updates for mod .bams, options relating to ridge plots in segmeth, bu…

…gfixes for multi-mod plots

methylartist

@@ -83,6 +83,10 @@ class Read:
 
     def add_mod(self, cpg_loc, stat, methcall, modname):
         assert methcall in (-1,0,1)
+
+        if cpg_loc in self.llrs:
+            logger.warning('warning: collision between mods, this is a bug! report me!')
+
         self.llrs[cpg_loc]       = stat
         self.meth_calls[cpg_loc] = methcall
         self.mod_names[cpg_loc]  = modname
@@ -697,10 +701,10 @@ def get_segmeth_calls(args, bam_fn, mod_names, meth_dbs, chrom, seg_start, seg_e
         reads = [r for r in reads if r in phased_reads_dict and phased_reads_dict[r] == phase]
 
     reads = set(reads)
-    seg_reads = {}
+    seg_reads = dd(dict)
 
     if methbam:
-        for row in parse_methbam(bam_fn, reads, chrom, seg_start, seg_end, meth_thresh=0.8, can_thresh=0.8):
+        for row in parse_methbam(bam_fn, reads, chrom, seg_start, seg_end, meth_thresh=0.8, can_thresh=0.8, restrict_ref=args.ref, restrict_motif=args.motif):
             index, cg_chrom, cg_start, stat, methstate, modname = row
 
             if chrom != cg_chrom:
@@ -711,10 +715,10 @@ def get_segmeth_calls(args, bam_fn, mod_names, meth_dbs, chrom, seg_start, seg_e
 
             cg_seg_start = cg_start - seg_start
 
-            if index not in seg_reads:
-                seg_reads[index] = Read(index, cg_seg_start, stat, methstate, modname)
+            if index not in seg_reads[modname]:
+                seg_reads[modname][index] = Read(index, cg_seg_start, stat, methstate, modname)
             else:
-                seg_reads[index].add_mod(cg_seg_start, stat, methstate, modname)
+                seg_reads[modname][index].add_mod(cg_seg_start, stat, methstate, modname)
 
     else:
         for index in reads:
@@ -732,27 +736,30 @@ def get_segmeth_calls(args, bam_fn, mod_names, meth_dbs, chrom, seg_start, seg_e
 
                     cg_seg_start = cg_start - seg_start
 
-                    if index not in seg_reads:
-                        seg_reads[index] = Read(index, cg_seg_start, stat, methstate, modname)
+                    if index not in seg_reads[modname]:
+                        seg_reads[modname][index] = Read(index, cg_seg_start, stat, methstate, modname)
                     else:
-                        seg_reads[index].add_mod(cg_seg_start, stat, methstate, modname)
+                        seg_reads[modname][index].add_mod(cg_seg_start, stat, methstate, modname)
 
     if args.max_read_density is not None:
-        seg_reads = densecall_filter(seg_reads, max_density=float(args.max_read_density))
+        seg_reads[modname] = densecall_filter(seg_reads[modname], max_density=float(args.max_read_density))
 
     seg_result = {}
+    seen_reads = {}
 
     for modname in mod_names:
         seg_meth_calls = dd(int)
 
-        for name, read in seg_reads.items():
+        for name, read in seg_reads[modname].items():
+            seen_reads[name] = 1
+
             for loc, call in read.meth_calls.items():
                 if read.mod_names[loc] == modname:
                     seg_meth_calls[call] += 1
 
         seg_result[modname] = seg_meth_calls
 
-    read_count = len(seg_reads)
+    read_count = len(seen_reads)
 
     return seg_result, (chrom, seg_start, seg_end, seg_name, seg_strand, read_count)
 
