RAxML_bipartitions.$sequences.tree3 $directory/_trees/$sequences.raxml.tree" -done -done -done -done - -#-------- CREATE TREES USING PHYML --------# -# CREATE TREE -for sequences in -do -for directory in -do -for substitution_model in GTR # HKY85, JC69, K80, F81, F84, TN93 -do -for bootstraps in 500 -do -bsub -n 1 -R "span[hosts=1]" -q week -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.pm "/idi/sabeti-scratch/kandersen/bin/phyml/PhyML-3.1_linux64 -i $directory/_msa/$sequences.pruned.phy -d nt -b $bootstraps -m $substitution_model --pinv 0 --nclasses 4 -s BEST --rand_start --n_rand_starts 10 --r_seed 1553 -f m --no_memory_check && mv $directory/_msa/$sequences.pruned.phy_phyml* $directory/_trees/ && /idi/sabeti-scratch/kandersen/bin/raxml/raxmlHPC-SSE3 -f b -t $directory/_trees/$sequences.pruned.phy_phyml_tree.txt -z $directory/_trees/$sequences.pruned.phy_phyml_boot_trees.txt -m GTRGAMMA -s $directory/_msa/$sequences.pruned.phy -n $sequences.phyml.tree -w $directory/_trees/ && mv $directory/_trees/RAxML_bipartitions.$sequences.phyml.tree $directory/_trees/$sequences.phyml.tree" -done -done -done -done - -#-------- CREATE TREES USING MR BAYES --------# -# OUTPUT NEXUS FILE -for sequences in -do -for directory in -do -bsub -R "rusage[mem=2]" -n 1 -R "span[hosts=1]" -W 4:00 -q hour -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.pr "/idi/sabeti-scratch/kandersen/bin/trimal/trimal -automated1 -in $directory/_msa/$sequences.pruned.phy -nexus -out $directory/_msa/$sequences.pruned.nex" -done -done - -# RUN MR BAYES -for sequences in -do -for directory in -do -bsub -R "rusage[mem=2]" -n 1 -R "span[hosts=1]" -q week -o $directory/_logs/$sequences.log.bsub.txt -P sabeti_trees -J $sequences.mb "cp $directory/_msa/$sequences.pruned.nex $directory/_trees/mr-bayes/$sequences.nex && /idi/sabeti-scratch/kandersen/bin/mrbayes/run_mrbayes.sh $directory/_trees/mr-bayes/$sequences.nex" -done -done \ No newline at end of file From c410ba27d9e105cc7426e623f1432f2159cec025 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 10:33:12 -0400 Subject: [PATCH 04/20] disable table for now temporarily [ci skip] --- pipes/rules/intrahost.rules | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/pipes/rules/intrahost.rules b/pipes/rules/intrahost.rules index 7dabe6197..91d2d75f0 100644 --- a/pipes/rules/intrahost.rules +++ b/pipes/rules/intrahost.rules @@ -35,8 +35,8 @@ rule isnvs_vcf: sample=read_samples_file(config["samples_assembly"])) output: config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.vcf.gz', - config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.vcf.gz', - config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.txt.gz' + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.vcf.gz' + #config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.txt.gz' resources: mem=4 params: LSF=config.get('LSF_queues', {}).get('short', '-W 4:00'), logid="all", @@ -51,5 +51,5 @@ rule isnvs_vcf: + " --strip_chr_version" ) shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]}") - shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") + #shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") From 72f37ce06d7f18bdbc56771761af1c9966c29875 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 14:30:20 -0400 Subject: [PATCH 05/20] fix iSNV table handling of ANN field --- intrahost.py | 24 ++++++++++++++++++++---- 1 file changed, 20 insertions(+), 4 deletions(-) diff --git a/intrahost.py b/intrahost.py index cca701009..2b756a8e3 100755 --- a/intrahost.py +++ b/intrahost.py @@ -666,10 +666,26 @@ def parse_eff(eff_field): def parse_ann(ann_field, alleles, transcript_blacklist=set(('GP.2','GP.3'))): ''' parse the new snpEff "ANN" INFO field ''' + if not all(len(a)==1 for a in alleles): - reflen = len(alleles[0]) - alleles = list(a[min(reflen,len(a)):] for a in alleles) - alleles = alleles[1:] + ''' This is incredibly annoying: instead of talking about indel alleles using + the VCF allele definitions or in the VCF order, the ANN field recomputes the + part of the allele that looks distinct from the reference and talks about + that instead. Note that this means some alleles (particularly deletions) + will collapse and become non-unique. + Oh, and py2 and py3 have different names for the same function, just for fun. + ''' + ziplong = itertools.zip_longest if 'zip_longest' in dir(itertools) else itertools.izip_longest + outalleles = [] + for a in alleles[1:]: + out_a = [] + for ref, alt in ziplong(alleles[0], a, fillvalue=''): + if ref!=alt or out_a: + out_a.append(alt) + outalleles.append(''.join(out_a)) + alleles = outalleles + else: + alleles = alleles[1:] effs = [eff.split('|') for eff in ann_field.split(',')] effs = [(eff[0], dict((k,eff[i]) for k,i in @@ -683,7 +699,7 @@ def parse_ann(ann_field, alleles, transcript_blacklist=set(('GP.2','GP.3'))): return {} if len(effs) != len(effs_dict): raise Exception() - if len(effs) != len(alleles): + if len(effs) != len(set(alleles)): raise Exception() for a in alleles: if a not in effs_dict: From 3f83a45dc3e46e158ce5a18258f74aea3b95744d Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 16:30:23 -0400 Subject: [PATCH 06/20] fix remaining snpEff ANN parsing bugs --- intrahost.py | 15 +++++++++++---- pipes/rules/intrahost.rules | 6 +++--- 2 files changed, 14 insertions(+), 7 deletions(-) diff --git a/intrahost.py b/intrahost.py index 2b756a8e3..ff318c2b9 100755 --- a/intrahost.py +++ b/intrahost.py @@ -678,6 +678,12 @@ def parse_ann(ann_field, alleles, transcript_blacklist=set(('GP.2','GP.3'))): ziplong = itertools.zip_longest if 'zip_longest' in dir(itertools) else itertools.izip_longest outalleles = [] for a in alleles[1:]: + if len(a)==len(alleles[0]): + out_a = [] + for ref, alt in reversed(list(zip(alleles[0], a))): + if ref!