From ad043e39e20820b1a7a70b5f71b01c3e89a2c53c Mon Sep 17 00:00:00 2001 From: "Mitchell R. Vollger" Date: Wed, 18 Jun 2025 11:50:04 -0700 Subject: [PATCH 1/6] drop filtering from mosdepth, allow it to only be done by the input user controlled input filtering --- workflow/rules/coverages.smk | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflow/rules/coverages.smk b/workflow/rules/coverages.smk index c354c76cc..9a0aa1394 100644 --- a/workflow/rules/coverages.smk +++ b/workflow/rules/coverages.smk @@ -17,7 +17,7 @@ rule genome_bedgraph: DEFAULT_ENV shell: """ - mosdepth -f {input.ref} -t {threads} tmp {input.cram} + mosdepth -F 4 -f {input.ref} -t {threads} tmp {input.cram} bgzip -cd tmp.per-base.bed.gz \ | LC_ALL=C sort --parallel={threads} -k1,1 -k2,2n -k3,3n -k4,4 \ | bgzip -@ {threads} \ From 30018c0a61117e4cdefb4f8fe5bde277579a04ad Mon Sep 17 00:00:00 2001 From: minkinaa Date: Tue, 28 Oct 2025 15:06:15 -0700 Subject: [PATCH 2/6] Allow multiple inputs with different reference files and get chr names from input bam instead of fasta --- pixi.lock | 3434 ++++++++-------------------- pixi.toml | 1 + workflow/Snakefile | 80 +- workflow/envs/env.yaml | 1 + workflow/envs/python.yaml | 2 +- workflow/envs/runner.yaml | 2 +- workflow/rules/apply-model.smk | 5 +- workflow/rules/common.smk | 177 +- workflow/rules/coverages.smk | 14 +- workflow/rules/decorated-reads.smk | 8 +- workflow/rules/fire-peaks.smk | 10 +- workflow/rules/stats.smk | 2 +- workflow/rules/track-hub.smk | 22 +- 13 files changed, 1244 insertions(+), 2514 deletions(-) diff --git a/pixi.lock b/pixi.lock index d798a10b3..9a206a92e 100644 --- a/pixi.lock +++ b/pixi.lock @@ -1,4 +1,4 @@ -version: 5 +version: 6 environments: default: channels: @@ -31,7 +31,7 @@ environments: - 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purls: [] - size: 498900 - timestamp: 1714723303098 -- kind: conda - name: zstd - version: 1.5.6 - build: ha6fb4c9_0 - subdir: linux-64 - url: https://conda.anaconda.org/conda-forge/linux-64/zstd-1.5.6-ha6fb4c9_0.conda - sha256: c558b9cc01d9c1444031bd1ce4b9cff86f9085765f17627a6cd85fc623c8a02b - md5: 4d056880988120e29d75bfff282e0f45 + size: 567578 + timestamp: 1742433379869 +- conda: https://conda.anaconda.org/conda-forge/osx-64/zstd-1.5.7-h8210216_2.conda + sha256: c171c43d0c47eed45085112cb00c8c7d4f0caa5a32d47f2daca727e45fb98dca + md5: cd60a4a5a8d6a476b30d8aa4bb49251a depends: - - libgcc-ng >=12 - - libstdcxx-ng >=12 - - libzlib >=1.2.13,<2.0.0a0 + - __osx >=10.13 + - libzlib >=1.3.1,<2.0a0 license: BSD-3-Clause license_family: BSD - purls: [] - size: 554846 - timestamp: 1714722996770 + size: 485754 + timestamp: 1742433356230 diff --git a/pixi.toml b/pixi.toml index 431ab05e7..b832d9ca0 100644 --- a/pixi.toml +++ b/pixi.toml @@ -65,5 +65,6 @@ snakefmt = "*" ruff = "*" awscli = "2.22.*" taplo = "*" +pysam = "*" [pypi-dependencies] diff --git a/workflow/Snakefile b/workflow/Snakefile index f36e0da1c..0dae5ee70 100644 --- a/workflow/Snakefile +++ b/workflow/Snakefile @@ -32,22 +32,30 @@ if config.get("full-version", False): MAX_THREADS = config.get("max_threads", 4) SORT_THREADS = config.get("sort_threads", 8) +# sample, haplotype, and chromosome wildcard building +#FAI_DF = get_fai_df() +DEFAULT_ENV = config.get("env", "../envs/env.yaml") +MANIFEST = get_manifest() +MIN_FIRE_FDR = config.get("min_fire_fdr", 0.10) + # reference genome and reference regions -REF = get_ref() -FAI = get_fai() -REF_NAME = config["ref_name"] -EXCLUDES = get_excludes() +#REF = get_ref() +#FAI = get_fai() +#REF_NAME = config["ref_name"] +#REF_NAME = get_ref_name() +#EXCLUDES = get_excludes() KEEP_CHRS = config.get("keep_chromosomes", ".