@@ -908,7 +915,7 @@ def get_meth_profile_composite(args, data, methbam, seg_chrom, seg_start, seg_en
         seg_reads = {}
 
         if methbam:
-            for row in parse_methbam(bam, reads, seg_chrom, seg_start, seg_end, motifsize=len(args.motif), meth_thresh=0.8, can_thresh=0.8):
+            for row in parse_methbam(bam, reads, seg_chrom, seg_start, seg_end, motifsize=len(args.motif), meth_thresh=0.8, can_thresh=0.8, restrict_motif=args.motif, restrict_ref=args.ref):
                 index, cg_chrom, cg_start, stat, methstate, modname = row
 
                 if seg_chrom != cg_chrom:
@@ -917,6 +924,9 @@ def get_meth_profile_composite(args, data, methbam, seg_chrom, seg_start, seg_en
                 if cg_start < seg_start or cg_start > seg_end:
                     continue
 
+                if modname != use_mod:
+                    continue
+
                 cg_seg_start = cg_start - seg_start
 
                 if index not in seg_reads:
@@ -1106,7 +1116,7 @@ def get_meth_calls_wg(args, bam_fn, meth_fn, chrom, seg_start, seg_end, phased,
     seg_reads = {}
 
     if methbam:
-        for row in parse_methbam(bam_fn, set(reads), chrom, seg_start, seg_end, meth_thresh=0.8, can_thresh=0.8):
+        for row in parse_methbam(bam_fn, set(reads), chrom, seg_start, seg_end, meth_thresh=0.8, can_thresh=0.8, restrict_ref=args.ref, restrict_motif=args.motif):
             index, cg_chrom, cg_start, stat, methstate, modname = row
 
             if mod is not None and mod != modname:
@@ -2035,6 +2045,10 @@ def segmeth(args):
                         mod_names.append(m)
 
     if args.bams is not None:
+        if None in (args.ref, args.motif):
+            logger.warning('--ref and --motif are required when using --bams')
+            sys.exit(1)
+
         methbam = True
         bams = []
 
@@ -2256,7 +2270,7 @@ def segplot(args):
 
         mods = user_mods
 
-    samples_mods = [s + '_' + m for s, m in itertools.product(samples, mods)]
+    samples_mods = list(set([s + '_' + m for s, m in itertools.product(samples, mods)]))
 
     logger.info('sample + mod permutations: %s' % ','.join(samples_mods))
 
@@ -2349,8 +2363,8 @@ def segplot(args):
             plot_data = plot_data.sort_values(['group','sample'])
 
         sns_plot = sns.FacetGrid(plot_data, row='samplegroup', hue='sample', aspect=15, height=.5, palette=args.palette)
-        sns_plot.map(sns.kdeplot, 'modbase', bw_adjust=.5, clip_on=False, fill=True, alpha=float(args.ridge_alpha), linewidth=1.5)
-        sns_plot.map(sns.kdeplot, 'modbase', clip_on=False, color='w', lw=2, bw_adjust=.5)
+        sns_plot.map(sns.kdeplot, 'modbase', bw_adjust=float(args.ridge_smoothing), clip_on=False, fill=True, alpha=float(args.ridge_alpha), linewidth=1.5)
+        sns_plot.map(sns.kdeplot, 'modbase', clip_on=False, color='w', lw=2, bw_adjust=float(args.ridge_smoothing), alpha=float(args.ridge_alpha))
         sns_plot.refline(y=0, linewidth=2, linestyle="-", color=None, clip_on=False)
         sns_plot.figure.subplots_adjust(hspace=float(args.ridge_spacing))
 
@@ -2465,24 +2479,17 @@ def locus(args):
                 if len(c) == 3:
                     user_colours[bam] = c[2]
 
-    if args.restrict_ref is not None and args.restrict_motif is None:
-        logger.warning('must specify --restrict_motif with --restrict_ref e.g. --restrict_motif CG for CpG methylation')
-        sys.exit(1)
-
-    if args.restrict_ref is None and args.restrict_motif is not None:
-        logger.warning('must specify --restrict_ref with --restrict_motif')
-        sys.exit(1)
-
-    if args.restrict_motif is not None:
-        if len(args.restrict_motif) != int(args.motifsize):
-            logger.warning('motif size (set with --motifsize) %d does not match length of --restrict_motif (%s)' % (int(args.motifsize), args.restrict_motif))
-            sys.exit(1)
+    if args.motif is not None:
+        if len(args.motif) != int(args.motifsize):
+            logger.warning('motif size (set with --motifsize) %d does not match length of --motif (%s), changed --motifsize' % (int(args.motifsize), args.motif))
+            args.motifsize = len(args.motif)
 
     if args.bams is not None:
         logger.info('mod motif size (--motifsize) = %d (ensure this is correct for your data)' % int(args.motifsize))
 
