iqbal-lab-org · martinghunt · Aug 16, 2023 · Aug 11, 2023 · Aug 11, 2023
diff --git a/python/clockwork/gvcf.py b/python/clockwork/gvcf.py
@@ -1,4 +1,7 @@
+from collections import Counter
 import datetime
+import logging
+import subprocess
 
 from cluster_vcf_records import vcf_file_read, vcf_record
 import pyfastaq
@@ -15,6 +18,7 @@ def _combine_minos_and_samtools_header(minos_header, samtools_header, ref_seqs):
     new_header = [
         '##FILTER=<ID=NO_DATA,Description="No information from minos or samtools">',
         '##INFO=<ID=CALLER,Number=1,Description="Origin of call, one of minos, samtools, or none if there was no depth">',
+        '##FORMAT=<ID=DP_ACGT,Number=8,Type=Integer,Description="Number of A-forward, A-reverse, C-forward, C-reverse, G-forward, G-reverse, T-forward, T-reverse bases">',
     ]
     new_header.extend(
         [f"##contig=<ID={k},length={len(v)}>" for k, v in ref_seqs.items()]
@@ -126,16 +130,61 @@ def _finish_contig(ref_pos, ref_seq, minos_record, minos_iter, filehandle):
             ref_pos += 1
 
 
-def gvcf_from_minos_vcf_and_samtools_gvcf(ref_fasta, minos_vcf, samtools_vcf, out_vcf):
+def _samtools_acgt_depths(bam_file):
+    command = f"samtools mpileup --no-output-ins --no-output-ins --no-output-del --no-output-del --no-output-ends -aa {bam_file}"
+    logging.info(f"Gathering per-position A/C/G/T depths ({command})")
+    p = subprocess.Popen(
+        command, shell=True, stdout=subprocess.PIPE, universal_newlines=True
+    )
+    acgt_depths = {}
+    acgt = ("A", "a", "C", "c", "G", "g", "T", "t")
+
+    for line in p.stdout:
+        ref, pos, _, _, depths, _ = line.split("\t")
+        pos = int(pos) - 1
+        if ref not in acgt_depths:
+            acgt_depths[ref] = {}
+        assert pos not in acgt_depths[ref]
+        counts = Counter(depths)
+        acgt_depths[ref][pos] = ",".join(str(counts[x]) for x in acgt)
+
+    p.wait()
+    if p.returncode != 0:
+        raise Exception(f"Error running samtools mpileup: {command}")
+
+    logging.info(f"Finished per-position A/C/G/T depths ({command})")
+    return acgt_depths
+
+
+def _add_acgt_depths_to_minos(minos_records, acgt_depths):
+    for ref in minos_records:
+        if ref not in acgt_depths:
+            continue
+        depths = acgt_depths[ref]
+
+        for record in minos_records[ref]:
+            if record.POS in depths:
+                record.set_format_key_value("DP_ACGT", depths[record.POS])
+
+
+def gvcf_from_minos_vcf_and_samtools_gvcf(
+    ref_fasta, minos_vcf, samtools_vcf, out_vcf, bam_file=None
+):
     minos_header, minos_records = vcf_file_read.vcf_file_to_dict(minos_vcf)
     samtools_header = vcf_file_read.get_header_lines_from_vcf_file(samtools_vcf)
+    if bam_file is not None:
+        acgt_depths = _samtools_acgt_depths(bam_file)
+        _add_acgt_depths_to_minos(minos_records, acgt_depths)
+    else:
+        acgt_depths = {}
 
     ref_seqs = {}
     pyfastaq.tasks.file_to_dict(ref_fasta, ref_seqs)
     used_ref_seqs = set()
     ref_seq = None
     ref_pos = -1
     minos_record = None
+    logging.info(f"Making GVCF file {out_vcf} from {minos_vcf} {samtools_vcf}")
 
     with open(out_vcf, "w") as f_out, open(samtools_vcf) as f_samtools:
         print(
@@ -161,6 +210,12 @@ def gvcf_from_minos_vcf_and_samtools_gvcf(ref_fasta, minos_vcf, samtools_vcf, ou
                 continue
 
             samtools_record = vcf_record.VcfRecord(line)
+            try:
+                samtools_record.set_format_key_value(
+                    "DP_ACGT", acgt_depths[samtools_record.CHROM][samtools_record.POS]
+                )
+            except KeyError:
+                pass
 