*") +#FAI_DF = get_fai_df() # coverage requirements MIN_COVERAGE = config.get("min_coverage", 4) COVERAGE_WITHIN_N_SD = config.get("coverage_within_n_sd", 5) # sample, haplotype, and chromosome wildcard building -FAI_DF = get_fai_df() -DEFAULT_ENV = config.get("env", "../envs/env.yaml") -MANIFEST = get_manifest() -MIN_FIRE_FDR = config.get("min_fire_fdr", 0.10) +#FAI_DF = get_fai_df() +#DEFAULT_ENV = config.get("env", "../envs/env.yaml") +#MANIFEST = get_manifest() +#MIN_FIRE_FDR = config.get("min_fire_fdr", 0.10) # FDR / peak calling thresholds MAX_PEAK_FDR = config.get("max_peak_fdr", 0.05) @@ -109,8 +117,62 @@ if DSA: include: "rules/levio.smk" +## mod manifest to include reference +if "ref" not in MANIFEST.columns: + if "ref" not in config or str(config.get("ref")) == "None": + raise ValueError("FIRE: ref parameter is missing in config.yaml") + else: + temp_ref = config.get("ref") + MANIFEST["ref"] = temp_ref + if "ref_name" not in config or str(config.get("ref_name")) == "None": + raise ValueError("FIRE: ref_name parameter is missing in config.yaml") + else: + temp_ref_name = config.get("ref_name") + MANIFEST["ref_name"] = temp_ref_name + +# make list of all chrs for +chroms_list_joined = "" + +for index, row in MANIFEST.iterrows(): + first_report_var = True + min_contig_length = config.get("min_contig_length", 0) + fai = row['ref'] + ".fai" + fai_df = pd.read_csv(fai, sep="\t", names=["chr", "length", "x", "y", "z"]) + + skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] + if len(skipped_contigs) > 0 and first_report_var: + print( + f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", + file=sys.stderr, + ) + + chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] + chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) + chroms = [chrom for chrom in chroms if "_random" not in chrom] + chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] + + if first_report_var: + first_report_var = False + print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) + + if len(chroms) == 0: + raise ValueError( + f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " + f"Your fai file contains the following chromosomes: {fai_df['chr']}" + ) + temp_chroms_list="|".join(chroms) + if chroms_list_joined == "": + chroms_list_joined = temp_chroms_list + else: + chroms_list_joined = chroms_list_joined + "|" + temp_chroms_list + +unique_chroms_list_joined = "|".join(set(chroms_list_joined.split("|"))) + +#print(unique_chroms_list_joined) + wildcard_constraints: - chrom="|".join(get_chroms()), + #chrom="|".join(get_chroms()), + chrom=unique_chroms_list_joined, call="|".join(["msp", "m6a"]), sm="|".join(MANIFEST.index), types="|".join(types), diff --git a/workflow/envs/env.yaml b/workflow/envs/env.yaml index 07984df4c..eeefa678e 100644 --- a/workflow/envs/env.yaml +++ b/workflow/envs/env.yaml @@ -14,3 +14,4 @@ dependencies: - csvtk - mosdepth==0.3.7 - bioconda::bigtools==0.5.5 + #- pysam==0.22 diff --git a/workflow/envs/python.yaml b/workflow/envs/python.yaml index b72cd8234..7978fbad1 100644 --- a/workflow/envs/python.yaml +++ b/workflow/envs/python.yaml @@ -12,6 +12,6 @@ dependencies: - pip - pip: - polars-lts-cpu[pyarrow]==1.7.1 +#- pysam==0.22 #- tqdm #- numba::numba==0.60 -#- pysam==0.21 diff --git a/workflow/envs/runner.yaml b/workflow/envs/runner.yaml index dddf584ec..39c693dcf 100644 --- a/workflow/envs/runner.yaml +++ b/workflow/envs/runner.yaml @@ -8,4 +8,4 @@ dependencies: #- tqdm #- pip #- pip: -#- pysam==0.22.1 + - pysam==0.22.1 diff --git a/workflow/rules/apply-model.smk b/workflow/rules/apply-model.smk index 8a2a6bf7d..e53b6ec1f 100644 --- a/workflow/rules/apply-model.smk +++ b/workflow/rules/apply-model.smk @@ -4,7 +4,8 @@ rule fire: input: bam=ancient(get_input_bam), - ref=ancient(REF), + ref=ancient(get_ref) + #ref=ancient(REF), output: cram="results/{sm}/{sm}-fire-{v}-filtered.cram", crai="results/{sm}/{sm}-fire-{v}-filtered.cram.crai", @@ -75,7 +76,7 @@ rule fire_sites_chrom: rule fire_sites: input: beds=expand( - rules.fire_sites_chrom.output.bed, chrom=get_chroms(), allow_missing=True + rules.fire_sites_chrom.output.bed, chrom=get_chroms, allow_missing=True ), output: bed="results/{sm}/additional-outputs-{v}/fire-peaks/{sm}-{v}-fire-elements.bed.gz", diff --git a/workflow/rules/common.smk b/workflow/rules/common.smk index 2dec22b79..dd67f0fa2 100644 --- a/workflow/rules/common.smk +++ b/workflow/rules/common.smk @@ -1,11 +1,12 @@ import re import logging import sys +import pysam FIRST_REPORT = True -def get_ref(): +def get_ref_orig(): if "ref" not in config: raise ValueError("FIRE: ref parameter is missing in config.yaml") ref = config["ref"] @@ -13,17 +14,60 @@ def get_ref(): raise ValueError(f"FIRE: reference file {ref} does not exist") return os.path.abspath(ref) +def get_ref_old(wc): + if "ref" in config: + ref = config["ref"] + if "ref" not in config: + if "ref" in MANIFEST.columns: + ref = get_input_ref() + else: + raise ValueError("FIRE: ref parameter is missing in config.yaml and no ref column in manifest") + #ref = config["ref"] + if not os.path.exists(ref): + raise ValueError(f"FIRE: reference file {ref} does not exist") + return os.path.abspath(ref) -def get_fai(): - fai = f"{get_ref()}.fai" +def get_ref(wc): + ref = MANIFEST.loc[wc.sm, "ref"] + if not os.path.exists(ref): + raise ValueError(f"FIRE: reference file {ref} does not exist") + return os.path.abspath(ref) + +def get_ref_name_old(wc): + if "ref_name" in config: + ref_name=config["ref_name"] + else: + if "ref_name" in MANIFEST.columns: + ref_name= get_input_ref_name() + else: + raise ValueError("FIRE: ref_name parameter is missing in config.yaml and no ref_name column in manifest") + #ref=get_ref() + #temp_split=ref.strip().split('/')[-1] + #fa_split=temp_split.split('.fa') + #ref_name = '.fa'.join(fa_split[:-1]) + return(ref_name) + +def get_ref_name(wc): + return MANIFEST.loc[wc.sm, "ref_name"] + +def get_fai_orig(wc): + fai = f"{get_ref(wc)}.fai" if not os.path.exists(fai): raise ValueError(f"FIRE: reference index file {fai} does not exist") return fai +def get_fai(wc): + ref = MANIFEST.loc[wc.sm, "ref"] + #fai= ref + ".fai" + fai = str(ref) + ".fai" + if not os.path.exists(fai): + raise ValueError(f"FIRE: reference index file {fai} does not exist") + return fai -def get_excludes(): +def get_excludes(wc): excludes = config.get("excludes", []) - if REF_NAME == "hg38" or REF_NAME == "GRCh38": + ref_name = get_ref_name(wc) + if ref_name == "hg38" or ref_name == "GRCh38": files = [ "../annotations/hg38.gap.bed.gz", "../annotations/hg38.blacklist.ENCFF356LFX.bed.gz", @@ -33,22 +77,23 @@ def get_excludes(): return excludes -def get_fai_df(): - fai = get_fai() +def get_fai_df(wc): + fai = get_fai(wc) return pd.read_csv(fai, sep="\t", names=["chr", "length", "x", "y", "z"]) -def get_chroms(): +def get_chroms_orig(wc): global FIRST_REPORT min_contig_length = config.get("min_contig_length", 0) - skipped_contigs = FAI_DF["chr"][FAI_DF["length"] < min_contig_length] + fai_df=get_fai_df(wc) + skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] if len(skipped_contigs) > 0 and FIRST_REPORT: print( f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", file=sys.stderr, ) - chroms = FAI_DF["chr"][FAI_DF["length"] >= min_contig_length] + chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) chroms = [chrom for chrom in chroms if "_random" not in chrom] chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] @@ -60,10 +105,115 @@ def get_chroms(): if len(chroms) == 0: raise ValueError( f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " - f"Your fai file contains the following chromosomes: {FAI_DF['chr']}" + f"Your fai file contains the following chromosomes: {fai_df['chr']}" ) return chroms +def get_chroms(wc): + global FIRST_REPORT + min_contig_length = config.get("min_contig_length", 0) + fai_df=get_fai_df(wc) + skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] + if len(skipped_contigs) > 0 and FIRST_REPORT: + print( + f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", + file=sys.stderr, + ) + + bam_chr_list=[] + input_bam_path=get_input_bam(wc) + input_bam = pysam.AlignmentFile(input_bam_path, "rc", threads=MAX_THREADS) + bam_header_dict = input_bam.header.to_dict() + + for line in bam_header_dict['SQ']: + chr_name=line['SN'] + bam_chr_list.append(chr_name) + + input_bam.close() + + chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] + chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) + chroms = [chrom for chrom in chroms if "_random" not in chrom] + chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] + chroms = [chrom for chrom in chroms if chrom in bam_chr_list] + + if FIRST_REPORT: + FIRST_REPORT = False + print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) + + if len(chroms) == 0: + raise ValueError( + f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " + f"Your fai file contains the following chromosomes: {fai_df['chr']}" + ) + return chroms + +def get_chroms_first_element(wc): + global FIRST_REPORT + min_contig_length = config.get("min_contig_length", 0) + fai_df=get_fai_df(wc) + skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] + if len(skipped_contigs) > 0 and FIRST_REPORT: + print( + f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", + file=sys.stderr, + ) + + bam_chr_list=[] + input_bam_path=get_input_bam(wc) + input_bam = pysam.AlignmentFile(input_bam_path, "rc", threads=MAX_THREADS) + bam_header_dict = input_bam.header.to_dict() + + for line in bam_header_dict['SQ']: + chr_name=line['SN'] + bam_chr_list.append(chr_name) + + input_bam.close() + + chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] + chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) + chroms = [chrom for chrom in chroms if "_random" not in chrom] + chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] + chroms = [chrom for chrom in chroms if chrom in bam_chr_list] + + if FIRST_REPORT: + FIRST_REPORT = False + print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) + + if len(chroms) == 0: + raise ValueError( + f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " + f"Your fai file contains the following chromosomes: {fai_df['chr']}" + ) + return chroms[0] + +def get_chroms_first_element_orig(wc): + global FIRST_REPORT + min_contig_length = config.get("min_contig_length", 0) + fai_df=get_fai_df(wc) + skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] + if len(skipped_contigs) > 0 and FIRST_REPORT: + print( + f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", + file=sys.stderr, + ) + + chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] + chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) + chroms = [chrom for chrom in chroms if "_random" not in chrom] + chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] + + if FIRST_REPORT: + FIRST_REPORT = False + print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) + + if len(chroms) == 0: + raise ValueError( + f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " + f"Your fai file contains the following chromosomes: {fai_df['chr']}" + ) + return chroms[0] + def get_manifest(): manifest = config.get("manifest") @@ -80,6 +230,11 @@ def get_manifest(): def get_input_bam(wc): return MANIFEST.loc[wc.sm, "bam"] +def get_input_ref(wc): + return MANIFEST.loc[wc.sm, "ref"] + +def get_input_ref_name(wc): + return MANIFEST.loc[wc.sm, "ref_name"] def get_mem_mb(wildcards, attempt): if attempt < 3: diff --git a/workflow/rules/coverages.smk b/workflow/rules/coverages.smk index 9a0aa1394..a9410305c 100644 --- a/workflow/rules/coverages.smk +++ b/workflow/rules/coverages.smk @@ -3,8 +3,8 @@ # rule genome_bedgraph: input: - ref=ancient(REF), - fai=ancient(FAI), + ref=ancient(get_ref), + fai=ancient(get_fai), cram=rules.fire.output.cram, crai=rules.fire.output.crai, output: @@ -43,7 +43,7 @@ rule coverage: params: coverage_within_n_sd=COVERAGE_WITHIN_N_SD, min_coverage=MIN_COVERAGE, - chroms=get_chroms(), + chroms=get_chroms, script: "../scripts/cov.py" @@ -76,7 +76,7 @@ rule fiber_locations: input: fibers=expand( rules.fiber_locations_chromosome.output.bed, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), bg=rules.genome_bedgraph.output.bg, @@ -119,14 +119,14 @@ rule fiber_locations: rule exclude_from_shuffle: input: filtered=rules.fiber_locations.output.filtered, - fai=ancient(FAI), + fai=ancient(get_fai), output: bed="results/{sm}/additional-outputs-{v}/coverage/exclude-from-shuffles.bed.gz", threads: 4 conda: DEFAULT_ENV params: - exclude=EXCLUDES, + exclude=get_excludes, shell: """ @@ -147,7 +147,7 @@ rule unreliable_coverage_regions: bg=rules.genome_bedgraph.output.bg, minimum=rules.coverage.output.minimum, maximum=rules.coverage.output.maximum, - fai=ancient(FAI), + fai=ancient(get_fai), output: bed="results/{sm}/additional-outputs-{v}/coverage/unreliable-coverage-regions.bed.gz", bed_tbi="results/{sm}/additional-outputs-{v}/coverage/unreliable-coverage-regions.bed.gz.tbi", diff --git a/workflow/rules/decorated-reads.smk b/workflow/rules/decorated-reads.smk index b39eb5f6f..7ec86bbe8 100644 --- a/workflow/rules/decorated-reads.smk +++ b/workflow/rules/decorated-reads.smk @@ -32,10 +32,10 @@ rule decorate_fibers_1: input: bed=expand( rules.decorate_fibers_chromosome.output.bed, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), - fai=ancient(FAI), + fai=ancient(get_fai), output: #bed=temp("temp/{sm}/fiber-calls/fire-fibers.bed.gz"), bb="results/{sm}/trackHub-{v}/bb/fire-fibers.bb", @@ -70,10 +70,10 @@ rule decorate_fibers_2: input: decorated=expand( rules.decorate_fibers_chromosome.output.decorated, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), - fai=ancient(FAI), + fai=ancient(get_fai), output: bb="results/{sm}/trackHub-{v}/bb/fire-fiber-decorators.bb", #bed=temp("temp/{sm}/trackHub-{v}/bb/fire-fiber-decorators.bed.gz"), diff --git a/workflow/rules/fire-peaks.smk b/workflow/rules/fire-peaks.smk index 07d15e0f6..e37e5ca7f 100644 --- a/workflow/rules/fire-peaks.smk +++ b/workflow/rules/fire-peaks.smk @@ -3,7 +3,7 @@ rule filtered_and_shuffled_fiber_locations_chromosome: filtered=rules.fiber_locations.output.filtered, filtered_tbi=rules.fiber_locations.output.filtered_tbi, exclude=rules.exclude_from_shuffle.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: shuffled=temp("temp/{sm}/shuffle/{v}-{chrom}.fiber-locations-shuffled.bed.gz"), threads: 4 @@ -46,7 +46,7 @@ rule shuffled_pileup: input: beds=expand( rules.shuffled_pileup_chromosome.output.bed, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), output: @@ -134,7 +134,7 @@ rule pileup: input: beds=expand( rules.fdr_track_chromosome.output.bed, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), output: @@ -245,7 +245,7 @@ rule fire_peaks: input: beds=expand( rules.fdr_peaks_by_fire_elements_chromosome.output.bed, - chrom=get_chroms(), + chrom=get_chroms, allow_missing=True, ), output: @@ -277,7 +277,7 @@ rule wide_fire_peaks: input: bed=rules.fire_peaks.output.bed, track=rules.pileup.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: bed="results/{sm}/additional-outputs-{v}/fire-peaks/{sm}-fire-{v}-wide-peaks.bed.gz", tbi="results/{sm}/additional-outputs-{v}/fire-peaks/{sm}-fire-{v}-wide-peaks.bed.gz.tbi", diff --git a/workflow/rules/stats.smk b/workflow/rules/stats.smk index 366eca87e..dadcd8495 100644 --- a/workflow/rules/stats.smk +++ b/workflow/rules/stats.smk @@ -4,7 +4,7 @@ rule clustering_vs_null: input: bed=rules.fire_sites.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: tmp=temp("temp/{sm}/tmp.pre.calls.bed"), null=temp("temp/{sm}/null.calls.bed"), diff --git a/workflow/rules/track-hub.smk b/workflow/rules/track-hub.smk index 422fde256..fcd6ed398 100644 --- a/workflow/rules/track-hub.smk +++ b/workflow/rules/track-hub.smk @@ -1,7 +1,7 @@ rule percent_accessible: input: bed=rules.pileup.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: tmp=temp("temp/{sm}/{hp}/{v}-percent.accessible.bed"), bw="results/{sm}/trackHub-{v}/bw/{hp}.percent.accessible.bw", @@ -13,9 +13,15 @@ rule percent_accessible: params: suffix=get_hap_col_suffix, nzooms=NZOOMS, - chrom=get_chroms()[0], + #chroms=get_chroms, + #chrom=get_chroms[0], + #chrom=lambda wc: get_chroms(get_fai(wc))[0], + #chrom=lambda wc: get_chroms(get_fai(wc))[0], + #chrom=lambda wc, input: get_chroms(input.fai)[0] + chrom=get_chroms_first_element shell: """ + bgzip -cd {input.bed} \ | bioawk -tc hdr '$coverage{params.suffix}>0' \ | bioawk -tc hdr \ @@ -40,7 +46,7 @@ rule percent_accessible: rule element_coverages_bw: input: bed=rules.pileup.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: bw="results/{sm}/trackHub-{v}/bw/{hp}.{el_type}.coverage.bw", conda: @@ -62,7 +68,7 @@ rule element_coverages_bw: rule fdr_track_to_bw: input: bed=rules.pileup.