=alt or out_a: + out_a.append(alt) + a = ''.join(reversed(out_a)) out_a = [] for ref, alt in ziplong(alleles[0], a, fillvalue=''): if ref!=alt or out_a: @@ -698,12 +704,13 @@ def parse_ann(ann_field, alleles, transcript_blacklist=set(('GP.2','GP.3'))): if not effs: return {} if len(effs) != len(effs_dict): - raise Exception() - if len(effs) != len(set(alleles)): - raise Exception() + raise Exception("ANN field has non-unique alleles") for a in alleles: if a not in effs_dict: - raise Exception("missing allele: " + a) + raise Exception("ANN field is missing ALT allele: " + a) + if len(effs) != len(set(alleles)): + raise Exception("ANN field has %s entries, but ALT field has %s unique alleles: %s" % ( + len(effs), len(set(alleles)), ','.join(alleles))) out = {} for k in ('eff_type', 'eff_codon_dna', 'eff_aa', 'eff_aa_pos', 'eff_prot_len', 'eff_gene', 'eff_protein'): diff --git a/pipes/rules/intrahost.rules b/pipes/rules/intrahost.rules index 91d2d75f0..7dabe6197 100644 --- a/pipes/rules/intrahost.rules +++ b/pipes/rules/intrahost.rules @@ -35,8 +35,8 @@ rule isnvs_vcf: sample=read_samples_file(config["samples_assembly"])) output: config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.vcf.gz', - config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.vcf.gz' - #config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.txt.gz' + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.vcf.gz', + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.annot.txt.gz' resources: mem=4 params: LSF=config.get('LSF_queues', {}).get('short', '-W 4:00'), logid="all", @@ -51,5 +51,5 @@ rule isnvs_vcf: + " --strip_chr_version" ) shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]}") - #shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") + shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") From 219969ecbcc95222ec352c59d13c3bd578ab0a2b Mon Sep 17 00:00:00 2001 From: Danny Park Date: Thu, 12 Mar 2015 17:16:17 -0400 Subject: [PATCH 07/20] initial work on incorporating lib counts and bias --- intrahost.py | 17 ++++++++++++++--- 1 file changed, 14 insertions(+), 3 deletions(-) diff --git a/intrahost.py b/intrahost.py index ff318c2b9..015c7ca42 100755 --- a/intrahost.py +++ b/intrahost.py @@ -369,6 +369,8 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version outf.write('##fileformat=VCFv4.1\n') outf.write('##FORMAT=\n') outf.write('##FORMAT=\n') + outf.write('##FORMAT=\n') + outf.write('##FORMAT=\n') for c, clen in ref_chrlens: if strip_chr_version: c = strip_accession_version(c) @@ -387,11 +389,16 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version for row in util.file.read_tabfile(samp_to_isnv[s]): s_chrom = samp_to_cmap[s].mapBtoA(ref_sequence.id) if row[0] == s_chrom: + allele_fields = list(AlleleFieldParser(x) for x in row[7:] if x) row = {'sample':s, 'CHROM':ref_sequence.id, 's_chrom':s_chrom, 's_pos':int(row[1]), 's_alt':row[2], 's_ref': row[3], - 'alleles':list(AlleleFieldParser(x).allele_and_strand_counts() - for x in row[7:] if x)} + 'alleles':list(x.allele_and_strand_counts() for x in allele_fields), + 'n_libs':list( + sum(1 for f,r in x.lib_counts() if f+r>0) + for x in allele_fields), + 'lib_bias':list(x.lib_bias_pval() for x in allele_fields), + } # make a sorted allele list row['allele_counts'] = list(sorted( [(a,int(f)+int(r)) for a,f,r in row['alleles']], @@ -466,6 +473,8 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version # define genotypes and fractions iSNVs = {} # {sample : {allele : fraction, ...}, ...} + iSNVs_n_libs = {} # {sample : {allele : n libraries > 0, ...}, ...} + iSNVs_lib_bias = {} # {sample : {allele : pval, ...}, ...} for s in samples: # get all rows for this sample and merge allele counts together @@ -474,7 +483,7 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version if 'i' in acounts and 'd' in acounts: # This sample has both an insertion line and a deletion line at the same spot! # To keep the reference allele from be counted twice, once as an i and once - # as a d, averge the counts and get rid of one of them. + # as a d, average the counts and get rid of one of them. acounts['i'] = int(round((acounts['i'] + acounts['d'])/2.0,0)) del acounts['d'] @@ -483,6 +492,8 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version consAllele = consAlleles[s] tot_n = sum(acounts.values()) iSNVs[s] = {} # {allele : fraction, ...} + iSNVs_n_libs[s] = {} + iSNVs_lib_bias[s] = {} for a,n in acounts.items(): f = float(n)/tot_n if a.startswith('I'): From 2da08d469a2c2578925dd2ede13b70dd39f02028 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Fri, 13 Mar 2015 10:08:42 -0400 Subject: [PATCH 08/20] speed optimization: dont re-index fasta file hundreds of times --- intrahost.py | 15 +++++++++------ 1 file changed, 9 insertions(+), 6 deletions(-) diff --git a/intrahost.py b/intrahost.py index 015c7ca42..4c7255407 100755 --- a/intrahost.py +++ b/intrahost.py @@ -349,10 +349,11 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version # setup if not (len(samples) == len(isnvs) == len(assemblies)): raise LookupError("samples, isnvs, and assemblies must have the same number of elements") - samp_to_fasta = dict(zip(samples, assemblies)) + samp_to_seqIndex = dict((s, Bio.SeqIO.index(fasta, 'fasta')) + for s, fasta in zip(samples, assemblies)) samp_to_isnv = dict(zip(samples, isnvs)) samp_to_cmap = dict((s, CoordMapper(genome, refFasta)) - for s, genome in