             # If we've found a new CHROM in the samtools VCF file
             if ref_seq is None or ref_seq.id != samtools_record.CHROM:
@@ -203,7 +258,7 @@ def gvcf_from_minos_vcf_and_samtools_gvcf(ref_fasta, minos_vcf, samtools_vcf, ou
                 print(samtools_record, file=f_out)
                 ref_pos = samtools_record.POS + 1
 
-        if ref_seq is not None: # happens if the samtools VCF was empty
+        if ref_seq is not None:  # happens if the samtools VCF was empty
             _finish_contig(ref_pos, ref_seq, minos_record, minos_iter, f_out)
         _print_non_samtools_seqs(ref_seqs, used_ref_seqs, minos_records, f_out)
 

diff --git a/python/clockwork/tasks/gvcf_from_minos_and_samtools.py b/python/clockwork/tasks/gvcf_from_minos_and_samtools.py
@@ -3,5 +3,9 @@
 
 def run(options):
     gvcf.gvcf_from_minos_vcf_and_samtools_gvcf(
-        options.ref_fasta, options.minos_vcf, options.samtools_vcf, options.outfile,
+        options.ref_fasta,
+        options.minos_vcf,
+        options.samtools_vcf,
+        options.outfile,
+        bam_file=options.bam_file,
     )
diff --git a/python/clockwork/tests/data/gvcf/gvcf_from_minos_vcf_and_samtools_gvcf.out.vcf b/python/clockwork/tests/data/gvcf/gvcf_from_minos_vcf_and_samtools_gvcf.out.vcf
@@ -16,6 +16,7 @@
 ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="total read depth from gramtools">
 ##FORMAT=<ID=DP4,Number=4,Type=Integer,Description="Number of high-quality ref-forward , ref-reverse, alt-forward and alt-reverse bases">
 ##FORMAT=<ID=DPF,Number=1,Type=Float,Description="Depth Fraction, defined as DP divided by mean depth">
+##FORMAT=<ID=DP_ACGT,Number=8,Type=Integer,Description="Number of A-forward, A-reverse, C-forward, C-reverse, G-forward, G-reverse, T-forward, T-reverse bases">
 ##FORMAT=<ID=FQ,Number=1,Type=Float,Description="Phred probability of all samples being the same">
 ##FORMAT=<ID=FRS,Number=1,Type=Float,Description="Fraction of reads that support the genotype call">
 ##FORMAT=<ID=G3,Number=3,Type=Float,Description="ML estimate of genotype frequencies">

diff --git a/python/clockwork/var_call_one_sample_pipeline.py b/python/clockwork/var_call_one_sample_pipeline.py
@@ -34,9 +34,13 @@ def run(
             "Must give same number of forward and reverse reads files. Got:\nForward:{reads1_list}\nReverse:{reads2_list}"
         )
     if not check_reads_files(reads1_list + reads2_list):
-        raise Exception("Reads file(s) not found or are empty. See previous error messages. Cannot continue")
+        raise Exception(
+            "Reads file(s) not found or are empty. See previous error messages. Cannot continue"
+        )
     if not os.path.exists(ref_dir):
-        raise FileNotFoundError(f"Reference directory not found: {ref_dir}. Cannot continue")
+        raise FileNotFoundError(
+            f"Reference directory not found: {ref_dir}. Cannot continue"
+        )
 
     os.mkdir(outdir)
 
@@ -51,10 +55,7 @@ def run(
         trimmed_reads_1.append(os.path.join(outdir, f"trimmed_reads.{i}.1.fq.gz"))
         trimmed_reads_2.append(os.path.join(outdir, f"trimmed_reads.{i}.2.fq.gz"))
         read_trim.run_trimmomatic(
-            reads1_list[i],
-            reads2_list[i],
-            trimmed_reads_1[-1],
-            trimmed_reads_2[-1],
+            reads1_list[i], reads2_list[i], trimmed_reads_1[-1], trimmed_reads_2[-1]
         )
 
