From b2b768929dfc1317af694ad166b6153f48e3422c Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Wed, 8 Jan 2025 09:18:09 -0800 Subject: [PATCH 01/21] DM-229 Consolidating parsing logic for load_processed functions. Still need to apply to export functions, and need to check that performance is unimpacted. --- dimelo/load_processed.py | 189 ++++++++++++++++++++------------------- 1 file changed, 95 insertions(+), 94 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 1c1e1a8..3e850eb 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -76,6 +76,60 @@ def regions_to_list( return results +def process_pileup_row( + row: str, + parsed_motif: utils.ParsedMotif, + region_start: int, + region_end: int, + region_strand: str, + single_strand: bool, + regions_5to3prime: bool, +) -> tuple[bool, int, int, int]: + """ + Returns: keep_basemod, pileup_coord_relative, modified_in_row, valid_in_row + """ + tabix_fields = row.split("\t") + pileup_basemod = tabix_fields[3] + pileup_strand = tabix_fields[5] + keep_basemod = False + if single_strand and pileup_strand.strip() != region_strand: + # We are on the wrong strand, skip the rest of the steps for this row + return keep_basemod, None, None, None + elif len(pileup_basemod.split(",")) == 3: + pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split(",") + if ( + pileup_motif == parsed_motif.motif_seq + and int(pileup_mod_coord) == parsed_motif.modified_pos + and pileup_modname in parsed_motif.mod_codes + ): + keep_basemod = True + elif len(pileup_basemod.split(",")) == 1: + if pileup_basemod in parsed_motif.mod_codes: + keep_basemod = True + else: + raise ValueError( + f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." + ) + if keep_basemod: + pileup_info = tabix_fields[9].split(" ") + genomic_coord = int(tabix_fields[1]) + if regions_5to3prime and region_strand == "-": + # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction + # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal + pileup_coord_relative = region_end - genomic_coord - 1 + else: + # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case + pileup_coord_relative = genomic_coord - region_start + + valid_in_row = int(pileup_info[0]) + modified_in_row = int(pileup_info[2]) + + return keep_basemod, pileup_coord_relative, modified_in_row, valid_in_row + + else: + return keep_basemod, None, None, None + + def pileup_counts_from_bedmethyl( bedmethyl_file: str | Path, motif: str, @@ -129,64 +183,35 @@ def pileup_counts_from_bedmethyl( # TODO: change to try-except if chromosome in source_tabix.contigs: for row in source_tabix.fetch(chromosome, start_coord, end_coord): - # TODO Consider using csv module - # TODO: probably this whole block should share logic with vectors_from_bedmethyl AND from export module functions - tabix_fields = row.split("\t") - pileup_basemod = tabix_fields[3] - pileup_strand = tabix_fields[5] - keep_basemod = False - if single_strand and pileup_strand != strand: - # This entry is on the wrong strand - skip it - continue - elif len(pileup_basemod.split(",")) == 3: - pileup_modname, pileup_motif, pileup_mod_coord = ( - pileup_basemod.split(",") - ) - if ( - pileup_motif == parsed_motif.motif_seq - and int(pileup_mod_coord) == parsed_motif.modified_pos - and pileup_modname in parsed_motif.mod_codes - ): - keep_basemod = True - elif len(pileup_basemod.split(",")) == 1: - if pileup_basemod in parsed_motif.mod_codes: - keep_basemod = True - else: - raise ValueError( - f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." + keep_basemod, _, modified_in_row, valid_in_row = ( + process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=False, ) - # TODO: consolidate the above into a function; just do adding outside + ) if keep_basemod: - pileup_info = tabix_fields[9].split(" ") - valid_base_count += int(pileup_info[0]) - modified_base_count += int(pileup_info[2]) + valid_base_count += valid_in_row + modified_base_count += modified_in_row else: # Get counts from the whole input file for row in source_tabix.fetch(): - tabix_fields = row.split("\t") - pileup_basemod = tabix_fields[3] - keep_basemod = False - if len(pileup_basemod.split(",")) == 3: - pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split( - "," - ) - if ( - pileup_motif == parsed_motif.motif_seq - and int(pileup_mod_coord) == parsed_motif.modified_pos - and pileup_modname in parsed_motif.mod_codes - ): - keep_basemod = True - elif len(pileup_basemod.split(",")) == 1: - if pileup_basemod in parsed_motif.mod_codes: - keep_basemod = True - else: - raise ValueError( - f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." - ) + keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=False, + ) if keep_basemod: - pileup_info = tabix_fields[9].split(" ") - valid_base_count += int(pileup_info[0]) - modified_base_count += int(pileup_info[2]) + valid_base_count += valid_in_row + modified_base_count += modified_in_row return (modified_base_count, valid_base_count) @@ -267,55 +292,31 @@ def pileup_vectors_from_bedmethyl( # center_coord = (start_coord+end_coord)//2 if chromosome in source_tabix.contigs: for row in source_tabix.fetch(chromosome, start_coord, end_coord): - # TODO: can we consolidate this with pileup_counts_from_bedmethyl? - # Just the checks? - # TODO: probably this whole block should share logic with counts_from_bedmethyl AND from export functions - tabix_fields = row.split("\t") - pileup_basemod = tabix_fields[3] - pileup_strand = tabix_fields[5] - keep_basemod = False - if single_strand and pileup_strand.strip() != strand: - # We are on the wrong strand, skip the rest of the steps for this row - continue - elif len(pileup_basemod.split(",")) == 3: - pileup_modname, pileup_motif, pileup_mod_coord = ( - pileup_basemod.split(",") - ) - if ( - pileup_motif == parsed_motif.motif_seq - and int(pileup_mod_coord) == parsed_motif.modified_pos - and pileup_modname in parsed_motif.mod_codes - ): - keep_basemod = True - elif len(pileup_basemod.split(",")) == 1: - if pileup_basemod in parsed_motif.mod_codes: - keep_basemod = True - else: - raise ValueError( - f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." - ) + ( + keep_basemod, + pileup_coord_relative, + modified_in_row, + valid_in_row, + ) = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=regions_5to3prime, + ) if keep_basemod: - pileup_info = tabix_fields[9].split(" ") - genomic_coord = int(tabix_fields[1]) - if regions_5to3prime and strand == "-": - # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction - # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal - pileup_coord_relative = end_coord - genomic_coord - 1 - else: - # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case - pileup_coord_relative = genomic_coord - start_coord if pileup_coord_relative > region_len: print( f"WARNING: You have specified a region {chromosome}:{start_coord}-{end_coord} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." ) - break else: - valid_base_counts[pileup_coord_relative] += int( - pileup_info[0] - ) - modified_base_counts[pileup_coord_relative] += int( - pileup_info[2] + valid_base_counts[pileup_coord_relative] += valid_in_row + modified_base_counts[pileup_coord_relative] += ( + modified_in_row ) + return modified_base_counts, valid_base_counts From 3f3ec8549c1b66c99cfb8f46f92c7f0e36d03f15 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Wed, 8 Jan 2025 09:30:39 -0800 Subject: [PATCH 02/21] DM-231 Removed regions=None case for pileup_counts_from_bedmethyl because it will complicate parallelization and is not clearly useful. --- dimelo/load_processed.py | 64 +++++++++++++++------------------------- 1 file changed, 23 insertions(+), 41 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 3e850eb..95ee18d 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -133,7 +133,7 @@ def process_pileup_row( def pileup_counts_from_bedmethyl( bedmethyl_file: str | Path, motif: str, - regions: str | Path | list[str | Path] | None = None, + regions: str | Path | list[str | Path], window_size: int | None = None, single_strand: bool = False, cores: int | None = None, # currently unused @@ -172,46 +172,28 @@ def pileup_counts_from_bedmethyl( parsed_motif = utils.ParsedMotif(motif) - if regions is not None: - # Get counts from the specified regions - regions_dict = utils.regions_dict_from_input( - regions, - window_size, - ) - for chromosome, region_list in regions_dict.items(): - for start_coord, end_coord, strand in region_list: - # TODO: change to try-except - if chromosome in source_tabix.contigs: - for row in source_tabix.fetch(chromosome, start_coord, end_coord): - keep_basemod, _, modified_in_row, valid_in_row = ( - process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - regions_5to3prime=False, - ) - ) - if keep_basemod: - valid_base_count += valid_in_row - modified_base_count += modified_in_row - else: - # Get counts from the whole input file - for row in source_tabix.fetch(): - keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - regions_5to3prime=False, - ) - if keep_basemod: - valid_base_count += valid_in_row - modified_base_count += modified_in_row + # Get counts from the specified regions + regions_dict = utils.regions_dict_from_input( + regions, + window_size, + ) + for chromosome, region_list in regions_dict.items(): + for start_coord, end_coord, strand in region_list: + # TODO: change to try-except + if chromosome in source_tabix.contigs: + for row in source_tabix.fetch(chromosome, start_coord, end_coord): + keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=False, + ) + if keep_basemod: + valid_base_count += valid_in_row + modified_base_count += modified_in_row return (modified_base_count, valid_base_count) From 91fb5e4abc317debb1a03716ec47b7f4c483773a Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Wed, 8 Jan 2025 09:40:23 -0800 Subject: [PATCH 03/21] DM-229 Adjusted export.pileup_to_bigwig to use new load_processed.process_pileup_row and renamed/configured a few variables to support this. --- dimelo/export.py | 88 +++++++++++++++++++----------------------------- 1 file changed, 34 insertions(+), 54 deletions(-) diff --git a/dimelo/export.py b/dimelo/export.py index c55010b..d3dabab 100644 --- a/dimelo/export.py +++ b/dimelo/export.py @@ -6,7 +6,7 @@ import pysam from tqdm.auto import tqdm -from . import utils +from . import load_processed, utils """ This module contains code to export indexed and compressed parse output files to other formats that may be helpful for downstream analysis. @@ -57,8 +57,8 @@ def pileup_to_bigwig( # Because we need to set up the bigwig header for we start writing data to it, we need to pre-index the length of each contig tabix = pysam.TabixFile(str(bedmethyl_file)) - contig_lengths_tuples = [] - lines_by_contig = {} + contig_lengths = {} + contig_lines = {} parsed_motif = utils.ParsedMotif(motif) @@ -84,11 +84,11 @@ def pileup_to_bigwig( )[0] fields = last_row.split("\t") max_coord = int(fields[2]) - contig_lengths_tuples.append((contig, max_coord)) - lines_by_contig[contig] = rows_count + contig_lengths[contig] = max_coord + contig_lines[contig] = rows_count with pyBigWig.open(str(output_file_path), "w") as bw: - bw.addHeader(contig_lengths_tuples) + bw.addHeader([(contig, length) for contig, length in contig_lengths.items()]) for contig in tqdm( tabix.contigs, desc=f"Step 2: Writing {Path(bedmethyl_file).name} contents to {Path(output_file_path).name}", @@ -100,57 +100,37 @@ def pileup_to_bigwig( for row in tqdm( tabix.fetch(contig), desc=f"Writing {contig}.", - total=lines_by_contig[contig], + total=contig_lines[contig], leave=False, ): - # TODO: This code is copied from load_processed.pileup_counts_from_bedmethyl and should probably be consolidated at some point - tabix_fields = row.split("\t") - pileup_basemod = tabix_fields[3] - pileup_strand = tabix_fields[5] - keep_basemod = False - if (strand != ".") and (pileup_strand != strand): - # This entry is on the wrong strand - skip it - continue - elif len(pileup_basemod.split(",")) == 3: - pileup_modname, pileup_motif, pileup_mod_coord = ( - pileup_basemod.split(",") + keep_basemod, genomic_coord, modified_in_row, valid_in_row = ( + load_processed.process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=0, + region_end=contig_lengths[contig], + region_strand=strand, + single_strand=(strand != "."), + regions_5to3prime=False, ) - if ( - pileup_motif == parsed_motif.motif_seq - and int(pileup_mod_coord) == parsed_motif.modified_pos - and pileup_modname in parsed_motif.mod_codes - ): - keep_basemod = True - elif len(pileup_basemod.split(",")) == 1: - if pileup_basemod in parsed_motif.mod_codes: - keep_basemod = True - else: - raise ValueError( - f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." - ) - # TODO: consolidate the above into a function; just do adding outside - if keep_basemod: - pileup_info = tabix_fields[9].split(" ") - valid_base_counts = int(pileup_info[0]) - modified_base_counts = int(pileup_info[2]) - if valid_base_counts > 0: - genomic_coord = int(tabix_fields[1]) - contig_list.append(contig) - start_list.append(genomic_coord) - end_list.append(genomic_coord + 1) - values_list.append(modified_base_counts / valid_base_counts) - - if len(values_list) > chunk_size: - bw.addEntries( - contig_list, # Contig names - start_list, # Start positions - ends=end_list, # End positions - values=values_list, # Corresponding values - ) - contig_list = [] - start_list = [] - end_list = [] - values_list = [] + ) + if keep_basemod and valid_in_row > 0: + contig_list.append(contig) + start_list.append(genomic_coord) + end_list.append(genomic_coord + 1) + values_list.append(modified_in_row / valid_in_row) + + if len(values_list) > chunk_size: + bw.addEntries( + contig_list, # Contig names + start_list, # Start positions + ends=end_list, # End positions + values=values_list, # Corresponding values + ) + contig_list = [] + start_list = [] + end_list = [] + values_list = [] bw.addEntries( contig_list, # Contig names start_list, # Start positions From 53d039bcebc71e0bbcfb7dee46f3d23b2cdf43d9 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 10:01:12 -0800 Subject: [PATCH 04/21] DM-192 Implemented parallelization core including shared memory and chunk generation, currently only for load_processed.pileup_vectors_from_bedmethyl. pileup_counts_from_bedmethyl is a simpler case. Passes pre-existing tests including when chunk_size 1: - with ProcessPoolExecutor(max_workers=cores_to_run) as executor: + with concurrent.futures.ProcessPoolExecutor( + max_workers=cores_to_run + ) as executor: # Use functools.partial to pre-fill arguments process_partial = partial( process_region, function_handle=function_handle, cores=1, **kwargs @@ -82,19 +86,25 @@ def process_pileup_row( region_start: int, region_end: int, region_strand: str, - single_strand: bool, - regions_5to3prime: bool, -) -> tuple[bool, int, int, int]: + subregion_start: int | None = None, + subregion_end: int | None = None, + single_strand: bool = False, + regions_5to3prime: bool = False, +) -> tuple[bool, int, int, int, int]: """ Returns: keep_basemod, pileup_coord_relative, modified_in_row, valid_in_row """ + if subregion_start is None: + subregion_start = region_start + if subregion_end is None: + subregion_end = region_end tabix_fields = row.split("\t") pileup_basemod = tabix_fields[3] pileup_strand = tabix_fields[5] keep_basemod = False if single_strand and pileup_strand.strip() != region_strand: # We are on the wrong strand, skip the rest of the steps for this row - return keep_basemod, None, None, None + return keep_basemod, 0, 0, 0, 0 elif len(pileup_basemod.split(",")) == 3: pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split(",") if ( @@ -110,24 +120,28 @@ def process_pileup_row( raise ValueError( f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." ) - if keep_basemod: - pileup_info = tabix_fields[9].split(" ") - genomic_coord = int(tabix_fields[1]) - if regions_5to3prime and region_strand == "-": - # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction - # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal - pileup_coord_relative = region_end - genomic_coord - 1 - else: - # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case - pileup_coord_relative = genomic_coord - region_start - - valid_in_row = int(pileup_info[0]) - modified_in_row = int(pileup_info[2]) - - return keep_basemod, pileup_coord_relative, modified_in_row, valid_in_row + pileup_info = tabix_fields[9].split(" ") + genomic_coord = int(tabix_fields[1]) + if regions_5to3prime and region_strand == "-": + # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction + # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal + pileup_coord_relative = subregion_end - genomic_coord - 1 + subregion_offset = region_end - subregion_end else: - return keep_basemod, None, None, None + # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case + pileup_coord_relative = genomic_coord - subregion_start + subregion_offset = subregion_start - region_start + valid_in_row = int(pileup_info[0]) + modified_in_row = int(pileup_info[2]) + + return ( + keep_basemod, + pileup_coord_relative, + modified_in_row, + valid_in_row, + subregion_offset, + ) def pileup_counts_from_bedmethyl( @@ -177,19 +191,25 @@ def pileup_counts_from_bedmethyl( regions, window_size, ) + + cores_to_run = utils.cores_to_run(cores) + _ = cores_to_run + for chromosome, region_list in regions_dict.items(): for start_coord, end_coord, strand in region_list: # TODO: change to try-except if chromosome in source_tabix.contigs: for row in source_tabix.fetch(chromosome, start_coord, end_coord): - keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - regions_5to3prime=False, + keep_basemod, _, modified_in_row, valid_in_row, _ = ( + process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=False, + ) ) if keep_basemod: valid_base_count += valid_in_row @@ -209,6 +229,76 @@ def counts_from_fake(*args, **kwargs) -> tuple[int, int]: return test_data.fake_peak_enrichment(halfsize=window_halfsize, peak_height=0.15) +def pileup_vectors_process_chunk( + bedmethyl_file, + parsed_motif, + chunk, + region_len, + shm_name_modified, + shm_name_valid, + lock, + single_strand, + regions_5to3prime, +) -> None: + source_tabix = pysam.TabixFile(str(bedmethyl_file)) + existing_valid = shared_memory.SharedMemory(name=shm_name_valid) + existing_modified = shared_memory.SharedMemory(name=shm_name_modified) + valid_base_counts = np.ndarray( + (region_len,), dtype=np.int32, buffer=existing_valid.buf + ) + modified_base_counts = np.ndarray( + (region_len,), dtype=np.int32, buffer=existing_modified.buf + ) + + chromosome = chunk["chromosome"] + region_start = chunk["region_start"] + region_end = chunk["region_end"] + subregion_start = chunk["subregion_start"] + subregion_end = chunk["subregion_end"] + strand = chunk["strand"] + + subregion_offset = 0 + + valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) + modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) + + for row in source_tabix.fetch(chunk["chromosome"], subregion_start, subregion_end): + ( + keep_basemod, + pileup_coord_relative, + modified_in_row, + valid_in_row, + subregion_offset, + ) = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=region_start, + region_end=region_end, + subregion_start=subregion_start, + subregion_end=subregion_end, + region_strand=strand, + single_strand=single_strand, + regions_5to3prime=regions_5to3prime, + ) + if keep_basemod: + if pileup_coord_relative > region_len: + print( + f"WARNING: You have specified a region starting at {chromosome}:{subregion_start - subregion_offset} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." + ) + else: + valid_base_subregion[pileup_coord_relative] += valid_in_row + modified_base_subregion[pileup_coord_relative] += modified_in_row + + with lock: + print(subregion_offset) + valid_base_counts[ + subregion_offset : subregion_offset + abs(subregion_end - subregion_start) + ] += valid_base_subregion + modified_base_counts[ + subregion_offset : subregion_offset + abs(subregion_end - subregion_start) + ] += modified_base_subregion + + def pileup_vectors_from_bedmethyl( bedmethyl_file: str | Path, motif: str, @@ -254,52 +344,65 @@ def pileup_vectors_from_bedmethyl( tuple containing (modified_base_counts, valid_base_counts) """ - source_tabix = pysam.TabixFile(str(bedmethyl_file)) + print(f"function call: {bedmethyl_file}, {regions}, {motif}") parsed_motif = utils.ParsedMotif(motif) regions_dict = utils.regions_dict_from_input(regions, window_size) + chunks_list = utils.process_chunks_from_regions_dict( + regions_dict, chunk_size=10_000 + ) + + print(regions_dict) + print(chunks_list) + + cores_to_run = utils.cores_to_run(cores) # Peek at a region to figure out what size the vectors should be first_key = next(iter(regions_dict)) first_tuple = regions_dict[first_key][0] region_len = first_tuple[1] - first_tuple[0] - valid_base_counts = np.zeros(region_len, dtype=int) - modified_base_counts = np.zeros(region_len, dtype=int) + shm_valid = shared_memory.SharedMemory( + create=True, size=(region_len) * np.dtype(np.int32).itemsize + ) + shm_modified = shared_memory.SharedMemory( + create=True, size=(region_len) * np.dtype(np.int32).itemsize + ) - for chromosome, region_list in regions_dict.items(): - for start_coord, end_coord, strand in region_list: - # TODO: This is not used anywhere; disabling for now - # center_coord = (start_coord+end_coord)//2 - if chromosome in source_tabix.contigs: - for row in source_tabix.fetch(chromosome, start_coord, end_coord): - ( - keep_basemod, - pileup_coord_relative, - modified_in_row, - valid_in_row, - ) = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - regions_5to3prime=regions_5to3prime, - ) - if keep_basemod: - if pileup_coord_relative > region_len: - print( - f"WARNING: You have specified a region {chromosome}:{start_coord}-{end_coord} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." - ) - else: - valid_base_counts[pileup_coord_relative] += valid_in_row - modified_base_counts[pileup_coord_relative] += ( - modified_in_row - ) - - return modified_base_counts, valid_base_counts + manager = multiprocessing.Manager() + lock = manager.Lock() + + with concurrent.futures.ProcessPoolExecutor(max_workers=cores_to_run) as executor: + futures = [ + executor.submit( + pileup_vectors_process_chunk, + bedmethyl_file, + parsed_motif, + chunk, + region_len, + shm_modified.name, + shm_valid.name, + lock, + single_strand, + regions_5to3prime, + ) + for chunk in chunks_list + ] + for future in tqdm( + concurrent.futures.as_completed(futures), total=len(futures) + ): + try: + future.result() + except Exception as err: + raise RuntimeError("Subprocess failed.") from err + + modified_base_counts = np.ndarray( + (region_len,), dtype=np.int32, buffer=shm_modified.buf + ) + valid_base_counts = np.ndarray((region_len,), dtype=np.int32, buffer=shm_valid.buf) + + return np.copy(modified_base_counts), np.copy(valid_base_counts) def vector_from_fake(window_size: int, *args, **kwargs) -> np.ndarray: diff --git a/dimelo/test/dimelo_test.py b/dimelo/test/dimelo_test.py index 787aa83..87ff71e 100644 --- a/dimelo/test/dimelo_test.py +++ b/dimelo/test/dimelo_test.py @@ -317,7 +317,7 @@ class TestLoadProcessed: outputs section of dimelo/test/generate_test_targets.ipynb. """ - def test_unit__regions_list_list( + def test_unit__regions_to_list( self, test_case, kwargs, diff --git a/dimelo/utils.py b/dimelo/utils.py index aa85e55..83e9ce1 100644 --- a/dimelo/utils.py +++ b/dimelo/utils.py @@ -98,6 +98,28 @@ def adjust_threshold( return thresh +def process_chunks_from_regions_dict( + regions_dict: dict, + chunk_size: int = 10_000, +): + chunk_list = [] + for chromosome, region_list in regions_dict.items(): + for start_coord, end_coord, strand in region_list: + for subregion_start in range(start_coord, end_coord, chunk_size): + subregion_end = min(end_coord, subregion_start + chunk_size) + chunk_list.append( + { + "chromosome": chromosome, + "region_start": start_coord, + "region_end": end_coord, + "subregion_start": subregion_start, + "subregion_end": subregion_end, + "strand": strand, + } + ) + return chunk_list + + def regions_dict_from_input( regions: str | Path | list[str | Path] | None = None, window_size: int | None = None, From caf9dcdb988cce84333eef49acef03b5393e24b9 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 10:41:09 -0800 Subject: [PATCH 05/21] DM-232 Added strand information to regions .bed files. Re-generated read_vectors_from_hdf5 test targets to pass tests; other test targets are the same for now. --- dimelo/test/data/ctcf_demo_not_peak.bed | 400 +++++++++--------- dimelo/test/data/ctcf_demo_peak.bed | 200 ++++----- .../test/data/test_targets/test_matrix.pickle | Bin 3144815 -> 3144853 bytes 3 files changed, 300 insertions(+), 300 deletions(-) diff --git a/dimelo/test/data/ctcf_demo_not_peak.bed b/dimelo/test/data/ctcf_demo_not_peak.bed index 920b114..edfe622 100644 --- a/dimelo/test/data/ctcf_demo_not_peak.bed +++ b/dimelo/test/data/ctcf_demo_not_peak.bed @@ -1,200 +1,200 @@ -chr14 44123158 44123308 + 465.63275 . -chr14 44127026 44127176 + 465.63275 . -chr14 44123159 44123308 - 465.63275 . -chr14 44127026 44127175 - 465.63275 . -chr1 114356587 114356736 - 441.00102 . -chr1 114360454 114360603 - 441.00102 . -chr1 114356586 114356736 + 441.00102 . -chr1 114360454 114360604 + 441.00102 . -chrX 9700334 9700484 - 418.31102 . -chrX 9704202 9704352 - 418.31102 . -chr15 54632158 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Adjusted generate_targets.py to properly handle copying over new kwargs from cases.py when re-running some but not all targets: if all targets are re-run, then the old test_matrix.pkl is discarded. If only some are re-run, we need the old test_matrix.pkl, but still need to copy over all the kwargs for all cases that won't be re-run and still need to copy over old results, which will be overridden for re-run targets. --- dimelo/test/cases.py | 10 ++++++++++ .../test/data/test_targets/test_matrix.pickle | Bin 3144853 -> 3144762 bytes dimelo/test/generate_targets.py | 18 ++++++++++++++++-- 3 files changed, 26 insertions(+), 2 deletions(-) diff --git a/dimelo/test/cases.py b/dimelo/test/cases.py index 2a717ec..85366d9 100644 --- a/dimelo/test/cases.py +++ b/dimelo/test/cases.py @@ -31,6 +31,8 @@ "sort_by": ["read_start", "read_name", "motif"], "smooth_window": 1, "title": "megalodon_peaks_190", + "single_strand": False, + "regions_5to3prime": False, }, # outputs dict function:values {}, # populated in subsequent cells @@ -48,6 +50,8 @@ "sort_by": ["read_start", "read_name", "motif"], "smooth_window": 10, "title": "megalodon_single_190", + "single_strand": False, + "regions_5to3prime": False, }, # outputs dict function:values {}, # populated in subsequent cells @@ -65,6 +69,8 @@ "sort_by": ["read_start", "read_name", "motif"], "smooth_window": 100, "title": "megalodon_single_and_peaks_190", + "single_strand": True, + "regions_5to3prime": True, }, # outputs dict function:values {}, # populated in subsequent cells @@ -82,6 +88,8 @@ "sort_by": ["read_start", "read_name", "motif"], "smooth_window": 100, "title": "megalodon_peaks_nothresh", + "single_strand": True, + "regions_5to3prime": False, }, # outputs dict function:values {}, # populated in subsequent cells @@ -99,6 +107,8 @@ "sort_by": ["read_start", "read_name", "motif"], "smooth_window": 1, "title": "megalodon_single_nothresh", + "single_strand": False, + "regions_5to3prime": True, }, # outputs dict function:values {}, # populated in subsequent cells diff --git a/dimelo/test/data/test_targets/test_matrix.pickle b/dimelo/test/data/test_targets/test_matrix.pickle index 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open(RelativePath(args.initial_target_pickle), "rb") as file: old_test_matrix = pickle.load(file) - for key, value in old_test_matrix.items(): + # loop through the old test matrix + for key, (old_kwargs, old_results) in old_test_matrix.items(): + # if the old test case is in the new test matrix, bring over the results + # either the test targets will be regenerated, and the results replaced, or they won't be regenerated, and we'll need the old results if key in test_matrix: - test_matrix[key] = value + new_kwargs = test_matrix[key][0] + # if case will be covered, bring in new kwargs + if args.case_subset is None or key in args.case_subset: + test_matrix[key] = (new_kwargs, old_results) + # if case will not be covered, keep old kwargs + else: + test_matrix[key] = (old_kwargs, old_results) print( f"Running {'all cases' if args.case_subset is None else args.case_subset} through {args.target_subset} to supplement test targets from {args.initial_target_pickle}. Any new tests in cases.py will be added to the test matrix." ) @@ -171,6 +180,11 @@ def main(test_matrix): f"Cannot run subset {args.target_subset} without a pre-existing complete set of generated targets, {args.initial_target_pickle} does not exist. Either specify an --initial-target-pickle that does exist or run without subsetting." ) + print("Generating targets for the following test matrix kwargs") + for test_name, (kwargs, _) in test_matrix.items(): + print(test_name) + print(kwargs) + for subset in args.target_subset: function_name = f"generate_{subset}" globals()[function_name](test_matrix, args.case_subset) From caa3e59a9863e9214f5d15333027dbd0b7a2e37f Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 12:36:21 -0800 Subject: [PATCH 07/21] DM-192 Fixed logic to properly handle single_strand case within process_pileup_row. Passing all tests. --- dimelo/export.py | 2 +- dimelo/load_processed.py | 21 +++++++++------------ 2 files changed, 10 insertions(+), 13 deletions(-) diff --git a/dimelo/export.py b/dimelo/export.py index d3dabab..4256954 100644 --- a/dimelo/export.py +++ b/dimelo/export.py @@ -103,7 +103,7 @@ def pileup_to_bigwig( total=contig_lines[contig], leave=False, ): - keep_basemod, genomic_coord, modified_in_row, valid_in_row = ( + keep_basemod, genomic_coord, modified_in_row, valid_in_row, _ = ( load_processed.process_pileup_row( row=row, parsed_motif=parsed_motif, diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 537345e..e951d10 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -101,10 +101,10 @@ def process_pileup_row( tabix_fields = row.split("\t") pileup_basemod = tabix_fields[3] pileup_strand = tabix_fields[5] - keep_basemod = False + if single_strand and pileup_strand.strip() != region_strand: - # We are on the wrong strand, skip the rest of the steps for this row - return keep_basemod, 0, 0, 0, 0 + # We are on the wrong strand, can't keep this position + keep_basemod = False elif len(pileup_basemod.split(",")) == 3: pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split(",") if ( @@ -113,9 +113,10 @@ def process_pileup_row( and pileup_modname in parsed_motif.mod_codes ): keep_basemod = True + else: + keep_basemod = False elif len(pileup_basemod.split(",")) == 1: - if pileup_basemod in parsed_motif.mod_codes: - keep_basemod = True + keep_basemod = pileup_basemod in parsed_motif.mod_codes else: raise ValueError( f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." @@ -123,6 +124,8 @@ def process_pileup_row( pileup_info = tabix_fields[9].split(" ") genomic_coord = int(tabix_fields[1]) + # TODO: consider moving this logic back outside of process_pileup_row, to avoid unecessary operations for both pileup_counts + # and for long regions that only need the offset value once if regions_5to3prime and region_strand == "-": # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal @@ -290,7 +293,6 @@ def pileup_vectors_process_chunk( modified_base_subregion[pileup_coord_relative] += modified_in_row with lock: - print(subregion_offset) valid_base_counts[ subregion_offset : subregion_offset + abs(subregion_end - subregion_start) ] += valid_base_subregion @@ -349,12 +351,7 @@ def pileup_vectors_from_bedmethyl( parsed_motif = utils.ParsedMotif(motif) regions_dict = utils.regions_dict_from_input(regions, window_size) - chunks_list = utils.process_chunks_from_regions_dict( - regions_dict, chunk_size=10_000 - ) - - print(regions_dict) - print(chunks_list) + chunks_list = utils.process_chunks_from_regions_dict(regions_dict, chunk_size=1_000) cores_to_run = utils.cores_to_run(cores) From ac3657e7aaf4abbe049931b9dbf67255dd8d9bb3 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 13:04:23 -0800 Subject: [PATCH 08/21] DM-192 Further refactor to simplify process_pileup_row and keep regions_5to3prime logic within the pileup_vectors_from_bedmethyl function, which is the only one that actually needs it. Runs faster, still passes all tests. --- dimelo/export.py | 3 +- dimelo/load_processed.py | 86 ++++++++++++++++++---------------------- 2 files changed, 39 insertions(+), 50 deletions(-) diff --git a/dimelo/export.py b/dimelo/export.py index 4256954..c3fd6c1 100644 --- a/dimelo/export.py +++ b/dimelo/export.py @@ -103,7 +103,7 @@ def pileup_to_bigwig( total=contig_lines[contig], leave=False, ): - keep_basemod, genomic_coord, modified_in_row, valid_in_row, _ = ( + keep_basemod, genomic_coord, modified_in_row, valid_in_row = ( load_processed.process_pileup_row( row=row, parsed_motif=parsed_motif, @@ -111,7 +111,6 @@ def pileup_to_bigwig( region_end=contig_lengths[contig], region_strand=strand, single_strand=(strand != "."), - regions_5to3prime=False, ) ) if keep_basemod and valid_in_row > 0: diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index e951d10..e9441c7 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -86,18 +86,11 @@ def process_pileup_row( region_start: int, region_end: int, region_strand: str, - subregion_start: int | None = None, - subregion_end: int | None = None, single_strand: bool = False, - regions_5to3prime: bool = False, -) -> tuple[bool, int, int, int, int]: +) -> tuple[bool, int, int, int]: """ - Returns: keep_basemod, pileup_coord_relative, modified_in_row, valid_in_row + Returns: keep_basemod, genomic_coord, modified_in_row, valid_in_row """ - if subregion_start is None: - subregion_start = region_start - if subregion_end is None: - subregion_end = region_end tabix_fields = row.split("\t") pileup_basemod = tabix_fields[3] pileup_strand = tabix_fields[5] @@ -124,26 +117,14 @@ def process_pileup_row( pileup_info = tabix_fields[9].split(" ") genomic_coord = int(tabix_fields[1]) - # TODO: consider moving this logic back outside of process_pileup_row, to avoid unecessary operations for both pileup_counts - # and for long regions that only need the offset value once - if regions_5to3prime and region_strand == "-": - # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction - # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal - pileup_coord_relative = subregion_end - genomic_coord - 1 - subregion_offset = region_end - subregion_end - else: - # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case - pileup_coord_relative = genomic_coord - subregion_start - subregion_offset = subregion_start - region_start valid_in_row = int(pileup_info[0]) modified_in_row = int(pileup_info[2]) return ( keep_basemod, - pileup_coord_relative, + genomic_coord, modified_in_row, valid_in_row, - subregion_offset, ) @@ -203,16 +184,13 @@ def pileup_counts_from_bedmethyl( # TODO: change to try-except if chromosome in source_tabix.contigs: for row in source_tabix.fetch(chromosome, start_coord, end_coord): - keep_basemod, _, modified_in_row, valid_in_row, _ = ( - process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - regions_5to3prime=False, - ) + keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=start_coord, + region_end=end_coord, + region_strand=strand, + single_strand=single_strand, ) if keep_basemod: valid_base_count += valid_in_row @@ -260,7 +238,17 @@ def pileup_vectors_process_chunk( subregion_end = chunk["subregion_end"] strand = chunk["strand"] - subregion_offset = 0 + flip_coords = regions_5to3prime and strand == "-" + + if flip_coords: + subregion_offset = region_end - subregion_end + else: + subregion_offset = subregion_start - region_start + + if region_end - region_start > region_len: + print( + f"WARNING: You have specified a region at {chromosome}:{region_start}-{region_end} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." + ) valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) @@ -268,29 +256,28 @@ def pileup_vectors_process_chunk( for row in source_tabix.fetch(chunk["chromosome"], subregion_start, subregion_end): ( keep_basemod, - pileup_coord_relative, + genomic_coord, modified_in_row, valid_in_row, - subregion_offset, ) = process_pileup_row( row=row, parsed_motif=parsed_motif, - region_start=region_start, - region_end=region_end, - subregion_start=subregion_start, - subregion_end=subregion_end, + region_start=subregion_start, + region_end=subregion_end, region_strand=strand, single_strand=single_strand, - regions_5to3prime=regions_5to3prime, ) if keep_basemod: - if pileup_coord_relative > region_len: - print( - f"WARNING: You have specified a region starting at {chromosome}:{subregion_start - subregion_offset} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." - ) + if flip_coords: + # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction + # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal + pileup_coord_in_subregion = subregion_end - genomic_coord - 1 else: - valid_base_subregion[pileup_coord_relative] += valid_in_row - modified_base_subregion[pileup_coord_relative] += modified_in_row + # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case + pileup_coord_in_subregion = genomic_coord - subregion_start + if pileup_coord_in_subregion < (subregion_end - subregion_start): + valid_base_subregion[pileup_coord_in_subregion] += valid_in_row + modified_base_subregion[pileup_coord_in_subregion] += modified_in_row with lock: valid_base_counts[ @@ -308,7 +295,8 @@ def pileup_vectors_from_bedmethyl( window_size: int | None = None, single_strand: bool = False, regions_5to3prime: bool = False, - cores: int | None = None, # currently unused + cores: int | None = None, + chunk_size: int = 100, ) -> tuple[np.ndarray, np.ndarray]: """ Extract per-position pileup counts at valid motifs across one or more superimposed regions. @@ -351,7 +339,9 @@ def pileup_vectors_from_bedmethyl( parsed_motif = utils.ParsedMotif(motif) regions_dict = utils.regions_dict_from_input(regions, window_size) - chunks_list = utils.process_chunks_from_regions_dict(regions_dict, chunk_size=1_000) + chunks_list = utils.process_chunks_from_regions_dict( + regions_dict, chunk_size=chunk_size + ) cores_to_run = utils.cores_to_run(cores) From a9648dfa8b1e88154db8c2a87b56c62f4e853460 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 13:08:56 -0800 Subject: [PATCH 09/21] DM-192 Added chunk_size for load_processes parallelization to cases.py, re-ran targets for pileup_counts_from_bedmethyl and pileup_vectors_from_bedmethyl. --- dimelo/test/cases.py | 5 +++++ .../test/data/test_targets/test_matrix.pickle | Bin 3144762 -> 3144835 bytes 2 files changed, 5 insertions(+) diff --git a/dimelo/test/cases.py b/dimelo/test/cases.py index 85366d9..001238c 100644 --- a/dimelo/test/cases.py +++ b/dimelo/test/cases.py @@ -33,6 +33,7 @@ "title": "megalodon_peaks_190", "single_strand": False, "regions_5to3prime": False, + "chunk_size": 1_000_000, }, # outputs dict function:values {}, # populated in subsequent cells @@ -52,6 +53,7 @@ "title": "megalodon_single_190", "single_strand": False, "regions_5to3prime": False, + "chunk_size": 100, }, # outputs dict function:values {}, # populated in subsequent cells @@ -71,6 +73,7 @@ "title": "megalodon_single_and_peaks_190", "single_strand": True, "regions_5to3prime": True, + "chunk_size": 10_000, }, # outputs dict function:values {}, # populated in subsequent cells @@ -90,6 +93,7 @@ "title": "megalodon_peaks_nothresh", "single_strand": True, "regions_5to3prime": False, + "chunk_size": 1000, }, # outputs dict function:values {}, # populated in subsequent cells @@ -109,6 +113,7 @@ "title": "megalodon_single_nothresh", "single_strand": False, "regions_5to3prime": True, + "chunk_size": 100, }, # outputs dict function:values {}, # populated in subsequent cells diff --git a/dimelo/test/data/test_targets/test_matrix.pickle b/dimelo/test/data/test_targets/test_matrix.pickle index f2cd6f3550a117c60cd672ee1a92dfb19c61ffec..b40735983ebb5f6b9f70d35e6d15f6ae2a655c08 100644 GIT binary patch delta 19922 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Added progress bars and quiet toggles for pileup loaders. Set default chunk size to 1 MB after whole-chromosome speed testing. Added option to parallelize within rather than between regions with regions_to_list. --- dimelo/load_processed.py | 200 +++++++++++++++++++++++++---------- dimelo/plot_depth_profile.py | 1 + 2 files changed, 144 insertions(+), 57 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index e9441c7..4da5f64 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -25,7 +25,9 @@ def regions_to_list( function_handle, regions, window_size: int | None = None, + quiet: bool = True, cores: int | None = None, + parallelize_within_regions: bool = False, **kwargs, ): """ @@ -53,29 +55,26 @@ def regions_to_list( cores_to_run = utils.cores_to_run(cores) - if cores_to_run > 1: - with concurrent.futures.ProcessPoolExecutor( - max_workers=cores_to_run - ) as executor: - # Use functools.partial to pre-fill arguments - process_partial = partial( - process_region, function_handle=function_handle, cores=1, **kwargs - ) + with concurrent.futures.ProcessPoolExecutor( + max_workers=1 if parallelize_within_regions else cores_to_run + ) as executor: + # Use functools.partial to pre-fill arguments + process_partial = partial( + process_region, + function_handle=function_handle, + quiet=quiet or not parallelize_within_regions, + cores=cores_to_run if parallelize_within_regions else 1, + **kwargs, + ) - # Use executor.map without lambda - results = list( - tqdm( - executor.map(process_partial, region_tuples), - total=len(region_tuples), - desc=f"Processing regions in parallel across {cores_to_run}", - ) + results = list( + tqdm( + executor.map(process_partial, region_tuples), + total=len(region_tuples), + desc=f"Processing regions in parallel across {cores_to_run}", + disable=quiet or parallelize_within_regions, ) - else: - # Single-threaded fallback - results = [ - process_region(region, function_handle, cores=1, **kwargs) - for region in tqdm(region_tuples, desc="Processing regions") - ] + ) return results @@ -128,13 +127,63 @@ def process_pileup_row( ) +def pileup_counts_process_chunk( + bedmethyl_file, + parsed_motif, + chunk, + shm_name_modified, + shm_name_valid, + lock, + single_strand, +) -> None: + source_tabix = pysam.TabixFile(str(bedmethyl_file)) + existing_valid = shared_memory.SharedMemory(name=shm_name_valid) + existing_modified = shared_memory.SharedMemory(name=shm_name_modified) + valid_base_counts = np.ndarray((1,), dtype=np.int32, buffer=existing_valid.buf) + modified_base_counts = np.ndarray( + (1,), dtype=np.int32, buffer=existing_modified.buf + ) + + chromosome = chunk["chromosome"] + subregion_start = chunk["subregion_start"] + subregion_end = chunk["subregion_end"] + strand = chunk["strand"] + + valid_base_subregion_counts = 0 + modified_base_subregion_counts = 0 + + for row in source_tabix.fetch(chromosome, subregion_start, subregion_end): + ( + keep_basemod, + _, + modified_in_row, + valid_in_row, + ) = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_start=subregion_start, + region_end=subregion_end, + region_strand=strand, + single_strand=single_strand, + ) + if keep_basemod: + valid_base_subregion_counts += valid_in_row + modified_base_subregion_counts += modified_in_row + + with lock: + valid_base_counts[0] += valid_base_subregion_counts + modified_base_counts[0] += modified_base_subregion_counts + + def pileup_counts_from_bedmethyl( bedmethyl_file: str | Path, motif: str, regions: str | Path | list[str | Path], window_size: int | None = None, single_strand: bool = False, - cores: int | None = None, # currently unused + quiet: bool = True, + cores: int | None = None, + chunk_size: int = 1_000_000, ) -> tuple[int, int]: """ Extract number of modified bases and total number of bases from the given bedmethyl file. @@ -157,46 +206,74 @@ def pileup_counts_from_bedmethyl( window_size: (currently disabled) window around center of region, +-window_size single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions - cores: cores across which to parallelize processes (currently unused) + quiet: disables progress bars + cores: cores across which to parallelize processes + chunk_size: size of genomic subregions to assign out to each process Returns: tuple containing counts of (modified_bases, total_bases) """ - source_tabix = pysam.TabixFile(str(bedmethyl_file)) - # Don't need vectors, just need counts; also not guaranteed that windows are the same length - valid_base_count = 0 - modified_base_count = 0 - parsed_motif = utils.ParsedMotif(motif) - # Get counts from the specified regions - regions_dict = utils.regions_dict_from_input( - regions, - window_size, + regions_dict = utils.regions_dict_from_input(regions, window_size) + chunks_list = utils.process_chunks_from_regions_dict( + regions_dict, chunk_size=chunk_size ) cores_to_run = utils.cores_to_run(cores) - _ = cores_to_run - - for chromosome, region_list in regions_dict.items(): - for start_coord, end_coord, strand in region_list: - # TODO: change to try-except - if chromosome in source_tabix.contigs: - for row in source_tabix.fetch(chromosome, start_coord, end_coord): - keep_basemod, _, modified_in_row, valid_in_row = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=start_coord, - region_end=end_coord, - region_strand=strand, - single_strand=single_strand, - ) - if keep_basemod: - valid_base_count += valid_in_row - modified_base_count += modified_in_row - return (modified_base_count, valid_base_count) + shm_valid = shared_memory.SharedMemory( + create=True, size=np.dtype(np.int32).itemsize + ) + shm_modified = shared_memory.SharedMemory( + create=True, size=np.dtype(np.int32).itemsize + ) + + manager = multiprocessing.Manager() + lock = manager.Lock() + + with concurrent.futures.ProcessPoolExecutor(max_workers=cores_to_run) as executor: + futures = [ + executor.submit( + pileup_counts_process_chunk, + bedmethyl_file, + parsed_motif, + chunk, + shm_modified.name, + shm_valid.name, + lock, + single_strand, + ) + for chunk in chunks_list + ] + for future in tqdm( + concurrent.futures.as_completed(futures), + total=len(futures), + disable=quiet, + desc=f"Loading genomic chunks, approx. {chunk_size/1000}kb per chunk", + ): + try: + future.result() + except Exception as err: + raise RuntimeError("Subprocess failed.") from err + + # Directly convert shared memory buffers to integers + modified_base_count = int.from_bytes( + shm_modified.buf[:4], byteorder="little", signed=True + ) + valid_base_count = int.from_bytes( + shm_valid.buf[:4], byteorder="little", signed=True + ) + + return modified_base_count, valid_base_count + + # We need to convert these shared memory buffers to ints that are no longer tied to the buffer, + # so they can be passed up without any seg fault type errors + modified_base_counts = np.ndarray((1,), dtype=np.int32, buffer=shm_modified.buf) + valid_base_counts = np.ndarray((1,), dtype=np.int32, buffer=shm_valid.buf) + + return int(np.copy(modified_base_counts[0])), int(np.copy(valid_base_counts[0])) def counts_from_fake(*args, **kwargs) -> tuple[int, int]: @@ -253,7 +330,7 @@ def pileup_vectors_process_chunk( valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) - for row in source_tabix.fetch(chunk["chromosome"], subregion_start, subregion_end): + for row in source_tabix.fetch(chromosome, subregion_start, subregion_end): ( keep_basemod, genomic_coord, @@ -295,8 +372,9 @@ def pileup_vectors_from_bedmethyl( window_size: int | None = None, single_strand: bool = False, regions_5to3prime: bool = False, + quiet: bool = True, cores: int | None = None, - chunk_size: int = 100, + chunk_size: int = 1_000_000, ) -> tuple[np.ndarray, np.ndarray]: """ Extract per-position pileup counts at valid motifs across one or more superimposed regions. @@ -328,14 +406,14 @@ def pileup_vectors_from_bedmethyl( single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping - cores: cores across which to parallelize processes (currently unused) + quiet: disables progress bars + cores: cores across which to parallelize processes + chunk_size: size of genomic subregions to assign out to each process Returns: tuple containing (modified_base_counts, valid_base_counts) """ - print(f"function call: {bedmethyl_file}, {regions}, {motif}") - parsed_motif = utils.ParsedMotif(motif) regions_dict = utils.regions_dict_from_input(regions, window_size) @@ -377,13 +455,19 @@ def pileup_vectors_from_bedmethyl( for chunk in chunks_list ] for future in tqdm( - concurrent.futures.as_completed(futures), total=len(futures) + concurrent.futures.as_completed(futures), + total=len(futures), + disable=quiet, + desc=f"Loading genomic chunks, approx. {chunk_size/1000}kb per chunk", ): try: future.result() except Exception as err: raise RuntimeError("Subprocess failed.") from err + # We need to convert these shared memory buffers to numpy arrays which + # we then copy, so that they no longer reference the shared memory which + # will soon be de-allocated modified_base_counts = np.ndarray( (region_len,), dtype=np.int32, buffer=shm_modified.buf ) @@ -416,6 +500,7 @@ def read_vectors_from_hdf5( sort_by: str | list[str] = ["chromosome", "region_start", "read_start"], calculate_mod_fractions: bool = True, cores: int | None = None, # currently unused + quiet: bool = True, # currently unused ) -> tuple[list[tuple], list[str], dict | None]: """ Pulls a list of read data out of an .h5 file containing processed read vectors, formatted @@ -458,6 +543,7 @@ def read_vectors_from_hdf5( include chromosome, region_start, region_end, read_start, read_end, and motif. More to be added in future. cores: cores across which to parallelize processes (currently unused) + quiet: silences progress bars (currently unused) Returns: a list of tuples, each tuple containing all datasets corresponding to an individual read that diff --git a/dimelo/plot_depth_profile.py b/dimelo/plot_depth_profile.py index d2d9ef3..e4b3327 100644 --- a/dimelo/plot_depth_profile.py +++ b/dimelo/plot_depth_profile.py @@ -198,6 +198,7 @@ def get_depth_profiles( window_size=window_size, single_strand=single_strand, regions_5to3prime=regions_5to3prime, + quiet=False, ) trace = valid_base_counts.astype(float) trace[trace == 0] = np.nan From 56e6333d8c51b09496e9c13112af488a9e242039 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 20:25:59 -0800 Subject: [PATCH 11/21] DM-192,DM-236 Properly close and delink shared memory when appropriate to avoid intermittent crashes. Adjust tabix fetch to avoid negative start values. Tweak wording on progress bars. --- dimelo/load_processed.py | 41 +++++++++++++++++++++++++--------------- 1 file changed, 26 insertions(+), 15 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 4da5f64..cb0b018 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -152,7 +152,7 @@ def pileup_counts_process_chunk( valid_base_subregion_counts = 0 modified_base_subregion_counts = 0 - for row in source_tabix.fetch(chromosome, subregion_start, subregion_end): + for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): ( keep_basemod, _, @@ -173,6 +173,9 @@ def pileup_counts_process_chunk( with lock: valid_base_counts[0] += valid_base_subregion_counts modified_base_counts[0] += modified_base_subregion_counts + # Close the file descriptor/handle to the shared memory + existing_valid.close() + existing_modified.close() def pileup_counts_from_bedmethyl( @@ -251,7 +254,7 @@ def pileup_counts_from_bedmethyl( concurrent.futures.as_completed(futures), total=len(futures), disable=quiet, - desc=f"Loading genomic chunks, approx. {chunk_size/1000}kb per chunk", + desc=f"Loading genomic chunks, up to {chunk_size/1000}kb per chunk", ): try: future.result() @@ -265,16 +268,14 @@ def pileup_counts_from_bedmethyl( valid_base_count = int.from_bytes( shm_valid.buf[:4], byteorder="little", signed=True ) + # Close and unlink shared memory - not fully handled by garbage collection otherwise + shm_modified.close() + shm_modified.unlink() + shm_valid.close() + shm_valid.unlink() return modified_base_count, valid_base_count - # We need to convert these shared memory buffers to ints that are no longer tied to the buffer, - # so they can be passed up without any seg fault type errors - modified_base_counts = np.ndarray((1,), dtype=np.int32, buffer=shm_modified.buf) - valid_base_counts = np.ndarray((1,), dtype=np.int32, buffer=shm_valid.buf) - - return int(np.copy(modified_base_counts[0])), int(np.copy(valid_base_counts[0])) - def counts_from_fake(*args, **kwargs) -> tuple[int, int]: """ @@ -330,7 +331,7 @@ def pileup_vectors_process_chunk( valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) - for row in source_tabix.fetch(chromosome, subregion_start, subregion_end): + for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): ( keep_basemod, genomic_coord, @@ -363,6 +364,9 @@ def pileup_vectors_process_chunk( modified_base_counts[ subregion_offset : subregion_offset + abs(subregion_end - subregion_start) ] += modified_base_subregion + # Close the file descriptor/handle to the shared memory + existing_modified.close() + existing_valid.close() def pileup_vectors_from_bedmethyl( @@ -458,7 +462,7 @@ def pileup_vectors_from_bedmethyl( concurrent.futures.as_completed(futures), total=len(futures), disable=quiet, - desc=f"Loading genomic chunks, approx. {chunk_size/1000}kb per chunk", + desc=f"Loading genomic chunks, up to {chunk_size/1000}kb per chunk", ): try: future.result() @@ -468,12 +472,19 @@ def pileup_vectors_from_bedmethyl( # We need to convert these shared memory buffers to numpy arrays which # we then copy, so that they no longer reference the shared memory which # will soon be de-allocated - modified_base_counts = np.ndarray( - (region_len,), dtype=np.int32, buffer=shm_modified.buf + modified_base_counts = np.copy( + np.ndarray((region_len,), dtype=np.int32, buffer=shm_modified.buf) + ) + valid_base_counts = np.copy( + np.ndarray((region_len,), dtype=np.int32, buffer=shm_valid.buf) ) - valid_base_counts = np.ndarray((region_len,), dtype=np.int32, buffer=shm_valid.buf) + # Close and unlink shared memory - not fully handled by garbage collection otherwise + shm_modified.close() + shm_modified.unlink() + shm_valid.close() + shm_valid.unlink() - return np.copy(modified_base_counts), np.copy(valid_base_counts) + return modified_base_counts, valid_base_counts def vector_from_fake(window_size: int, *args, **kwargs) -> np.ndarray: From 97efec1bd44e5e0baac70e63f83191814f5c4be3 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Thu, 9 Jan 2025 21:08:40 -0800 Subject: [PATCH 12/21] DM-191 Re-arrange contents of load_processed for better clarity. Added some comments. --- dimelo/export.py | 2 - dimelo/load_processed.py | 483 +++++++++++++++++++++++---------------- 2 files changed, 291 insertions(+), 194 deletions(-) diff --git a/dimelo/export.py b/dimelo/export.py index c3fd6c1..ce64027 100644 --- a/dimelo/export.py +++ b/dimelo/export.py @@ -107,8 +107,6 @@ def pileup_to_bigwig( load_processed.process_pileup_row( row=row, parsed_motif=parsed_motif, - region_start=0, - region_end=contig_lengths[contig], region_strand=strand, single_strand=(strand != "."), ) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index cb0b018..509cc4b 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -14,11 +14,9 @@ from . import test_data, utils - -def process_region(region_tuple, function_handle, **kwargs): - chromosome, start_coord, end_coord, strand = region_tuple - single_region_str = f"{chromosome}:{start_coord}-{end_coord},{strand}" - return function_handle(regions=single_region_str, **kwargs) +################################################################################################################ +#### Loader wrappers #### +################################################################################################################ def regions_to_list( @@ -31,6 +29,8 @@ def regions_to_list( **kwargs, ): """ + User-facing function. + Run any standard load_processed pileup or extract loader loading each region from the region specifier into a new element of a list. @@ -40,6 +40,9 @@ def regions_to_list( window_size: window around centers of regions, defaults to None cores: process count across which to parallelize. Each individual region will only ever get one core. **kwargs: all necessary keyword arguments to pass down to the loader + + Returns: + list([function_handle returns for region in regions]) """ regions_dict = utils.regions_dict_from_input( regions, @@ -79,103 +82,26 @@ def regions_to_list( return results -def process_pileup_row( - row: str, - parsed_motif: utils.ParsedMotif, - region_start: int, - region_end: int, - region_strand: str, - single_strand: bool = False, -) -> tuple[bool, int, int, int]: - """ - Returns: keep_basemod, genomic_coord, modified_in_row, valid_in_row +def process_region(region_tuple, function_handle, **kwargs): """ - tabix_fields = row.split("\t") - pileup_basemod = tabix_fields[3] - pileup_strand = tabix_fields[5] - - if single_strand and pileup_strand.strip() != region_strand: - # We are on the wrong strand, can't keep this position - keep_basemod = False - elif len(pileup_basemod.split(",")) == 3: - pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split(",") - if ( - pileup_motif == parsed_motif.motif_seq - and int(pileup_mod_coord) == parsed_motif.modified_pos - and pileup_modname in parsed_motif.mod_codes - ): - keep_basemod = True - else: - keep_basemod = False - elif len(pileup_basemod.split(",")) == 1: - keep_basemod = pileup_basemod in parsed_motif.mod_codes - else: - raise ValueError( - f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." - ) - - pileup_info = tabix_fields[9].split(" ") - genomic_coord = int(tabix_fields[1]) - valid_in_row = int(pileup_info[0]) - modified_in_row = int(pileup_info[2]) - - return ( - keep_basemod, - genomic_coord, - modified_in_row, - valid_in_row, - ) + Helper function for regions_to_list. Takes in the region tuple, creates a string, and runs the loader function. + Args: + region_tuple: chromosome, start, end, strand + function_handle: a function that takes a regions specifier and loads processed data + **kwargs: the remaining kwargs necessary to run the function in question -def pileup_counts_process_chunk( - bedmethyl_file, - parsed_motif, - chunk, - shm_name_modified, - shm_name_valid, - lock, - single_strand, -) -> None: - source_tabix = pysam.TabixFile(str(bedmethyl_file)) - existing_valid = shared_memory.SharedMemory(name=shm_name_valid) - existing_modified = shared_memory.SharedMemory(name=shm_name_modified) - valid_base_counts = np.ndarray((1,), dtype=np.int32, buffer=existing_valid.buf) - modified_base_counts = np.ndarray( - (1,), dtype=np.int32, buffer=existing_modified.buf - ) - - chromosome = chunk["chromosome"] - subregion_start = chunk["subregion_start"] - subregion_end = chunk["subregion_end"] - strand = chunk["strand"] - - valid_base_subregion_counts = 0 - modified_base_subregion_counts = 0 + Returns: + Whatever function_handle returns + """ + chromosome, start_coord, end_coord, strand = region_tuple + single_region_str = f"{chromosome}:{start_coord}-{end_coord},{strand}" + return function_handle(regions=single_region_str, **kwargs) - for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): - ( - keep_basemod, - _, - modified_in_row, - valid_in_row, - ) = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=subregion_start, - region_end=subregion_end, - region_strand=strand, - single_strand=single_strand, - ) - if keep_basemod: - valid_base_subregion_counts += valid_in_row - modified_base_subregion_counts += modified_in_row - with lock: - valid_base_counts[0] += valid_base_subregion_counts - modified_base_counts[0] += modified_base_subregion_counts - # Close the file descriptor/handle to the shared memory - existing_valid.close() - existing_modified.close() +################################################################################################################ +#### Pileup loaders #### +################################################################################################################ def pileup_counts_from_bedmethyl( @@ -189,6 +115,8 @@ def pileup_counts_from_bedmethyl( chunk_size: int = 1_000_000, ) -> tuple[int, int]: """ + User-facing function. + Extract number of modified bases and total number of bases from the given bedmethyl file. Called by plotters or by the user. @@ -259,7 +187,7 @@ def pileup_counts_from_bedmethyl( try: future.result() except Exception as err: - raise RuntimeError("Subprocess failed.") from err + raise RuntimeError("pileup_counts_process_chunk failed.") from err # Directly convert shared memory buffers to integers modified_base_count = int.from_bytes( @@ -277,98 +205,6 @@ def pileup_counts_from_bedmethyl( return modified_base_count, valid_base_count -def counts_from_fake(*args, **kwargs) -> tuple[int, int]: - """ - Generates a fake set of enrichment counts. Ignores all arguments. - - Returns: - tuple containing counts of (modified_bases, total_bases) - """ - window_halfsize = 500 - return test_data.fake_peak_enrichment(halfsize=window_halfsize, peak_height=0.15) - - -def pileup_vectors_process_chunk( - bedmethyl_file, - parsed_motif, - chunk, - region_len, - shm_name_modified, - shm_name_valid, - lock, - single_strand, - regions_5to3prime, -) -> None: - source_tabix = pysam.TabixFile(str(bedmethyl_file)) - existing_valid = shared_memory.SharedMemory(name=shm_name_valid) - existing_modified = shared_memory.SharedMemory(name=shm_name_modified) - valid_base_counts = np.ndarray( - (region_len,), dtype=np.int32, buffer=existing_valid.buf - ) - modified_base_counts = np.ndarray( - (region_len,), dtype=np.int32, buffer=existing_modified.buf - ) - - chromosome = chunk["chromosome"] - region_start = chunk["region_start"] - region_end = chunk["region_end"] - subregion_start = chunk["subregion_start"] - subregion_end = chunk["subregion_end"] - strand = chunk["strand"] - - flip_coords = regions_5to3prime and strand == "-" - - if flip_coords: - subregion_offset = region_end - subregion_end - else: - subregion_offset = subregion_start - region_start - - if region_end - region_start > region_len: - print( - f"WARNING: You have specified a region at {chromosome}:{region_start}-{region_end} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." - ) - - valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) - modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) - - for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): - ( - keep_basemod, - genomic_coord, - modified_in_row, - valid_in_row, - ) = process_pileup_row( - row=row, - parsed_motif=parsed_motif, - region_start=subregion_start, - region_end=subregion_end, - region_strand=strand, - single_strand=single_strand, - ) - if keep_basemod: - if flip_coords: - # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction - # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal - pileup_coord_in_subregion = subregion_end - genomic_coord - 1 - else: - # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case - pileup_coord_in_subregion = genomic_coord - subregion_start - if pileup_coord_in_subregion < (subregion_end - subregion_start): - valid_base_subregion[pileup_coord_in_subregion] += valid_in_row - modified_base_subregion[pileup_coord_in_subregion] += modified_in_row - - with lock: - valid_base_counts[ - subregion_offset : subregion_offset + abs(subregion_end - subregion_start) - ] += valid_base_subregion - modified_base_counts[ - subregion_offset : subregion_offset + abs(subregion_end - subregion_start) - ] += modified_base_subregion - # Close the file descriptor/handle to the shared memory - existing_modified.close() - existing_valid.close() - - def pileup_vectors_from_bedmethyl( bedmethyl_file: str | Path, motif: str, @@ -381,6 +217,8 @@ def pileup_vectors_from_bedmethyl( chunk_size: int = 1_000_000, ) -> tuple[np.ndarray, np.ndarray]: """ + User-facing function. + Extract per-position pileup counts at valid motifs across one or more superimposed regions. Called by profile plotters, can also be used by a user directly. @@ -467,7 +305,7 @@ def pileup_vectors_from_bedmethyl( try: future.result() except Exception as err: - raise RuntimeError("Subprocess failed.") from err + raise RuntimeError("pileup_vectors_process_chunk failed.") from err # We need to convert these shared memory buffers to numpy arrays which # we then copy, so that they no longer reference the shared memory which @@ -487,8 +325,23 @@ def pileup_vectors_from_bedmethyl( return modified_base_counts, valid_base_counts +def counts_from_fake(*args, **kwargs) -> tuple[int, int]: + """ + Test helper function. + + Generates a fake set of enrichment counts. Ignores all arguments. + + Returns: + tuple containing counts of (modified_bases, total_bases) + """ + window_halfsize = 500 + return test_data.fake_peak_enrichment(halfsize=window_halfsize, peak_height=0.15) + + def vector_from_fake(window_size: int, *args, **kwargs) -> np.ndarray: """ + Tes helper function. + Generates a fake peak trace. Ignores all arguments except window_size. Args: @@ -502,6 +355,233 @@ def vector_from_fake(window_size: int, *args, **kwargs) -> np.ndarray: ) +def pileup_vectors_process_chunk( + bedmethyl_file, + parsed_motif, + chunk, + region_len, + shm_name_modified, + shm_name_valid, + lock, + single_strand, + regions_5to3prime, +) -> None: + """ + Helper function to allow pileup_vectors_from_bedmethyl to operate in a parallized fashion. + + Sum up modified and valid counts for a subregion chunk in a bedmethyl file. + + Args: + bedmethyl_file: Path to bedmethyl file + parsed_motif: ParsedMotif object + chunk: a dict containing subregion chunk information + shm_name_modified: the name string for the shared memory location containing the modified counts array + shm_name_valid: the name string for the shared memory location containing the valid counts array + lock: a manager.Lock object to allow synchronization in accessing shared memory + single_strand: True if only single-strand mods are desired + + Returns: + None. Counts are added in-place to shared memory. + """ + source_tabix = pysam.TabixFile(str(bedmethyl_file)) + existing_valid = shared_memory.SharedMemory(name=shm_name_valid) + existing_modified = shared_memory.SharedMemory(name=shm_name_modified) + valid_base_counts = np.ndarray( + (region_len,), dtype=np.int32, buffer=existing_valid.buf + ) + modified_base_counts = np.ndarray( + (region_len,), dtype=np.int32, buffer=existing_modified.buf + ) + + chromosome = chunk["chromosome"] + region_start = chunk["region_start"] + region_end = chunk["region_end"] + subregion_start = chunk["subregion_start"] + subregion_end = chunk["subregion_end"] + strand = chunk["strand"] + + flip_coords = regions_5to3prime and strand == "-" + + if flip_coords: + subregion_offset = region_end - subregion_end + else: + subregion_offset = subregion_start - region_start + + if region_end - region_start > region_len: + print( + f"WARNING: You have specified a region at {chromosome}:{region_start}-{region_end} that is longer than the first region; the end of the region will be skipped. To make a profile plot with differently-sized region, consider using the window_size parameter to make a profile across centered windows." + ) + + valid_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) + modified_base_subregion = np.zeros(subregion_end - subregion_start, dtype=int) + + for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): + ( + keep_basemod, + genomic_coord, + modified_in_row, + valid_in_row, + ) = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_strand=strand, + single_strand=single_strand, + ) + if keep_basemod: + if flip_coords: + # We want to flip the coordinates for this region so that it is recorded along the 5 prime to 3 prime direction + # This will enable analyses where the orientation of protein binding / transcriptional dynamics / etc is relevant for our pileup signal + pileup_coord_in_subregion = subregion_end - genomic_coord - 1 + else: + # Normal coordinates are the default. This will be used both for the '+' case and the '.' (no strand specified) case + pileup_coord_in_subregion = genomic_coord - subregion_start + if pileup_coord_in_subregion < (subregion_end - subregion_start): + valid_base_subregion[pileup_coord_in_subregion] += valid_in_row + modified_base_subregion[pileup_coord_in_subregion] += modified_in_row + + with lock: + valid_base_counts[ + subregion_offset : subregion_offset + abs(subregion_end - subregion_start) + ] += valid_base_subregion + modified_base_counts[ + subregion_offset : subregion_offset + abs(subregion_end - subregion_start) + ] += modified_base_subregion + # Close the file descriptor/handle to the shared memory + existing_modified.close() + existing_valid.close() + + +def pileup_counts_process_chunk( + bedmethyl_file, + parsed_motif, + chunk, + shm_name_modified, + shm_name_valid, + lock, + single_strand, +) -> None: + """ + Helper function to allow pileup_counts_from_bedmethyl to operate in a parallized fashion. + + Sum up modified