samp_to_fasta.items()) + for s, genome in zip(samples, assemblies)) with open(refFasta, 'rU') as inf: ref_chrlens = list((seq.id, len(seq)) for seq in Bio.SeqIO.parse(inf, 'fasta')) @@ -425,7 +426,7 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version # process one reference position at a time for pos, rows in data: rows = list(rows) - + # define the length of this variation based on the largest deletion end = pos for row in rows: @@ -460,10 +461,8 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version log.warning("dropping consensus because allele is outside " "consensus for %s at %s-%s." % (s, pos, end)) continue - seqIndex = Bio.SeqIO.index(samp_to_fasta[s], 'fasta') - cons = seqIndex[samp_to_cmap[s].mapBtoA(ref_sequence.id)] + cons = samp_to_seqIndex[s][samp_to_cmap[s].mapBtoA(ref_sequence.id)] allele = str(cons[cons_start-1:cons_stop].seq).upper() - seqIndex.close() if s in samp_offsets: samp_offsets[s] -= cons_start if all(a in set(('A','C','T','G')) for a in allele): @@ -568,6 +567,10 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version out = out + list(map(':'.join, zip(genos, freqs))) outf.write('\t'.join(map(str, out))+'\n') + # close fasta files + for idx in samp_to_seqIndex.values(): + idx.close() + # compress output if requested if outVcf.endswith('.vcf.gz'): pysam.tabix_compress(tmpVcf, outVcf, force=True) From 5d0b37b769834c926efd734583a672349eb56d31 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Fri, 13 Mar 2015 11:06:15 -0400 Subject: [PATCH 09/20] add crude filter --- intrahost.py | 49 ++++++++++++++++++++---- pipes/rules/intrahost.rules | 34 ++++++++++++++++- test/unit/test_intrahost.py | 74 ++++++++++++++++++------------------- 3 files changed, 112 insertions(+), 45 deletions(-) diff --git a/intrahost.py b/intrahost.py index 4c7255407..f5214d379 100755 --- a/intrahost.py +++ b/intrahost.py @@ -341,7 +341,7 @@ def strip_accession_version(acc): acc = m.group(1) return acc -def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version=False): +def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version=False, naive_filter=False): ''' Combine and convert vPhaser2 parsed filtered output text files into VCF format. Assumption: consensus assemblies do not extend beyond ends of reference. ''' @@ -364,6 +364,7 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version else: raise ValueError("outVcf must end in .vcf or .vcf.gz") + log.info("loaded CoordMapper for all genomes, starting VCF merge...") with open(tmpVcf, 'wt') as outf: # write header @@ -395,15 +396,23 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version 's_chrom':s_chrom, 's_pos':int(row[1]), 's_alt':row[2], 's_ref': row[3], 'alleles':list(x.allele_and_strand_counts() for x in allele_fields), - 'n_libs':list( - sum(1 for f,r in x.lib_counts() if f+r>0) + 'n_libs':dict( + (x.allele(), sum(1 for f,r in x.lib_counts() if f+r>0)) + for x in allele_fields), + 'lib_bias':dict( + (x.allele(), x.lib_bias_pval()) for x in allele_fields), - 'lib_bias':list(x.lib_bias_pval() for x in allele_fields), } # make a sorted allele list row['allele_counts'] = list(sorted( [(a,int(f)+int(r)) for a,f,r in row['alleles']], key=(lambda x:x[1]), reverse=True)) + # naive filter (quick and dirty) + if naive_filter: + tot_n = sum(f+r for a,f,r in row['allele_counts']) + row['allele_counts'] = list((a,f,r) + for a,f,r in row['allele_counts'] + if row['n_libs'][a]>=2 and float(f+r)/tot_n >= 0.005) # reposition vphaser deletions minus one to be consistent with # VCF conventions if row['s_alt'].startswith('D'): @@ -479,12 +488,18 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version # get all rows for this sample and merge allele counts together acounts = dict(itertools.chain.from_iterable(row['allele_counts'] for row in rows if row['sample'] == s)) + nlibs = dict(itertools.chain.from_iterable(row['n_libs'].items() + for row in rows if row['sample'] == s)) + libbias = dict(itertools.chain.from_iterable(row['lib_bias'].items() + for row in rows if row['sample'] == s)) if 'i' in acounts and 'd' in acounts: # This sample has both an insertion line and a deletion line at the same spot! # To keep the reference allele from be counted twice, once as an i and once # as a d, average the counts and get rid of one of them. acounts['i'] = int(round((acounts['i'] + acounts['d'])/2.0,0)) del acounts['d'] + nlibs['i'] = max(nlibs['i'], nlibs['d']) + libbias['i'] = max(libbias['i'], libbias['d']) if acounts and s in consAlleles: # we have iSNV data on this sample @@ -493,8 +508,9 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version iSNVs[s] = {} # {allele : fraction, ...} iSNVs_n_libs[s] = {} iSNVs_lib_bias[s] = {} - for a,n in acounts.items(): + for orig_a,n in acounts.items(): f = float(n)/tot_n + a = orig_a if a.startswith('I'): # insertion point is relative to each sample insert_point = samp_offsets[s]+1 @@ -523,6 +539,8 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version if not (a and a==a.upper()): raise Exception() iSNVs[s][a] = f + iSNVs_n_libs[s][a] = nlibs[orig_a] + iSNVs_lib_bias[s][a] = libbias[orig_a] if all(len(a)==1 for a in iSNVs[s].keys()): if consAllele not in iSNVs[s]: raise Exception() @@ -545,17 +563,29 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version alleles_isnv2 = [] for a in set(a for a,n in alleles_isnv): counts = list(x[1] for x in alleles_isnv if x[0]==a) - alleles_isnv2.append((len(counts),sum(counts),a)) + if len(counts)>0 and sum(counts)>0: + # if we filtered any alleles above, make sure to omit absent alleles + alleles_isnv2.append((len(counts),sum(counts),a)) + else: + log.info("dropped allele {} at position {}:{}".format(a, ref_sequence.id, pos)) alleles_isnv = list(allele for n_samples, n_reads, allele in reversed(sorted(alleles_isnv2))) alleles = list(util.misc.unique([refAllele] + alleles_cons + alleles_isnv)) # map alleles from strings to numeric indexes if not alleles: raise Exception() + elif len(alleles)==1: + # if we filtered any alleles above, skip this position if there is no variation left here + log.info("dropped position {}:{} due to lack of variation".format(ref_sequence.id, pos)) + continue alleleMap = dict((a,i) for i,a in enumerate(alleles)) genos = [str(alleleMap.get(consAlleles.get(s),'.')) for s in samples] freqs = [(s in iSNVs) and ','.join(map(str, [iSNVs[s].get(a,0.0) for a in alleles[1:]])) or '.' for s in samples] + nlibs = [(s in iSNVs_n_libs) and ','.join([str(iSNVs_n_libs[s].get(a,0)) for a in alleles]) or '.' + for s in samples] + pvals = [(s in iSNVs_lib_bias) and ','.join([str(iSNVs_lib_bias[s].get(a,'.')) for a in alleles]) or '.' + for s in samples] # prepare output row and write to file c = ref_sequence.id @@ -564,7 +594,7 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version out = [c, pos, '.', alleles[0], ','.join(alleles[1:]), '.', '.', '.', 'GT:AF'] - out = out + list(map(':'.join, zip(genos, freqs))) + out = out + list(map(':'.join, zip(genos, freqs, nlibs, pvals))) outf.write('\t'.join(map(str, out))+'\n') # close fasta files @@ -601,6 +631,11 @@ def parser_merge_to_vcf(parser=argparse.ArgumentParser()): often used by SnpEff databases downstream, but without the corresponding version number. Default is false (leave chromosome names untouched).""") + parser.add_argument("--naive_filter", + default=False, action="store_true", dest="naive_filter", + help="""If set, keep only the alleles that have at least + two independent libraries of support and allele freq > 0.005. + Default is false (do not filter at this stage).""") util.cmd.common_args(parser, (('loglevel',None), ('version',None))) util.cmd.attach_main(parser, merge_to_vcf, split_args=True) return parser diff --git a/pipes/rules/intrahost.rules b/pipes/rules/intrahost.rules index 7dabe6197..95bd3fed7 100644 --- a/pipes/rules/intrahost.rules +++ b/pipes/rules/intrahost.rules @@ -9,7 +9,9 @@ import os, os.path, time rule all_intrahost: - input: config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.vcf.gz' + input: + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.vcf.gz', + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.filtered.vcf.gz' rule isnvs_per_sample: input: @@ -53,3 +55,33 @@ rule isnvs_vcf: shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]}") shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") +rule isnvs_vcf_filtered: + input: + expand("{dataDir}/{subdir}/vphaser2.{sample}.txt.gz", + dataDir=config["dataDir"], + subdir=config["subdirs"]["intrahost"], + sample=read_samples_file(config["samples_assembly"])), + expand("{dataDir}/{subdir}/{sample}.fasta", + dataDir=config["dataDir"], + subdir=config["subdirs"]["assembly"], + sample=read_samples_file(config["samples_assembly"])) + output: + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.filtered.vcf.gz', + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.filtered.annot.vcf.gz', + config["dataDir"]+'/'+config["subdirs"]["intrahost"] +'/isnvs.filtered.annot.txt.gz' + resources: mem=4 + params: LSF=config.get('LSF_queues', {}).get('short', '-W 4:00'), + logid="all", + refGenome=config["ref_genome"], + snpEff_ref=config["snpEff_genome"], + samples=read_samples_file(config["samples_assembly"]) + run: + shell("{config[binDir]}/intrahost.py merge_to_vcf {params.refGenome} {output[0]}" + + " --samples {params.samples}" + + " --isnvs " + " ".join("{config[dataDir]}/{config[subdirs][intrahost]}/vphaser2."+s+".txt.gz" for s in params.samples) + + " --assemblies " + " ".join("{config[dataDir]}/{config[subdirs][assembly]}/"+s+".fasta" for s in params.samples) + + " --strip_chr_version --naive_filter" + ) + shell("{config[binDir]}/interhost.py snpEff {output[0]} {params.snpEff_ref} {output[1]}") + shell("{config[binDir]}/intrahost.py iSNV_table {output[1]} {output[2]}") + diff --git a/test/unit/test_intrahost.py b/test/unit/test_intrahost.py index f26db93b8..ddfbe7dc4 100644 --- a/test/unit/test_intrahost.py +++ b/test/unit/test_intrahost.py @@ -306,16 +306,16 @@ def test_simple_snps(self): self.assertEqual(rows[0].pos+1, 3) self.assertEqual(rows[0].ref, 'C') self.assertEqual(rows[0].alt, 'A') - self.assertEqual(rows[0][0], '0:0.2') - self.assertEqual(rows[0][1], '0:0.1') - self.assertEqual(rows[0][2], '0:0.0') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '0:0.2') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '0:0.1') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '0:0.0') self.assertEqual(rows[1].contig, 'ref1') self.assertEqual(rows[1].pos+1, 6) self.assertEqual(rows[1].ref, 'A') self.assertEqual(rows[1].alt, 'T') - self.assertEqual(rows[1][0], '0:0.0') - self.assertEqual(rows[1][1], '0:0.0') - self.assertEqual(rows[1][2], '0:0.3') + self.assertEqual(':'.join(rows[1][0].split(':')[:2]), '0:0.0') + self.assertEqual(':'.join(rows[1][1].split(':')[:2]), '0:0.0') + self.assertEqual(':'.join(rows[1][2].split(':')[:2]), '0:0.3') def test_snps_downstream_of_indels(self): merger = VcfMergeRunner([('ref1', 'ATCGGACT')]) @@ -331,9 +331,9 @@ def test_snps_downstream_of_indels(self): self.assertEqual(rows[0].pos+1, 6) self.assertEqual(rows[0].ref, 'A') self.assertEqual(rows[0].alt, 