-        if args.restrict_ref is None:
-            logger.warning('*** WARNING: specifying a reference genome (indexed via samtools faidx) with --restrict_ref is strongly recommended when using mod .bams ***')
+        if None in (args.ref, args.motif):
+            logger.warning('--ref and --motif are required when using --bams')
+            sys.exit(1)
 
         methbam = True
         bams = []
@@ -2549,7 +2556,7 @@ def locus(args):
                 for phase in phases[bam]:
                     bamname = '.'.join(os.path.basename(bam).split('.')[:-1]) + '.' + phase + '.' + mod
                     orig_bam[bamname] = bam
-                    reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, phase=phase, methbam=methbam, HP_only=args.ignore_ps, restrict_motif=args.restrict_motif, restrict_ref=args.restrict_ref)
+                    reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, phase=phase, methbam=methbam, HP_only=args.ignore_ps, restrict_motif=args.motif, restrict_ref=args.ref)
 
                     for name, read in reads[bamname].items():
                         for loc in read.llrs.keys():
@@ -2566,7 +2573,7 @@ def locus(args):
             else:
                 bamname = '.'.join(os.path.basename(bam).split('.')[:-1]) + '.' + mod
                 orig_bam[bamname] = bam
-                reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, methbam=methbam, restrict_motif=args.restrict_motif, restrict_ref=args.restrict_ref)
+                reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, methbam=methbam, restrict_motif=args.motif, restrict_ref=args.ref)
 
                 for name, read in reads[bamname].items():
                     for loc in read.llrs.keys():
@@ -3186,7 +3193,7 @@ def locus(args):
             logger.warning('%s:%d-%d (%s), skip sample %s due to --maxmaskedfrac %.3f' % (chrom, elt_start, elt_end, ''.join(use_mods), sample, float(args.maxmaskedfrac)))
             continue
 
-        if frac_masked > 0.1 and args.bams is not None and args.restrict_ref is None:
+        if frac_masked > 0.1 and args.bams is not None and args.ref is None:
             logger.warning('*** WARNING: specifying a reference genome (indexed via samtools faidx) with --restrict_ref is strongly recommended when using mod .bams ***')
 
         ax5.plot(list(windowed_methfrac.keys()), smoothed_methfrac, marker='', lw=4, color=sample_color[sample], alpha=smoothalpha)
@@ -3308,7 +3315,7 @@ def region(args):
         logger.warning('locus smaller than 0.5 Mbp, "methylartist locus" may yield better results')
 
     ref = pysam.FastaFile(args.ref)
-    motif = args.norm_motif.upper()
+    motif = args.motif.upper()
     region_seq = ref.fetch(chrom, start, end).upper()
     motif_count = region_seq.count(motif) + region_seq.count(rc(motif))
 
@@ -3352,7 +3359,7 @@ def region(args):
     w_ends.append(end)
 
     assert len(w_starts) == len(w_ends)
-    logger.info('using %d windows normalised for %s content' % (len(w_starts), args.norm_motif))
+    logger.info('using %d windows normalised for %s content' % (len(w_starts), args.motif))
 
     if args.smoothwindowsize is None:
         w = len(w_starts)
@@ -3452,7 +3459,7 @@ def region(args):
                     bamname += '.' + phase
 
                 orig_bam[bamname] = bam
-                reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, phase=phase, methbam=methbam)
+                reads[bamname] = get_meth_locus(args, bam, meth_dbs, mod, phase=phase, methbam=methbam, restrict_motif=args.motif, restrict_ref=args.ref)
 
     pool = mp.Pool(processes=int(args.procs))
 