     refdir = reference_dir.ReferenceDir(directory=ref_dir)
@@ -77,7 +78,9 @@ def run(
     utils.syscall(cmd)
     samtools_vcf_has_vars = utils.vcf_has_records(samtools_vcf)
     if not samtools_vcf_has_vars:
-        logging.warn("SAMTOOLS VCF FILE HAS ZERO VARIANTS. PLEASE CHECK THE QUALITY OF INPUT DATA")
+        logging.warn(
+            "SAMTOOLS VCF FILE HAS ZERO VARIANTS. PLEASE CHECK THE QUALITY OF INPUT DATA"
+        )
 
     samtools_gvcf = os.path.join(outdir, "samtools.gvcf")
     cmd = f"bcftools mpileup -I --output-type u -f {refdir.ref_fasta} {rmdup_bam} | bcftools call -c -O v -o {samtools_gvcf}"
@@ -94,7 +97,9 @@ def run(
     try:
         ctx.run(run_mccortex_view_kmers=False)
     except:
-        logging.error("ERROR RUNNING CORTEX. WILL TRY TO CONTINUE USING SAMTOOLS VCF ONLY (IF SAMTOOLS FOUND VARIANTS), BUT PLEASE CHECK CORTEX LOGS")
+        logging.error(
+            "ERROR RUNNING CORTEX. WILL TRY TO CONTINUE USING SAMTOOLS VCF ONLY (IF SAMTOOLS FOUND VARIANTS), BUT PLEASE CHECK CORTEX LOGS"
+        )
 
     ctx_vcf_dir = os.path.join(cortex_dir, "cortex.out", "vcfs")
     try:
@@ -108,24 +113,43 @@ def run(
 
     cortex_vcf_has_vars = False
     if len(cortex_vcfs) != 1:
-        logging.error("NO VCF FILE MADE BY CORTEX. WILL TRY TO CONTINUE USING SAMTOOLS VCF ONLY (IF SAMTOOLS FOUND VARIANTS), BUT PLEASE CHECK THE QUALITY OF INPUT DATA, AND CORTEX LOGS")
+        logging.error(
+            "NO VCF FILE MADE BY CORTEX. WILL TRY TO CONTINUE USING SAMTOOLS VCF ONLY (IF SAMTOOLS FOUND VARIANTS), BUT PLEASE CHECK THE QUALITY OF INPUT DATA, AND CORTEX LOGS"
+        )
         cortex_vcf = ""
     else:
         cortex_vcf = os.path.join(outdir, "cortex.vcf")
         os.rename(cortex_vcfs[0], cortex_vcf)
         cortex_vcf_has_vars = utils.vcf_has_records(cortex_vcf)
         if not cortex_vcf_has_vars:
-            logging.warn("CORTEX VCF FILE HAS ZERO VARIANTS. PLEASE CHECK THE QUALITY OF INPUT DATA")
+            logging.warn(
+                "CORTEX VCF FILE HAS ZERO VARIANTS. PLEASE CHECK THE QUALITY OF INPUT DATA"
+            )
         if not debug:
             utils.syscall(f"rm -rf {cortex_dir}")
 
     final_vcf = os.path.join(outdir, "final.vcf")
 
     if not (samtools_vcf_has_vars and cortex_vcf_has_vars):
-        logging.error("NO VARIANTS FOUND BY CORTEX OR SAMTOOLS. WRITING HEADER-ONLY FINAL VCF FILE INSTEAD OF RUNNING MINOS")
+        logging.error(
+            "NO VARIANTS FOUND BY CORTEX OR SAMTOOLS. WRITING HEADER-ONLY FINAL VCF FILE INSTEAD OF RUNNING MINOS"
+        )
         with open(final_vcf, "w") as f:
             print("##fileformat=VCFv4.2", file=f)
-            print("#CHROM", "POS", "ID", "REF", "ALT", "QUAL", "FILTER", "INFO", "FORMAT", "sample", sep="\t", file=f)
+            print(
+                "#CHROM",
+                "POS",
+                "ID",
+                "REF",
+                "ALT",
+                "QUAL",
+                "FILTER",
+                "INFO",
+                "FORMAT",
+                "sample",
+                sep="\t",
+                file=f,
+            )
         minos_vcf_has_vars = False
     else:
         minos_dir = os.path.join(outdir, "minos")
@@ -137,7 +161,7 @@ def run(
 