'C') - self.assertEqual(rows[0][0], '0:0.2') - self.assertEqual(rows[0][1], '0:0.1') - self.assertEqual(rows[0][2], '1:0.7') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '0:0.2') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '0:0.1') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '1:0.7') def test_sample_major_allele_not_ref_allele(self): # make sure we can invert the allele frequency of the isnv @@ -347,7 +347,7 @@ def test_sample_major_allele_not_ref_allele(self): self.assertEqual(rows[0].pos+1, 3) self.assertEqual(rows[0].ref, 'C') self.assertEqual(rows[0].alt, 'A') - self.assertEqual(rows[0][0], '1:0.9') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '1:0.9') def test_backfill_sample_from_assembly(self): # one sample has no isnv, but another does, so we fill it in @@ -367,9 +367,9 @@ def test_backfill_sample_from_assembly(self): self.assertEqual(rows[0].pos+1, 3) self.assertEqual(rows[0].ref, 'C') self.assertEqual(rows[0].alt, 'A') - self.assertEqual(rows[0][0], '0:0.1') - self.assertEqual(rows[0][1], '1:1.0') - self.assertEqual(rows[0][2], '.:.') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '0:0.1') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '1:1.0') + self.assertEqual(rows[0][2], '.:.:.:.') def test_simple_insertions(self): # IA, ITCG, etc @@ -394,9 +394,9 @@ def test_simple_insertions(self): self.assertEqual(rows[0].pos+1, 2) self.assertEqual(rows[0].ref, 'T') self.assertEqual(rows[0].alt, 'TAA,TGCC,TAT') - self.assertEqual(rows[0][0], '0:0.2,0.0,0.0') - self.assertEqual(rows[0][1], '0:0.0,0.0,0.1') - self.assertEqual(rows[0][2], '0:0.05,0.15,0.0') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '0:0.2,0.0,0.0') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '0:0.0,0.0,0.1') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '0:0.05,0.15,0.0') def test_simple_deletions(self): # D1, D2, etc... @@ -420,9 +420,9 @@ def test_simple_deletions(self): self.assertEqual(rows[0].pos+1, 2) self.assertEqual(rows[0].ref, 'TCG') self.assertEqual(rows[0].alt, 'TG,T') - self.assertEqual(rows[0][0], '0:0.0,0.2') - self.assertEqual(rows[0][1], '0:0.1,0.0') - self.assertEqual(rows[0][2], '0:0.15,0.05') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '0:0.0,0.2') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '0:0.1,0.0') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '0:0.15,0.05') def test_deletion_spans_deletion(self): # sample assembly has deletion against reference and isnv deletes even more @@ -439,7 +439,7 @@ def test_deletion_spans_deletion(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'GTTC') self.assertEqual(rows[0].alt, 'GC,G') - self.assertEqual(rows[0][0], '1:0.9,0.1') + self.assertEqual(rows[0][0], '1:0.9,0.1:0,1,1:.,1.0,1.0') def test_insertion_spans_deletion(self): # sample assembly has deletion against reference and isnv inserts back into it @@ -462,9 +462,9 @@ def test_insertion_spans_deletion(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'GTT') self.assertEqual(rows[0].alt, 'G,GT,GTTC') - self.assertEqual(rows[0][0], '1:0.7,0.3,0.0') - self.assertEqual(rows[0][1], '1:0.8,0.0,0.0') - self.assertEqual(rows[0][2], '1:0.9,0.0,0.1') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '1:0.7,0.3,0.0') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '1:0.8,0.0,0.0') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '1:0.9,0.0,0.1') def test_snp_within_insertion(self): # sample assembly has insertion against reference and isnp modifies it @@ -486,9 +486,9 @@ def test_snp_within_insertion(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'G') self.assertEqual(rows[0].alt, 'GTT,CTT,GCT,GTC') - self.assertEqual(rows[0][0], '1:0.7,0.3,0.0,0.0') - self.assertEqual(rows[0][1], '1:0.8,0.0,0.2,0.0') - self.assertEqual(rows[0][2], '1:0.9,0.0,0.0,0.1') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '1:0.7,0.3,0.0,0.0') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '1:0.8,0.0,0.2,0.0') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '1:0.9,0.0,0.0,0.1') def test_2snps_within_insertion_same_sample(self): # Sample assembly has insertion against reference containing two SNPs @@ -525,8 +525,8 @@ def test_deletion_past_end_of_some_consensus(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'GAA') self.assertEqual(rows[0].alt, 'G,A') - self.assertEqual(rows[0][0], '1:0.7,0.3') - self.assertEqual(rows[0][1], '.:.') + self.assertEqual(rows[0][0], '1:0.7,0.3:0,1,1:.,1.0,1.0') + self.assertEqual(rows[0][1], '.:.:.:.') def test_snp_past_end_of_some_consensus(self): # Some sample contains SNP beyond the end of the consensus @@ -546,8 +546,8 @@ def test_snp_past_end_of_some_consensus(self): self.assertEqual(rows[0].pos+1, 2) self.assertEqual(rows[0].ref, 'T') self.assertEqual(rows[0].alt, 'G') - self.assertEqual(rows[0][0], '0:0.3') - self.assertEqual(rows[0][1], '.:.') + self.assertEqual(rows[0][0], '0:0.3:1,1:1.0,1.0') + self.assertEqual(rows[0][1], '.:.:.:.') def test_deletion_within_insertion(self): # sample assembly has insertion against reference and isnv deletes from it @@ -574,10 +574,10 @@ def test_deletion_within_insertion(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'GG') self.assertEqual(rows[0].alt, 'GTTG,GTG,GTT,G,GT') - self.assertEqual(rows[0][0], '1:0.4,0.3,0.0,0.1,0.0') - self.assertEqual(rows[0][1], '1:0.8,0.2,0.0,0.0,0.0') - self.assertEqual(rows[0][2], '1:0.85,0.1,0.0,0.0,0.05') - self.assertEqual(rows[0][3], '1:0.9,0.0,0.1,0.0,0.0') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '1:0.4,0.3,0.0,0.1,0.0') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '1:0.8,0.2,0.0,0.0,0.0') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '1:0.85,0.1,0.0,0.0,0.05') + self.assertEqual(':'.join(rows[0][3].split(':')[:2]), '1:0.9,0.0,0.1,0.0,0.0') def