@@ -4365,6 +4372,10 @@ def wgmeth(args):
     methbam = False
 
     if args.methdb is None:
+        if None in (args.ref, args.motif):
+            logger.warning('--ref and --motif are required when using mod .bams (no --methdb)')
+            sys.exit(1)
+
         methbam = True
 
     if methbam:
@@ -4376,7 +4387,10 @@ def wgmeth(args):
         sys.exit('bam %s does not appear to contain Mm/Ml tags' % args.bam)
 
     if args.mod not in mods:
-        logger.warning('mod %s not in known mods for db: %s' % (args.mod, ','.join(mods)))
+        if args.mod is None:
+            logger.warning('more than one mod available, must specify which to use with --mod: %s' % ','.join(mods))
+        else:
+            logger.warning('mod %s not in known mods for db: %s' % (args.mod, ','.join(mods)))
         sys.exit()
 
     if len(mods) > 1 and args.mod is None:
@@ -4484,7 +4498,12 @@ def wgmeth(args):
             meth_table[0]['start']  = meth_table[0]['pos']-1
             meth_table[0]['end']    = meth_table[0]['pos']
             meth_table[0]['pct']    = meth_table[0]['X']/meth_table[0]['N']*100
-            meth_table[0]['name']   = '.'.join(args.methdb.split('.')[:-1])
+
+            if args.methdb:
+                meth_table[0]['name']   = '.'.join(args.methdb.split('.')[:-1])
+            else:
+                meth_table[0]['name']   = '.'.join(args.bam.split('.')[:-1])
+
             meth_table[0]['strand'] = '.'
             meth_table[0]['colour'] = '0,0,0'
 
@@ -4500,7 +4519,7 @@ if __name__ == '__main__':
     subparsers = parser.add_subparsers(title="tool", dest="tool")
     subparsers.required = True
 
-    __version__ = "1.2.1"
+    __version__ = "1.2.2"
     parser.add_argument('-v', '--version', action='version', version='%(prog)s {version}'.format(version=__version__))
 
     parser_nanopolish    = subparsers.add_parser('db-nanopolish')
@@ -4535,6 +4554,8 @@ if __name__ == '__main__':
     parser_segmeth.add_argument('-i', '--intervals', required=True, help='.bed file')
     parser_segmeth.add_argument('-p', '--procs', default=1, help='multiprocessing')
     parser_segmeth.add_argument('-q', '--min_mapq', default=10, help='minimum mapping quality (mapq), default = 10')
+    parser_segmeth.add_argument('--ref', default=None, help='reference genome .fa (build .fai index with samtools faidx) (required for mod bams)')
+    parser_segmeth.add_argument('--motif', default=None, help='expected modification motif (e.g. CG for 5mCpG required for mod bams)')
     parser_segmeth.add_argument('--max_read_density', default=None, help='filter reads with call density greater >= value, can be helpful in footprinting assays (default=None)')
     parser_segmeth.add_argument('--excl_ambig', action='store_true', default=False, help='do not consider reads that align entirely within segment')
     parser_segmeth.add_argument('--spanning_only', action='store_true', default=False, help='only consider reads that span segment')
@@ -4561,6 +4582,7 @@ if __name__ == '__main__':
     parser_segplot.add_argument('--palette', default="tab10", help='palette for phases (default = "tab10"), see https://seaborn.pydata.org/tutorial/color_palettes.html')
     parser_segplot.add_argument('--ridge_alpha', default=1.0, help='alpha (tranparency) for ridge plot fills (default = 1.0)')
     parser_segplot.add_argument('--ridge_spacing', default=-0.25, help='ridge plot spacing (generally negative, default = -0.25)')
+    parser_segplot.add_argument('--ridge_smoothing', default=0.5, help='smoothing parameter for ridge plot, bigger is smoother (default=0.5)')
     parser_segplot.add_argument('--svg', default=False, action='store_true')
 