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: bw="results/{sm}/trackHub-{v}/bw/{col}.bw", threads: 4 @@ -83,7 +89,7 @@ rule fdr_track_to_bw: rule fire_peaks_bb: input: bed=rules.fire_peaks.output.bed, - fai=ancient(FAI), + fai=ancient(get_fai), output: bb="results/{sm}/trackHub-{v}/bb/fire-peaks.bb", threads: 4 @@ -106,7 +112,7 @@ rule fire_peaks_bb: rule hap_differences_track: input: bed9=rules.hap_differences.output.bed9, - fai=ancient(FAI), + fai=ancient(get_fai), output: bb="results/{sm}/trackHub-{v}/bb/hap_differences.bb", threads: 4 @@ -115,7 +121,7 @@ rule hap_differences_track: conda: DEFAULT_ENV params: - chrom=get_chroms()[0], + chrom=get_chroms_first_element, bed9_as=workflow.source_path("../templates/bed9.as"), shell: """ @@ -141,7 +147,7 @@ rule trackhub: conda: "../envs/python.yaml" params: - ref=REF_NAME, + ref=get_ref, script=workflow.source_path("../scripts/trackhub.py"), description=workflow.source_path("../templates/fire-description.html"), shell: From 34dfa1e09f5ba833ea90ef01b7cbf1d19302e226 Mon Sep 17 00:00:00 2001 From: minkinaa Date: Wed, 29 Oct 2025 12:12:55 -0700 Subject: [PATCH 3/6] added example config files for running a group of samples with different assembly inputs --- config/config_4col.tbl | 3 +++ config/config_4col.yaml | 3 +++ 2 files changed, 6 insertions(+) create mode 100644 config/config_4col.tbl create mode 100644 config/config_4col.yaml diff --git a/config/config_4col.tbl b/config/config_4col.tbl new file mode 100644 index 000000000..a25aade74 --- /dev/null +++ b/config/config_4col.tbl @@ -0,0 +1,3 @@ +sample bam ref ref_name +test test.cram /mmfs1/gscratch/stergachislab/minkinaa/FIRE/fire-test-data/test.fa.gz hg38 +test2 test.cram /mmfs1/gscratch/stergachislab/minkinaa/FIRE/fire-test-data/test.fa.gz hg38 diff --git a/config/config_4col.yaml b/config/config_4col.yaml new file mode 100644 index 000000000..08a5e04ed --- /dev/null +++ b/config/config_4col.yaml @@ -0,0 +1,3 @@ +#ref: +#ref_name: +manifest: test_2row.tbl From 7e5b6bfad6a5d5a73028766875b5e198f1a53ce0 Mon Sep 17 00:00:00 2001 From: minkinaa Date: Wed, 29 Oct 2025 13:00:42 -0700 Subject: [PATCH 4/6] change pixi version to 0.46.0 --- .github/workflows/main.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/.github/workflows/main.yml b/.github/workflows/main.yml index 8af20c95b..ba83a6dc6 100644 --- a/.github/workflows/main.yml +++ b/.github/workflows/main.yml @@ -14,6 +14,6 @@ jobs: - uses: actions/checkout@v4 - uses: prefix-dev/setup-pixi@v0.8.1 with: - pixi-version: v0.37.0 + pixi-version: v0.46.0 cache: true - run: pixi run test From 7582e64ac4d015c87763b60b2377a7a4dcef41b2 Mon Sep 17 00:00:00 2001 From: minkinaa Date: Mon, 10 Nov 2025 09:03:39 -0800 Subject: [PATCH 5/6] removed dependence on .fai for chr names where bam already present and removed commented unused code --- workflow/Snakefile | 12 --- workflow/rules/common.smk | 162 +++++++---------------------------- workflow/rules/track-hub.smk | 5 -- 3 files changed, 31 insertions(+), 148 deletions(-) diff --git a/workflow/Snakefile b/workflow/Snakefile index 0dae5ee70..d310e56f5 100644 --- a/workflow/Snakefile +++ b/workflow/Snakefile @@ -27,35 +27,23 @@ VERSION = ".".join(FULL_VERSION.split(".")[:2]) if config.get("full-version", False): VERSION = FULL_VERSION - # thread options MAX_THREADS = config.get("max_threads", 4) SORT_THREADS = config.get("sort_threads", 8) # sample, haplotype, and chromosome wildcard building -#FAI_DF = get_fai_df() DEFAULT_ENV = config.get("env", "../envs/env.yaml") MANIFEST = get_manifest() MIN_FIRE_FDR = config.get("min_fire_fdr", 0.10) # reference genome and reference regions -#REF = get_ref() -#FAI = get_fai() -#REF_NAME = config["ref_name"] -#REF_NAME = get_ref_name() -#EXCLUDES = get_excludes() KEEP_CHRS = config.get("keep_chromosomes", ".*") -#FAI_DF = get_fai_df() # coverage requirements MIN_COVERAGE = config.get("min_coverage", 4) COVERAGE_WITHIN_N_SD = config.get("coverage_within_n_sd", 5) # sample, haplotype, and chromosome wildcard building -#FAI_DF = get_fai_df() -#DEFAULT_ENV = config.get("env", "../envs/env.yaml") -#MANIFEST = get_manifest() -#MIN_FIRE_FDR = config.get("min_fire_fdr", 0.10) # FDR / peak calling thresholds MAX_PEAK_FDR = config.get("max_peak_fdr", 0.05) diff --git a/workflow/rules/common.smk b/workflow/rules/common.smk index dd67f0fa2..8c61f8eb4 100644 --- a/workflow/rules/common.smk +++ b/workflow/rules/common.smk @@ -5,65 +5,15 @@ import pysam FIRST_REPORT = True - -def get_ref_orig(): - if "ref" not in config: - raise ValueError("FIRE: ref parameter is missing in config.yaml") - ref = config["ref"] - if not os.path.exists(ref): - raise ValueError(f"FIRE: reference file {ref} does not exist") - return os.path.abspath(ref) - -def get_ref_old(wc): - if "ref" in config: - ref = config["ref"] - if "ref" not in config: - if "ref" in MANIFEST.columns: - ref = get_input_ref() - else: - raise ValueError("FIRE: ref parameter is missing in config.yaml and no ref column in manifest") - #ref = config["ref"] - if not os.path.exists(ref): - raise ValueError(f"FIRE: reference file {ref} does not exist") - return os.path.abspath(ref) - def get_ref(wc): ref = MANIFEST.loc[wc.sm, "ref"] if not os.path.exists(ref): raise ValueError(f"FIRE: reference file {ref} does not exist") return os.path.abspath(ref) -def get_ref_name_old(wc): - if "ref_name" in config: - ref_name=config["ref_name"] - else: - if "ref_name" in MANIFEST.columns: - ref_name= get_input_ref_name() - else: - raise ValueError("FIRE: ref_name parameter is missing in config.yaml and no ref_name column in manifest") - #ref=get_ref() - #temp_split=ref.strip().split('/')[-1] - #fa_split=temp_split.split('.fa') - #ref_name = '.fa'.join(fa_split[:-1]) - return(ref_name) - def get_ref_name(wc): return MANIFEST.loc[wc.sm, "ref_name"] -def get_fai_orig(wc): - fai = f"{get_ref(wc)}.fai" - if not os.path.exists(fai): - raise ValueError(f"FIRE: reference index file {fai} does not exist") - return fai - -def get_fai(wc): - ref = MANIFEST.loc[wc.sm, "ref"] - #fai= ref + ".fai" - fai = str(ref) + ".fai" - if not os.path.exists(fai): - raise ValueError(f"FIRE: reference index file {fai} does not exist") - return fai - def get_excludes(wc): excludes = config.get("excludes", []) ref_name = get_ref_name(wc) @@ -76,66 +26,42 @@ def get_excludes(wc): excludes += [workflow.source_path(file) for file in files] return excludes +def get_fai(wc): + ref = get_ref(wc) + fai = f"{ref}.fai" + return fai def get_fai_df(wc): fai = get_fai(wc) return pd.read_csv(fai, sep="\t", names=["chr", "length", "x", "y", "z"]) - -def get_chroms_orig(wc): - global FIRST_REPORT - min_contig_length = config.get("min_contig_length", 0) - fai_df=get_fai_df(wc) - skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] - if len(skipped_contigs) > 0 and FIRST_REPORT: - print( - f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", - file=sys.stderr, - ) - - chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] - chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) - chroms = [chrom for chrom in chroms if "_random" not in chrom] - chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] - - if FIRST_REPORT: - FIRST_REPORT = False - print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) - - if len(chroms) == 0: - raise ValueError( - f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " - f"Your fai file contains the following chromosomes: {fai_df['chr']}" - ) - return chroms - def get_chroms(wc): global FIRST_REPORT min_contig_length = config.get("min_contig_length", 0) - fai_df=get_fai_df(wc) - skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] - if len(skipped_contigs) > 0 and FIRST_REPORT: - print( - f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", - file=sys.stderr, - ) - bam_chr_list=[] + chroms=[] + skipped_contigs=[] input_bam_path=get_input_bam(wc) - input_bam = pysam.AlignmentFile(input_bam_path, "rc", threads=MAX_THREADS) - bam_header_dict = input_bam.header.to_dict() + input_bam = pysam.AlignmentFile(input_bam_path) + name_length=zip(input_bam.references, input_bam.lengths) - for line in bam_header_dict['SQ']: - chr_name=line['SN'] - bam_chr_list.append(chr_name) + for chrom_name, chrom_length in name_length: + if chrom_length >=min_contig_length: + chroms.append(chrom_name) + else: + skipped_contigs.append(chrom_name) input_bam.close() - chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) chroms = [chrom for chrom in chroms if "_random" not in chrom] chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] - chroms = [chrom for chrom in chroms if chrom in bam_chr_list] + + if len(skipped_contigs) > 0 and FIRST_REPORT: + print( + f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", + file=sys.stderr, + ) if FIRST_REPORT: FIRST_REPORT = False @@ -144,65 +70,39 @@ def get_chroms(wc): if len(chroms) == 0: raise ValueError( f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " - f"Your fai file contains the following chromosomes: {fai_df['chr']}" + f"Your bam file contains the following chromosomes: {skipped_contigs}" ) return chroms def get_chroms_first_element(wc): global FIRST_REPORT min_contig_length = config.get("min_contig_length", 0) - fai_df=get_fai_df(wc) - skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] - if len(skipped_contigs) > 0 and FIRST_REPORT: - print( - f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", - file=sys.stderr, - ) - bam_chr_list=[] + chroms=[] + skipped_contigs=[] input_bam_path=get_input_bam(wc) - input_bam = pysam.AlignmentFile(input_bam_path, "rc", threads=MAX_THREADS) - bam_header_dict = input_bam.header.to_dict() + input_bam = pysam.AlignmentFile(input_bam_path) + name_length=zip(input_bam.references, input_bam.lengths) + + for chrom_name, chrom_length in name_length: + if chrom_length >=min_contig_length: + chroms.append(chrom_name) + else: + skipped_contigs.append(chrom_name) - for line in bam_header_dict['SQ']: - chr_name=line['SN'] - bam_chr_list.append(chr_name) input_bam.close() - chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) chroms = [chrom for chrom in chroms if "_random" not in chrom] chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] - chroms = [chrom for chrom in chroms if chrom in bam_chr_list] - - if FIRST_REPORT: - FIRST_REPORT = False - print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) - if len(chroms) == 0: - raise ValueError( - f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " - f"Your fai file contains the following chromosomes: {fai_df['chr']}" - ) - return chroms[0] - -def get_chroms_first_element_orig(wc): - global FIRST_REPORT - min_contig_length = config.get("min_contig_length", 0) - fai_df=get_fai_df(wc) - skipped_contigs = fai_df["chr"][fai_df["length"] < min_contig_length] if len(skipped_contigs) > 0 and FIRST_REPORT: print( f"WARNING: Skipping contigs with length < {min_contig_length:,}: {skipped_contigs}", file=sys.stderr, ) - chroms = fai_df["chr"][fai_df["length"] >= min_contig_length] - chroms = sorted([chrom for chrom in chroms if "chrUn_" not in chrom]) - chroms = [chrom for chrom in chroms if "_random" not in chrom] - chroms = [chrom for chrom in chroms if re.fullmatch(KEEP_CHRS, chrom)] - if FIRST_REPORT: FIRST_REPORT = False print(f"INFO: Using N chromosomes: {len(chroms)}", file=sys.stderr) @@ -210,7 +110,7 @@ def get_chroms_first_element_orig(wc): if len(chroms) == 0: raise ValueError( f"No chromosomes left after filtering. Check your keep_chromosomes parameter in config.yaml. " - f"Your fai file contains the following chromosomes: {fai_df['chr']}" + f"Your bam file contains the following chromosomes: {skipped_contigs}" ) return chroms[0] diff --git a/workflow/rules/track-hub.smk b/workflow/rules/track-hub.smk index fcd6ed398..9c0cb3873 100644 --- a/workflow/rules/track-hub.smk +++ b/workflow/rules/track-hub.smk @@ -13,11 +13,6 @@ rule percent_accessible: params: suffix=get_hap_col_suffix, nzooms=NZOOMS, - #chroms=get_chroms, - #chrom=get_chroms[0], - #chrom=lambda wc: get_chroms(get_fai(wc))[0], - #chrom=lambda wc: get_chroms(get_fai(wc))[0], - #chrom=lambda wc, input: get_chroms(input.fai)[0] chrom=get_chroms_first_element shell: """ From d881fcba1efb1a864c73b88354f0f4db47da9c02 Mon Sep 17 00:00:00 2001 From: minkinaa Date: Mon, 10 Nov 2025 10:40:27 -0800 Subject: [PATCH 6/6] added error message to get_fai --- workflow/rules/common.smk | 2 ++ 1 file changed, 2 insertions(+) diff --git a/workflow/rules/common.smk b/workflow/rules/common.smk index 8c61f8eb4..effe1b85e 100644 --- a/workflow/rules/common.smk +++ b/workflow/rules/common.smk @@ -29,6 +29,8 @@ def get_excludes(wc): def get_fai(wc): ref = get_ref(wc) fai = f"{ref}.fai" + if not os.path.exists(fai): + raise ValueError(f"FIRE: reference index file {fai} does not exist") return fai def get_fai_df(wc):