     final_gvcf = os.path.join(outdir, "final.gvcf")
     gvcf.gvcf_from_minos_vcf_and_samtools_gvcf(
-        refdir.ref_fasta, final_vcf, samtools_gvcf, final_gvcf,
+        refdir.ref_fasta, final_vcf, samtools_gvcf, final_gvcf, bam_file=rmdup_bam
     )
     if not debug:
         os.unlink(samtools_gvcf)

diff --git a/python/scripts/clockwork b/python/scripts/clockwork
@@ -477,20 +477,17 @@ subparser_gvcf_from_minos_and_samtools = subparsers.add_parser(
 )
 
 subparser_gvcf_from_minos_and_samtools.add_argument(
-    "ref_fasta",
-    help="Reference genome FASTA file",
+    "ref_fasta", help="Reference genome FASTA file"
 )
 subparser_gvcf_from_minos_and_samtools.add_argument(
-    "minos_vcf",
-    help="VCF file made by minos",
+    "minos_vcf", help="VCF file made by minos"
 )
 subparser_gvcf_from_minos_and_samtools.add_argument(
-    "samtools_vcf",
-    help="VCF file made by samtools",
+    "samtools_vcf", help="VCF file made by samtools"
 )
+subparser_gvcf_from_minos_and_samtools.add_argument("outfile", help="Output gVCF file")
 subparser_gvcf_from_minos_and_samtools.add_argument(
-    "outfile",
-    help="Output gVCF file",
+    "--bam_file", help="BAM file. If used, will add DP_ACGT to VCF"
 )
 subparser_gvcf_from_minos_and_samtools.set_defaults(
     func=clockwork.tasks.gvcf_from_minos_and_samtools.run
@@ -506,13 +503,9 @@ subparser_gvcf_to_fasta = subparsers.add_parser(
 )
 
 subparser_gvcf_to_fasta.add_argument(
-    "gvcf",
-    help="Input gVCF file made by gvcf_from_minos_and_samtools",
-)
-subparser_gvcf_to_fasta.add_argument(
-    "outfile",
-    help="Output FASTA file",
+    "gvcf", help="Input gVCF file made by gvcf_from_minos_and_samtools"
 )
+subparser_gvcf_to_fasta.add_argument("outfile", help="Output FASTA file")
 subparser_gvcf_to_fasta.add_argument(
     "--ignore_minos_pass",
     action="store_true",
@@ -840,9 +833,7 @@ subparser_samtools_qc = subparsers.add_parser(
     description="Runs BWA MEM, picard MarkDuplicates, then samtools stats and plots",
 )
 
-subparser_samtools_qc.add_argument(
-    "ref_fasta", help="Name of reference fasta file"
-)
+subparser_samtools_qc.add_argument("ref_fasta", help="Name of reference fasta file")
 subparser_samtools_qc.add_argument("reads1", help="Name of reads file 1")
 subparser_samtools_qc.add_argument("reads2", help="Name of reads file 2")
 subparser_samtools_qc.add_argument(
@@ -967,19 +958,13 @@ subparser_variant_call_one_sample.add_argument(
     metavar="INT",
 )
 subparser_variant_call_one_sample.add_argument(
-    "--force",
-    action="store_true",
-    help="Overwrite outdir if it already exists",
+    "--force", action="store_true", help="Overwrite outdir if it already exists"
 )
 subparser_variant_call_one_sample.add_argument(
-    "--keep_bam",
-    action="store_true",
-    help="Keep BAM file of rmdup reads",
+    "--keep_bam", action="store_true", help="Keep BAM file of rmdup reads"
 )
 subparser_variant_call_one_sample.add_argument(
-    "--debug",
-    action="store_true",
-    help="Debug mode: do not clean up any files",
+    "--debug", action="store_true", help="Debug mode: do not clean up any files"
 )
 subparser_variant_call_one_sample.add_argument(
     "--no_trim",
@@ -990,8 +975,7 @@ subparser_variant_call_one_sample.add_argument(
     "ref_dir", help="Directory of reference files, made by clockwork reference_prepare"
 )
 subparser_variant_call_one_sample.add_argument(
-    "outdir",
-    help="Output directory (must not exist, will be created)",
+    "outdir", help="Output directory (must not exist, will be created)"
 )
 subparser_variant_call_one_sample.add_argument(
     "reads_files",