test_insertion_within_insertion(self): # sample assembly has insertion against reference and isnv puts even more in @@ -599,9 +599,9 @@ def test_insertion_within_insertion(self): self.assertEqual(rows[0].pos+1, 4) self.assertEqual(rows[0].ref, 'G') self.assertEqual(rows[0].alt, 'GTT,GATT,GTAT,GTTA') - self.assertEqual(rows[0][0], '1:0.7,0.3,0.0,0.0') - self.assertEqual(rows[0][1], '1:0.8,0.0,0.2,0.0') - self.assertEqual(rows[0][2], '1:0.9,0.0,0.0,0.1') + self.assertEqual(':'.join(rows[0][0].split(':')[:2]), '1:0.7,0.3,0.0,0.0') + self.assertEqual(':'.join(rows[0][1].split(':')[:2]), '1:0.8,0.0,0.2,0.0') + self.assertEqual(':'.join(rows[0][2].split(':')[:2]), '1:0.9,0.0,0.0,0.1') def test_indel_collapse(self): # vphaser describes insertions and deletions separately From e8427bec3ca0af2166b9da6f3c33709e0cccd3f6 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Fri, 13 Mar 2015 12:18:53 -0400 Subject: [PATCH 10/20] fix naive_filter --- intrahost.py | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/intrahost.py b/intrahost.py index f5214d379..0f7df5816 100755 --- a/intrahost.py +++ b/intrahost.py @@ -409,10 +409,10 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version key=(lambda x:x[1]), reverse=True)) # naive filter (quick and dirty) if naive_filter: - tot_n = sum(f+r for a,f,r in row['allele_counts']) - row['allele_counts'] = list((a,f,r) - for a,f,r in row['allele_counts'] - if row['n_libs'][a]>=2 and float(f+r)/tot_n >= 0.005) + tot_n = sum(n for a,n in row['allele_counts']) + row['allele_counts'] = list((a,n) + for a,n in row['allele_counts'] + if row['n_libs'][a]>=2 and float(n)/tot_n >= 0.005) # reposition vphaser deletions minus one to be consistent with # VCF conventions if row['s_alt'].startswith('D'): From a6fb1a344f038ca7a7f449d9543445e03b797772 Mon Sep 17 00:00:00 2001 From: Daniel Park Date: Fri, 13 Mar 2015 21:47:57 -0400 Subject: [PATCH 11/20] fix format field --- intrahost.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/intrahost.py b/intrahost.py index 0f7df5816..729d4d83d 100755 --- a/intrahost.py +++ b/intrahost.py @@ -593,7 +593,7 @@ def merge_to_vcf(refFasta, outVcf, samples, isnvs, assemblies, strip_chr_version c = strip_accession_version(c) out = [c, pos, '.', alleles[0], ','.join(alleles[1:]), - '.', '.', '.', 'GT:AF'] + '.', '.', '.', 'GT:AF:NL:LB'] out = out + list(map(':'.join, zip(genos, freqs, nlibs, pvals))) outf.write('\t'.join(map(str, out))+'\n') From bf5592c9ab0517418a8774efb1a7b3be08564893 Mon Sep 17 00:00:00 2001 From: Daniel Park Date: Fri, 13 Mar 2015 22:10:29 -0400 Subject: [PATCH 12/20] fix lib stats to operate on LB, not ID --- intrahost.py | 21 ++++++++++++++------- 1 file changed, 14 insertions(+), 7 deletions(-) diff --git a/intrahost.py b/intrahost.py index 729d4d83d..9e98d0122 100755 --- a/intrahost.py +++ b/intrahost.py @@ -145,13 +145,19 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : ''' alleleCol = 7 # First column of output with allele counts samtoolsTool = SamtoolsTool() - readGroups = list(r for r in samtoolsTool.getReadGroups(inBam) - if samtoolsTool.count(inBam, ['-r', r])>0) - tempDir = tempfile.mkdtemp() - libBams = [os.path.join(tempDir, groupID + '.bam') for groupID in readGroups] - for groupID, libBam in zip(readGroups, libBams) : - samtoolsTool.view(['-r', groupID, '-b'], inBam, libBam) + rgs_by_lib = sorted((rg['LB'],rg['ID']) + for rg in samtoolsTool.getReadGroups(inBam).values()) + rgs_by_lib = itertools.groupby(rgs_by_lib, lambda x: x[0]) + libBams = [] + for lib,rgs in rgs_by_lib: + rgs = list(itertools.chain(('-r', id) for lib,id in rgs)) + libBam = util.file.mkstempfname('.bam') + samtoolsTool.view(['-b'] + rgs, inBam, libBam) samtoolsTool.index(libBam) + if samtoolsTool.count(libBam) > 0: + libBams.append(libBam) + else: + os.unlink(libBam) for row in isnvs : consensusAllele = row[3] pos = int(row[1]) if consensusAllele != 'i' else int(row[1]) - 1 @@ -185,7 +191,8 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : *(row[alleleCol + alleleInd].split(':') + [pval, libCountsByAllele[alleleInd]]))) yield row - shutil.rmtree(tempDir) + for bam in libBams: + os.unlink(bam) def parse_alleles_string(allelesStr) : # Return {allele : [forwardCount, reverseCount]} From d94730d9a162a49182a1eef47b8c2955898878ca Mon Sep 17 00:00:00 2001 From: Daniel Park Date: Fri, 13 Mar 2015 22:18:25 -0400 Subject: [PATCH 13/20] fix itertools.chain --- intrahost.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/intrahost.py b/intrahost.py index 9e98d0122..a4cc0fc73 100755 --- a/intrahost.py +++ b/intrahost.py @@ -150,7 +150,7 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : rgs_by_lib = itertools.groupby(rgs_by_lib, lambda x: x[0]) libBams = [] for lib,rgs in rgs_by_lib: - rgs = list(itertools.chain(('-r', id) for lib,id in rgs)) + rgs = list(itertools.chain.from_iterable(('-r', id) for lib,id in rgs)) libBam = util.file.mkstempfname('.bam') samtoolsTool.view(['-b'] + rgs, inBam, libBam) samtoolsTool.index(libBam) From 54af1e8c330497629dc9eba5ebb1edae321cf819 Mon Sep 17 00:00:00 2001 From: Daniel Park Date: Fri, 13 Mar 2015 22:54:49 -0400 Subject: [PATCH 14/20] some debug info --- intrahost.py | 12 ++++++++---- 1 file changed, 8 insertions(+), 4 deletions(-) diff --git a/intrahost.py b/intrahost.py index a4cc0fc73..63cf88c96 100755 --- a/intrahost.py +++ b/intrahost.py @@ -139,7 +139,7 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : ''' For each variant, compute read counts in each library and p-value for library