     # options for locus-specific plots
@@ -4574,8 +4596,8 @@ if __name__ == '__main__':
     parser_locus.add_argument('-t', '--slidingwindowstep', default=1, help='step size for initial sliding window (default=1)')
     parser_locus.add_argument('-p', '--panelratios',  default=None, help='Alter panel ratios: needs to be 5 comma-seperated integers. Default: 1,5,1,3,3')
     parser_locus.add_argument('-q', '--min_mapq', default=10, help='minimum mapping quality (mapq), default = 10')
-    parser_locus.add_argument('--restrict_ref', default=None, help='restrict modified motifs to those that agree with the reference genome (requires --restrict_motif, recommended for mod bams)')
-    parser_locus.add_argument('--restrict_motif', default=None, help='motifs to use with --restrict_reference (required if using --restrict_reference, recommended for mod bams)')
+    parser_locus.add_argument('--ref', default=None, help='reference genome .fa (build .fai index with samtools faidx) (required for mod bams)')
+    parser_locus.add_argument('--motif', default=None, help='expected modification motif (e.g. CG for 5mCpG required for mod bams)')
     parser_locus.add_argument('--bed', default=None, help='.bed file for additional annotations')
     parser_locus.add_argument('--highlight_bed', default=None, help='BED3+1 format (chrom, start, end, optional_colour) where colour (optional) must be intelligible to matplotlib')
     parser_locus.add_argument('--motifsize', default=2, help='mod motif size, only used with -b/--bams (default is 2 as "CG" is most common use case, e.g. set to 1 for 6mA)')
@@ -4622,7 +4644,7 @@ if __name__ == '__main__':
     parser_region.add_argument('-i', '--interval', required=True, help='chrom:start-end')
     parser_region.add_argument('-d', '--data', default=None, help='text file with .bam filename and corresponding methylation database per line(whitespace-delimited)')
     parser_region.add_argument('-b', '--bams', default=None, help='one or more .bams with Mm and Ml tags for modification calls (see samtags spec)')
-    parser_region.add_argument('-n', '--norm_motif', required=True, help='normalise window sizes to motif occurance')
+    parser_region.add_argument('-n', '--motif', required=True, help='normalise window sizes to motif occurance')
     parser_region.add_argument('-r', '--ref', required=True, help='ref genome fasta, required if normalising windows with -n/--norm_motif')
     parser_region.add_argument('-g', '--gtf', default=None, help='genes or intervals to display in gtf format')
     parser_region.add_argument('-l', '--highlight', default=None, help='format: start-end, (can be chrom:start-end but chrom is ignored) can comma-delimit multiple highlights')
@@ -4698,6 +4720,8 @@ if __name__ == '__main__':
     parser_wgmeth.add_argument('-p', '--procs', default=1, help='multiprocessing')
     parser_wgmeth.add_argument('-c', '--chrom', default=None, help='limit analysis to one chromosome')
     parser_wgmeth.add_argument('-q', '--min_mapq', default=10, help='minimum mapping quality (mapq), default = 10')
+    parser_wgmeth.add_argument('-r', '--ref', default=None, help='reference genome .fa (build .fai index with samtools faidx) (required for mod bams)')
+    parser_wgmeth.add_argument('--motif', default=None, help='expected modification motif (e.g. CG for 5mCpG required for mod bams)')
     parser_wgmeth.add_argument('--max_read_density', default=None, help='filter reads with call density greater >= value, can be helpful in footprinting assays (default=None)')
     parser_wgmeth.add_argument('--dss', default=False, action='store_true', help='output in DSS format (default = bedMethyl)')
     parser_wgmeth.add_argument('--phased', action='store_true', default=False, help='split output into phases (currently just 1,2)')

setup.py

@@ -4,13 +4,13 @@
 
 setup(
     name='methylartist',
-    version='1.2.1',
+    version='1.2.2',
     author='Adam Ewing',
     author_email='adam.ewing@gmail.com',
     description=("Tools for parsing and plotting nanopore methylation data"),
     license='MIT',
     url='https://github.com/adamewing/methylartist',
-    download_url='https://github.com/adamewing/methylartist/archive/refs/tags/1.2.1.tar.gz',
+    download_url='https://github.com/adamewing/methylartist/archive/refs/tags/1.2.2.tar.gz',
     scripts=['methylartist'],
     packages=find_packages(),
     install_requires = [