bias; append them to string for each variant. Format is allele:totalF:totalR:1stLibFCount:1stLibRCount:2ndLibFCount:...:p-val. - Library counts are in same order as in bam header. + Library counts are in alphabetical order of library IDs. Note: Total was computed by vphaser, library counts by samtools mpileup, so total might not be sum of library counts. ''' @@ -150,11 +150,15 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : rgs_by_lib = itertools.groupby(rgs_by_lib, lambda x: x[0]) libBams = [] for lib,rgs in rgs_by_lib: - rgs = list(itertools.chain.from_iterable(('-r', id) for lib,id in rgs)) + rgs = list(id for lb,id in rgs) + rgs_cmdline = itertools.chain.from_iterable(('-r', id) for id in rgs) libBam = util.file.mkstempfname('.bam') - samtoolsTool.view(['-b'] + rgs, inBam, libBam) + samtoolsTool.view(['-b'] + rgs_cmdline, inBam, libBam) samtoolsTool.index(libBam) - if samtoolsTool.count(libBam) > 0: + n_reads = samtoolsTool.count(libBam) + log.debug("LB:{} has {} reads in {} read groups ({})".format( + lib, n_reads, len(rgs), ', '.join(rgs))) + if n_reads > 0: libBams.append(libBam) else: os.unlink(libBam) From 2a8f42c6f091fcc87c66ec2532be8ec54e0aac87 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Fri, 13 Mar 2015 23:15:05 -0400 Subject: [PATCH 15/20] listify chain to append --- intrahost.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/intrahost.py b/intrahost.py index 63cf88c96..69205b223 100755 --- a/intrahost.py +++ b/intrahost.py @@ -151,7 +151,7 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : libBams = [] for lib,rgs in rgs_by_lib: rgs = list(id for lb,id in rgs) - rgs_cmdline = itertools.chain.from_iterable(('-r', id) for id in rgs) + rgs_cmdline = list(itertools.chain.from_iterable(('-r', id) for id in rgs)) libBam = util.file.mkstempfname('.bam') samtoolsTool.view(['-b'] + rgs_cmdline, inBam, libBam) samtoolsTool.index(libBam) From 7962a50641b6e646cc342aae7a8d79fe1d6fda6c Mon Sep 17 00:00:00 2001 From: Irwin Jungreis Date: Tue, 17 Mar 2015 15:45:13 -0400 Subject: [PATCH 16/20] Fixed bug in compute_library_bias that with samtools 0.1.19 only one readgroup per library was getting counted. There are still 4 tests failing in test_intrahost. --- intrahost.py | 16 ++++++++++++++-- tools/samtools.py | 7 +++++++ 2 files changed, 21 insertions(+), 2 deletions(-) diff --git a/intrahost.py b/intrahost.py index 69205b223..f78c9984c 100755 --- a/intrahost.py +++ b/intrahost.py @@ -151,9 +151,21 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : libBams = [] for lib,rgs in rgs_by_lib: rgs = list(id for lb,id in rgs) - rgs_cmdline = list(itertools.chain.from_iterable(('-r', id) for id in rgs)) + + # Create libBam containing all the readgroups in rgs. + # In samtools 1.1, this can be done by including -r multiple times on + # a single command line, but that doesn't work in 0.1.19, so instead + # extract readgroups one by one and then concatenate. + rgBams = [] + for id in rgs : + rgBam = util.file.mkstempfname('.bam') + rgBams.append(rgBam) + samtoolsTool.view(['-b', '-r', id], inBam, rgBam) libBam = util.file.mkstempfname('.bam') - samtoolsTool.view(['-b'] + rgs_cmdline, inBam, libBam) + samtoolsTool.merge(rgBams, libBam) + for bam in rgBams : + os.unlink(bam) + samtoolsTool.index(libBam) n_reads = samtoolsTool.count(libBam) log.debug("LB:{} has {} reads in {} read groups ({})".format( diff --git a/tools/samtools.py b/tools/samtools.py index 43a950aa2..a51b5d73b 100644 --- a/tools/samtools.py +++ b/tools/samtools.py @@ -54,6 +54,13 @@ def execute(self, command, args, stdin=None, stdout=None, stderr=None): def view(self, args, inFile, outFile, regions=[]): self.execute('view', args + ['-o', outFile, inFile] + regions) + def merge(self, inFiles, outFile): + "Merge a list of inFiles to create outFile." + # We are using -f for now because mkstempfname actually makes an empty + # file, and merge fails with that as output target without the -f. + # When mkstempfname is fixed, we should remove the -f. + self.execute('merge', ['-f'] + [outFile] + inFiles) + def index(self, inBam): self.execute('index', [inBam]) From cadea30240b573d13856f5d684cb369951b700a5 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Wed, 18 Mar 2015 15:14:34 -0400 Subject: [PATCH 17/20] add Hs field to tabular output --- intrahost.py | 9 +++++++-- 1 file changed, 7 insertions(+), 2 deletions(-) diff --git a/intrahost.py b/intrahost.py index f78c9984c..211e8296c 100755 --- a/intrahost.py +++ b/intrahost.py @@ -808,6 +808,11 @@ def iSNV_table(vcf_iter): for row in vcf_iter: info = dict(kv.split('=') for kv in row['INFO'].split(';') if kv and kv != '.') samples = [k for k in row.keys() if k not in set(('CHROM','POS','ID','REF','ALT','QUAL','FILTER','INFO','FORMAT'))] + # compute Hs: heterozygosity in population based on consensus genotypes alone + genos = [row[s].split(':')[0] for s in samples] + genos = util.misc.histogram(int(gt) for gt in genos if gt != '.') + n = sum(genos.values()) + Hs = 1.0 - sum(k*k/float(n*n) for k in genos.values()) try: for s in samples: f = row[s].split(':')[1] @@ -817,7 +822,7 @@ def iSNV_table(vcf_iter): Hw = 1.0 - sum(p*p for p in [1.0-f]+freqs) out = {'chr':row['CHROM'], 'pos':row['POS'], 'alleles':"%s,%s" %(row['REF'],row['ALT']), 'sample':s, - 'iSNV_freq':f, 'Hw':Hw} + 'iSNV_freq':f, 'Hw':Hw, 'Hs':Hs} if 'EFF' in info: for k,v in parse_eff(info['EFF']).items(): out[k] = v @@ -841,7 +846,7 @@ def parser_iSNV_table(parser=argparse.ArgumentParser()): return parser def main_iSNV_table(args): '''Convert VCF iSNV data to tabular text''' - header = ['chr','pos','sample','patient','time','alleles','iSNV_freq','Hw', + header = ['chr','pos','sample','patient','time','alleles','iSNV_freq','Hw','Hs', 'eff_type','eff_codon_dna','eff_aa','eff_aa_pos','eff_prot_len','eff_gene','eff_protein'] with util.file.open_or_gzopen(args.outFile, 'wt') as outf: outf.write('\t'.join(header)+'\n') From 14823ddabc63a9e74faf8463064942f1419a65db Mon Sep 17 00:00:00 2001 From: Danny Park Date: Wed, 18 Mar 2015 15:44:21 -0400 Subject: [PATCH 18/20] fix up test bams for vphaser to now include LB tags in the RG headers --- test/input/TestPerSample/in.2libs.bam | Bin 504905 -> 504942 bytes test/input/TestPerSample/in.2libs.bam.bai | Bin 176 -> 0 bytes test/input/TestPerSample/in.2libs3rgs.bam | Bin 0 -> 505309 bytes test/input/TestPerSample/in.3libs.bam | Bin 505271 -> 505312 bytes test/input/TestPerSample/in.3libs.bam.bai | Bin 176 -> 0 bytes test/input/TestPerSample/in.bam | Bin 503332 -> 503364 bytes test/input/TestPerSample/in.indels.bam | Bin 450861 -> 450893 bytes test/input/TestPerSample/in.indels.bam.bai | Bin 96 -> 0 bytes 8 files changed, 0 insertions(+), 0 deletions(-) delete mode 100644 test/input/TestPerSample/in.2libs.bam.bai create mode 100644 test/input/TestPerSample/in.2libs3rgs.bam delete mode 100644 test/input/TestPerSample/in.3libs.bam.bai delete mode 100644 test/input/TestPerSample/in.indels.bam.bai diff --git a/test/input/TestPerSample/in.2libs.bam b/test/input/TestPerSample/in.2libs.bam index ef2cf1938bcc91029b5d1f32cf93d50185706ec8..7f88d0234ae68f2deae19884de772b7578c69a1a 100644 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WEQ~A^kigYU|?VZVoxCk1`wNpL4^fOG3Y*I12ViOLFCcJd8Ht#U?i$uh$sLoodnYW From 24399b343dc1da26e7f40ab994a52079294a72b8 Mon Sep 17 00:00:00 2001 From: Danny Park Date: Wed, 18 Mar 2015 15:55:14 -0400 Subject: [PATCH 19/20] samtools merge fails with error if you do not provide the BAM header in a separate input file --- intrahost.py | 5 ++++- tools/samtools.py | 4 ++-- 2 files changed, 6 insertions(+), 3 deletions(-) diff --git a/intrahost.py b/intrahost.py index 211e8296c..988c23b2d 100755 --- a/intrahost.py +++ b/intrahost.py @@ -149,6 +149,8 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : for rg in samtoolsTool.getReadGroups(inBam).values()) rgs_by_lib = itertools.groupby(rgs_by_lib, lambda x: x[0]) libBams = [] + header_sam = util.file.mkstempfname('.sam') + samtoolsTool.dumpHeader(inBam, header_sam) for lib,rgs in rgs_by_lib: rgs = list(id for lb,id in rgs) @@ -162,7 +164,7 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : rgBams.append(rgBam) samtoolsTool.view(['-b', '-r', id], inBam, rgBam) libBam = util.file.mkstempfname('.bam') - samtoolsTool.merge(rgBams, libBam) + samtoolsTool.merge(rgBams, libBam, ['-f', '-1', '-h', header_sam]) for bam in rgBams : os.unlink(bam) @@ -209,6 +211,7 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : yield row for bam in libBams: os.unlink(bam) + os.unlink(header_sam) def parse_alleles_string(allelesStr) : # Return {allele : [forwardCount, reverseCount]} diff --git a/tools/samtools.py b/tools/samtools.py index a51b5d73b..7b80ecfa7 100644 --- a/tools/samtools.py +++ b/tools/samtools.py @@ -54,12 +54,12 @@ def execute(self, command, args, stdin=None, stdout=None, stderr=None): def view(self, args, inFile, outFile, regions=[]): self.execute('view', args + ['-o', outFile, inFile] + regions) - def merge(self, inFiles, outFile): + def merge(self, inFiles, outFile, options=['-f']): "Merge a list of inFiles to create outFile." # We are using -f for now because mkstempfname actually makes an empty # file, and merge fails with that as output target without the -f. # When mkstempfname is fixed, we should remove the -f. - self.execute('merge', ['-f'] + [outFile] + inFiles) + self.execute('merge', options + [outFile] + inFiles) def index(self, inBam): self.execute('index', [inBam]) From c9347223a7b7b0d3b820e19977d7456f3563d9bd Mon Sep 17 00:00:00 2001 From: Danny Park Date: Wed, 18 Mar 2015 16:14:03 -0400 Subject: [PATCH 20/20] workaround for solitary input bam --- intrahost.py | 33 ++++++++++++++++++++------------- 1 file changed, 20 insertions(+), 13 deletions(-) diff --git a/intrahost.py b/intrahost.py index 988c23b2d..41da2518a 100755 --- a/intrahost.py +++ b/intrahost.py @@ -161,21 +161,28 @@ def compute_library_bias(isnvs, inBam, inConsFasta) : rgBams = [] for id in rgs : rgBam = util.file.mkstempfname('.bam') - rgBams.append(rgBam) samtoolsTool.view(['-b', '-r', id], inBam, rgBam) - libBam = util.file.mkstempfname('.bam') - samtoolsTool.merge(rgBams, libBam, ['-f', '-1', '-h', header_sam]) - for bam in rgBams : - os.unlink(bam) - - samtoolsTool.index(libBam) - n_reads = samtoolsTool.count(libBam) - log.debug("LB:{} has {} reads in {} read groups ({})".format( - lib, n_reads, len(rgs), ', '.join(rgs))) - if n_reads > 0: + samtoolsTool.index(rgBam) + if samtoolsTool.count(rgBam) > 0: + rgBams.append(rgBam) + else: + # most samtools functions don't like empty input bams, so skip them + os.unlink(rgBam) + if rgBams: + if len(rgBams) > 1: + libBam = util.file.mkstempfname('.bam') + samtoolsTool.merge(rgBams, libBam, ['-f', '-1', '-h', header_sam]) + for bam in rgBams : + os.unlink(bam) + else: + # samtools merge cannot deal with only one (or zero) input bams + libBam = rgBams[0] + samtoolsTool.index(libBam) + n_reads = samtoolsTool.count(libBam) + log.debug("LB:{} has {} reads in {} read groups ({})".format( + lib, n_reads, len(rgs), ', '.join(rgs))) libBams.append(libBam) - else: - os.unlink(libBam) + for row in isnvs : consensusAllele = row[3] pos = int(row[1]) if consensusAllele != 'i' else int(row[1]) - 1