and valid counts for a subregion chunk in a bedmethyl file. + + Args: + bedmethyl_file: Path to bedmethyl file + parsed_motif: ParsedMotif object + chunk: a dict containing subregion chunk information + shm_name_modified: the name string for the shared memory location containing the modified counts sum + shm_name_valid: the name string for the shared memory location containing the valid counts sum + lock: a manager.Lock object to allow synchronization in accessing shared memory + single_strand: True if only single-strand mods are desired + + Returns: + None. Counts are added in-place to shared memory. + """ + source_tabix = pysam.TabixFile(str(bedmethyl_file)) + existing_valid = shared_memory.SharedMemory(name=shm_name_valid) + existing_modified = shared_memory.SharedMemory(name=shm_name_modified) + valid_base_counts = np.ndarray((1,), dtype=np.int32, buffer=existing_valid.buf) + modified_base_counts = np.ndarray( + (1,), dtype=np.int32, buffer=existing_modified.buf + ) + + chromosome = chunk["chromosome"] + subregion_start = chunk["subregion_start"] + subregion_end = chunk["subregion_end"] + strand = chunk["strand"] + + valid_base_subregion_counts = 0 + modified_base_subregion_counts = 0 + + for row in source_tabix.fetch(chromosome, max(subregion_start, 0), subregion_end): + ( + keep_basemod, + _, + modified_in_row, + valid_in_row, + ) = process_pileup_row( + row=row, + parsed_motif=parsed_motif, + region_strand=strand, + single_strand=single_strand, + ) + if keep_basemod: + valid_base_subregion_counts += valid_in_row + modified_base_subregion_counts += modified_in_row + + with lock: + valid_base_counts[0] += valid_base_subregion_counts + modified_base_counts[0] += modified_base_subregion_counts + + # Close the file descriptor/handle to the shared memory + existing_valid.close() + existing_modified.close() + + +def process_pileup_row( + row: str, + parsed_motif: utils.ParsedMotif, + region_strand: str, + single_strand: bool = False, +) -> tuple[bool, int, int, int]: + """ + Helper function designed for pileup_counts_from_bedmethyl via pileup_counts_process_chunk, pileup_vectors_from_bedmethyl + via pileup_vectors_process_chunk, and export.pileup_to_bigwig; changed to logic here may impact some or all of + these. + + Process a row from a pileup, determining whether the basemod is relevant and passing back its coordinate, + modification count, and valid read count. + + Args: + row: a string row from a bedmethyl file + parsed_motif: a ParsedMotif object + region_strand: the strand from the query region + single_strand: True if only mods on the region_strand are to be kept + + Returns: keep_basemod, genomic_coord, modified_in_row, valid_in_row + """ + tabix_fields = row.split("\t") + pileup_basemod = tabix_fields[3] + pileup_strand = tabix_fields[5] + + if single_strand and pileup_strand.strip() != region_strand: + # We are on the wrong strand, can't keep this position + keep_basemod = False + elif len(pileup_basemod.split(",")) == 3: + pileup_modname, pileup_motif, pileup_mod_coord = pileup_basemod.split(",") + if ( + pileup_motif == parsed_motif.motif_seq + and int(pileup_mod_coord) == parsed_motif.modified_pos + and pileup_modname in parsed_motif.mod_codes + ): + keep_basemod = True + else: + keep_basemod = False + elif len(pileup_basemod.split(",")) == 1: + keep_basemod = pileup_basemod in parsed_motif.mod_codes + else: + raise ValueError( + f"Unexpected format in bedmethyl file: {row} contains {pileup_basemod} which cannot be parsed." + ) + + pileup_info = tabix_fields[9].split(" ") + genomic_coord = int(tabix_fields[1]) + valid_in_row = int(pileup_info[0]) + modified_in_row = int(pileup_info[2]) + + return ( + keep_basemod, + genomic_coord, + modified_in_row, + valid_in_row, + ) + + +################################################################################################################ +#### Single read loaders #### +################################################################################################################ + + def read_vectors_from_hdf5( file: str | Path, motifs: list[str], @@ -514,6 +594,8 @@ def read_vectors_from_hdf5( quiet: bool = True, # currently unused ) -> tuple[list[tuple], list[str], dict | None]: """ + User-facing function. + Pulls a list of read data out of an .h5 file containing processed read vectors, formatted for read-by-read vector processing downstream use cases. @@ -747,6 +829,8 @@ def readwise_binary_modification_arrays( cores: int | None = None, # currently unused ) -> tuple[list[np.ndarray], np.ndarray[int], np.ndarray[str], dict | None]: """ + Primarily designed as a helper function for single-read plotting, but can be used by a user. + Pulls a list of read data out of a file containing processed read vectors, formatted with seaborn plotting in mind. Currently we only support .h5 files. @@ -900,6 +984,8 @@ def reads_from_fake( file: Path, regions: Path, motifs: list[str] ) -> tuple[list[np.ndarray], np.ndarray[int], np.ndarray[str], dict]: """ + Helper function to support testing. + TODO: What does the bed file represent in this method? This one is breaking my brain a bit. TODO: Variable names in this method stink. TODO: Currently assumes mod calling (thresholding probabilities) was already performed elsewhere @@ -952,17 +1038,27 @@ def reads_from_fake( def convert_bytes_to_strings(tup): - """Convert all bytes elements in a tuple to strings.""" + """ + Helper function for single read loading. + Convert all bytes elements in a tuple to strings. + """ return tuple(item.decode() if isinstance(item, bytes) else item for item in tup) # tuple(convert_bytes(item) for item in tup) def adjust_mod_probs_in_arrays(mod_array, val_array): + """ + Helper function to correct for an idiosyncracy in modkit single-read parsing wherein 0-255 + "mod quality" values are parsed as floating-point values from 1/512 to 511/512. + """ mod_array[np.flatnonzero(val_array)] += 1 / 512 return mod_array def adjust_mod_probs_in_tuples(tup, mod_idx, val_idx): + """ + Helper function to apply mod prob adjustments + """ return tuple( item if index != mod_idx else adjust_mod_probs_in_arrays(item, tup[val_idx]) for index, item in enumerate(tup) @@ -970,6 +1066,9 @@ def adjust_mod_probs_in_tuples(tup, mod_idx, val_idx): def binary_to_np_array(compressed_bytes, dtype, decompressor, binarized, int8tofloat): + """ + Helper function to decompress binary data to boolean or floating point arrays + """ if binarized: return np.frombuffer(decompressor(compressed_bytes), dtype=dtype).astype(bool) elif int8tofloat: From a8ed1ae5b598dc4b6dad87d92549375109151204 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 17 Jan 2025 12:09:33 -0800 Subject: [PATCH 13/21] DM-238 Add quiet and cores parameters and corresponding documentation to all pileup plotters. A few other small tweaks. --- dimelo/load_processed.py | 44 ++++++++++++++++++++++--------- dimelo/plot_depth_histogram.py | 12 +++++++-- dimelo/plot_depth_profile.py | 13 ++++++++- dimelo/plot_enrichment.py | 10 +++++++ dimelo/plot_enrichment_profile.py | 10 +++++++ 5 files changed, 73 insertions(+), 16 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 509cc4b..43ebef7 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -38,7 +38,11 @@ def regions_to_list( function_handle: the loader function you want to run. regions: the region specifier window_size: window around centers of regions, defaults to None - cores: process count across which to parallelize. Each individual region will only ever get one core. + quiet: disables progress bars + cores: CPU cores across which to parallelize processing + parallelize_within_regions: if True, regions will be run sequentially in parallelized chunks. If False, + each individual region's chunks will be run sequentially but there will be parallelization across + regions, i.e. each core will be assigned one region at a time by the executor **kwargs: all necessary keyword arguments to pass down to the loader Returns: @@ -57,7 +61,12 @@ def regions_to_list( ] cores_to_run = utils.cores_to_run(cores) - + # quiet and cores logic below is driven by the following: + # If the parallelization is within regions: + # (1) progress bars should happen within regions if at all, because we assume regions are + # large if they make sense to parallelize + # (2) the cores_to_run will be allocated to within-region parallelization, and the top-level + # jobs sequence is run sequentially with concurrent.futures.ProcessPoolExecutor( max_workers=1 if parallelize_within_regions else cores_to_run ) as executor: @@ -65,8 +74,10 @@ def regions_to_list( process_partial = partial( process_region, function_handle=function_handle, - quiet=quiet or not parallelize_within_regions, - cores=cores_to_run if parallelize_within_regions else 1, + quiet=quiet or not parallelize_within_regions, # + cores=cores_to_run + if parallelize_within_regions + else 1, # if parallelization is within region **kwargs, ) @@ -110,7 +121,7 @@ def pileup_counts_from_bedmethyl( regions: str | Path | list[str | Path], window_size: int | None = None, single_strand: bool = False, - quiet: bool = True, + quiet: bool = False, cores: int | None = None, chunk_size: int = 1_000_000, ) -> tuple[int, int]: @@ -138,7 +149,7 @@ def pileup_counts_from_bedmethyl( single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions quiet: disables progress bars - cores: cores across which to parallelize processes + cores: CPU cores across which to parallelize processing chunk_size: size of genomic subregions to assign out to each process Returns: @@ -212,7 +223,7 @@ def pileup_vectors_from_bedmethyl( window_size: int | None = None, single_strand: bool = False, regions_5to3prime: bool = False, - quiet: bool = True, + quiet: bool = False, cores: int | None = None, chunk_size: int = 1_000_000, ) -> tuple[np.ndarray, np.ndarray]: @@ -249,7 +260,7 @@ def pileup_vectors_from_bedmethyl( the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping quiet: disables progress bars - cores: cores across which to parallelize processes + cores: CPU cores across which to parallelize processing chunk_size: size of genomic subregions to assign out to each process Returns: @@ -379,9 +390,10 @@ def pileup_vectors_process_chunk( shm_name_valid: the name string for the shared memory location containing the valid counts array lock: a manager.Lock object to allow synchronization in accessing shared memory single_strand: True if only single-strand mods are desired + regions_5to3prime: True means negative strand regions get flipped, False means no flipping Returns: - None. Counts are added in-place to shared memory. + None. Counts are added to arrays in-place to shared memory. """ source_tabix = pysam.TabixFile(str(bedmethyl_file)) existing_valid = shared_memory.SharedMemory(name=shm_name_valid) @@ -526,7 +538,7 @@ def process_pileup_row( ) -> tuple[bool, int, int, int]: """ Helper function designed for pileup_counts_from_bedmethyl via pileup_counts_process_chunk, pileup_vectors_from_bedmethyl - via pileup_vectors_process_chunk, and export.pileup_to_bigwig; changed to logic here may impact some or all of + via pileup_vectors_process_chunk, and export.pileup_to_bigwig; changes to logic here may impact some or all of these. Process a row from a pileup, determining whether the basemod is relevant and passing back its coordinate, @@ -538,7 +550,7 @@ def process_pileup_row( region_strand: the strand from the query region single_strand: True if only mods on the region_strand are to be kept - Returns: keep_basemod, genomic_coord, modified_in_row, valid_in_row + Returns: keep_basemod, genomic_coord, modified_in_row, valid_in_row. Values are provided even if keep_basemod is False. """ tabix_fields = row.split("\t") pileup_basemod = tabix_fields[3] @@ -590,8 +602,8 @@ def read_vectors_from_hdf5( single_strand: bool = False, sort_by: str | list[str] = ["chromosome", "region_start", "read_start"], calculate_mod_fractions: bool = True, + quiet: bool = True, # currently unused; change to default False when pbars are implemented cores: int | None = None, # currently unused - quiet: bool = True, # currently unused ) -> tuple[list[tuple], list[str], dict | None]: """ User-facing function. @@ -613,6 +625,8 @@ def read_vectors_from_hdf5( After this processing, we calculate modification fractions, sort, and return. + TODO: Implement progress bars and parallelization as with pileup loaders + Args: file: Path to an hdf5 (.h5) file containing modification data for single reads, stored in datasets read_name, chromosome, read_start, @@ -635,8 +649,8 @@ def read_vectors_from_hdf5( sort_by: Read properties by which to sort, either one string or a list of strings. Options include chromosome, region_start, region_end, read_start, read_end, and motif. More to be added in future. - cores: cores across which to parallelize processes (currently unused) quiet: silences progress bars (currently unused) + cores: cores across which to parallelize processes (currently unused) Returns: a list of tuples, each tuple containing all datasets corresponding to an individual read that @@ -826,6 +840,7 @@ def readwise_binary_modification_arrays( sort_by: str | list[str] = ["chromosome", "region_start", "read_start"], thresh: float | None = None, relative: bool = True, + quiet: bool = True, # currently unused; change to default False when pbars are implemented cores: int | None = None, # currently unused ) -> tuple[list[np.ndarray], np.ndarray[int], np.ndarray[str], dict | None]: """ @@ -842,6 +857,8 @@ def readwise_binary_modification_arrays( coordinates. If positions are relative, regions_5to3prime can be used to show all regions as upstream-to-downstream along their respective strands. + TODO: Implement progress bars and parallelization as with pileup loaders + Args: file: Path to an hdf5 (.h5) file containing modification data for single reads, stored in datasets read_name, chromosome, read_start, @@ -870,6 +887,7 @@ def readwise_binary_modification_arrays( in the genomes, centered at the center of the region. If False, absolute coordinates are provided. There is not currently a check for all reads being on the same chromosome if relative=False, but this could create unexpected behaviour for a the standard visualizations. + quiet: silences progress bars (currently unused) cores: cores across which to parallelize processes (currently unused) Returns: diff --git a/dimelo/plot_depth_histogram.py b/dimelo/plot_depth_histogram.py index 655e163..949b4ec 100644 --- a/dimelo/plot_depth_histogram.py +++ b/dimelo/plot_depth_histogram.py @@ -14,7 +14,8 @@ def plot_depth_histogram( window_size: int, single_strand: bool = False, average_within_region: bool = False, - cores=None, + quiet: bool = False, + cores: int | None = None, **kwargs, ) -> Axes: """ @@ -40,6 +41,8 @@ def plot_depth_histogram( single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions average_within_region: if True, each region will only report a single depth value, averaging across all non-zero depths + quiet: disables progress bars + cores: CPU cores across which to parallelize processing kwargs: other keyword parameters passed through to utils.line_plot Returns: @@ -55,6 +58,7 @@ def plot_depth_histogram( window_size=window_size, single_strand=single_strand, average_within_region=average_within_region, + quiet=quiet, cores=cores, ) @@ -159,7 +163,8 @@ def get_depth_counts( window_size: int, single_strand: bool = False, average_within_region: bool = False, - cores=1, + quiet: bool = False, + cores: int | None = None, ) -> list[np.ndarray]: """ Get the depth counts, ready for plotting. @@ -180,6 +185,8 @@ def get_depth_counts( the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping smooth_window: size of the moving window to use for smoothing. If set to None, no smoothing is performed + quiet: disables progress bars + cores: CPU cores across which to parallelize processing Returns: List of depth histogram traces @@ -201,6 +208,7 @@ def get_depth_counts( motif=motif, window_size=window_size, single_strand=single_strand, + quiet=quiet, cores=cores, ) # places where read depth is zero are assumed to not have the motif present - this may not always be true, diff --git a/dimelo/plot_depth_profile.py b/dimelo/plot_depth_profile.py index e4b3327..0e59db1 100644 --- a/dimelo/plot_depth_profile.py +++ b/dimelo/plot_depth_profile.py @@ -15,6 +15,8 @@ def plot_depth_profile( single_strand: bool = False, regions_5to3prime: bool = False, smooth_window: int | None = None, + quiet: bool = False, + cores: int | None = None, **kwargs, ) -> Axes: """ @@ -41,6 +43,8 @@ def plot_depth_profile( the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping smooth_window: size of the moving window to use for smoothing. If set to None, no smoothing is performed + quiet: disables progress bars + cores: CPU cores across which to parallelize processing kwargs: other keyword parameters passed through to utils.line_plot Returns: @@ -57,6 +61,8 @@ def plot_depth_profile( single_strand=single_strand, regions_5to3prime=regions_5to3prime, smooth_window=smooth_window, + quiet=quiet, + cores=cores, ) axes = make_depth_profile_plot( @@ -157,6 +163,8 @@ def get_depth_profiles( single_strand: bool = False, regions_5to3prime: bool = False, smooth_window: int | None = None, + quiet: bool = False, + cores: int | None = None, ) -> list[np.ndarray]: """ Get the depth profile traces, ready for plotting. @@ -177,6 +185,8 @@ def get_depth_profiles( the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping smooth_window: size of the moving window to use for smoothing. If set to None, no smoothing is performed + quiet: disables progress bars + cores: CPU cores across which to parallelize processing Returns: List of depth profile traces @@ -198,7 +208,8 @@ def get_depth_profiles( window_size=window_size, single_strand=single_strand, regions_5to3prime=regions_5to3prime, - quiet=False, + quiet=quiet, + cores=cores, ) trace = valid_base_counts.astype(float) trace[trace == 0] = np.nan diff --git a/dimelo/plot_enrichment.py b/dimelo/plot_enrichment.py index 74c80d6..b30e8e4 100644 --- a/dimelo/plot_enrichment.py +++ b/dimelo/plot_enrichment.py @@ -12,6 +12,8 @@ def plot_enrichment( sample_names: list[str], window_size: int | None = None, single_strand: bool = False, + quiet: bool = False, + cores: int | None = None, **kwargs, ) -> Axes: """ @@ -31,6 +33,8 @@ def plot_enrichment( window_size: (currently disabled) window around center of region, +-window_size//2 single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions + quiet: disables progress bars + cores: CPU cores across which to parallelize processing kwargs: other keyword parameters passed through to utils.bar_plot Returns: @@ -143,6 +147,8 @@ def get_enrichments( motifs: list[str], window_size: int | None = None, single_strand: bool = False, + quiet: bool = False, + cores: int | None = None, ) -> list[float]: """ Get the enrichment values, ready for plotting. @@ -160,6 +166,8 @@ def get_enrichments( window_size: (currently disabled) window around center of region, +-window_size//2 single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions + quiet: disables progress bars + cores: CPU cores across which to parallelize processing Returns: List of modified fraction values. @@ -180,6 +188,8 @@ def get_enrichments( motif=motif, window_size=window_size, single_strand=single_strand, + quiet=quiet, + cores=cores, ) case ".fake": n_mod, n_total = load_processed.counts_from_fake( diff --git a/dimelo/plot_enrichment_profile.py b/dimelo/plot_enrichment_profile.py index 8a33cbf..0e339ec 100644 --- a/dimelo/plot_enrichment_profile.py +++ b/dimelo/plot_enrichment_profile.py @@ -15,6 +15,8 @@ def plot_enrichment_profile( single_strand: bool = False, regions_5to3prime: bool = False, smooth_window: int | None = None, + quiet: bool = False, + cores: int | None = None, **kwargs, ) -> Axes: """ @@ -41,6 +43,8 @@ def plot_enrichment_profile( the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping smooth_window: size of the moving window to use for smoothing. If set to None, no smoothing is performed + quiet: disables progress bars + cores: CPU cores across which to parallelize processing kwargs: other keyword parameters passed through to utils.line_plot Returns: @@ -157,6 +161,8 @@ def get_enrichment_profiles( single_strand: bool = False, regions_5to3prime: bool = False, smooth_window: int | None = None, + quiet: bool = False, + cores: int | None = None, ) -> list[np.ndarray]: """ Get the enrichment profile traces, ready for plotting. @@ -176,6 +182,8 @@ def get_enrichment_profiles( single_strand: True means we only grab counts from reads from the same strand as the region of interest, False means we always grab both strands within the regions regions_5to3prime: True means negative strand regions get flipped, False means no flipping + quiet: disables progress bars + cores: CPU cores across which to parallelize processing smooth_window: size of the moving window to use for smoothing. If set to None, no smoothing is performed Returns: @@ -199,6 +207,8 @@ def get_enrichment_profiles( window_size=window_size, single_strand=single_strand, regions_5to3prime=regions_5to3prime, + quiet=quiet, + cores=cores, ) ) # Default to nan so we can skip over unfilled values when plotting or doing a rolling average From f1b07586cb818cad0f586f7fbf8b20f7e10c15dd Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 17 Jan 2025 12:17:15 -0800 Subject: [PATCH 14/21] DM-239 Added test case coverage for 1-4 cores. GitHub jobs for Ubuntu and Macos ostensibly have 4 cores. This should not change any outputs because for extract the test is hardcoded to use 1 core, and that is the only one with any stochasticity in parallelization. --- dimelo/test/cases.py | 5 +++++ 1 file changed, 5 insertions(+) diff --git a/dimelo/test/cases.py b/dimelo/test/cases.py index 001238c..ee02c90 100644 --- a/dimelo/test/cases.py +++ b/dimelo/test/cases.py @@ -34,6 +34,7 @@ "single_strand": False, "regions_5to3prime": False, "chunk_size": 1_000_000, + "cores": 2, }, # outputs dict function:values {}, # populated in subsequent cells @@ -54,6 +55,7 @@ "single_strand": False, "regions_5to3prime": False, "chunk_size": 100, + "cores": 4, }, # outputs dict function:values {}, # populated in subsequent cells @@ -74,6 +76,7 @@ "single_strand": True, "regions_5to3prime": True, "chunk_size": 10_000, + "cores": 1, }, # outputs dict function:values {}, # populated in subsequent cells @@ -94,6 +97,7 @@ "single_strand": True, "regions_5to3prime": False, "chunk_size": 1000, + "cores": 4, }, # outputs dict function:values {}, # populated in subsequent cells @@ -114,6 +118,7 @@ "single_strand": False, "regions_5to3prime": True, "chunk_size": 100, + "cores": 3, }, # outputs dict function:values {}, # populated in subsequent cells From 0b68892af60eb9fbd5664ece2a04bc15adcb7e5c Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 17 Jan 2025 12:30:13 -0800 Subject: [PATCH 15/21] DM-239 Added cases.py support for cores=None. Tweaked dimelo_test.py to avoid ever sending the cores arg twice, and added a comment to explain the reason why extract is tested unparallelized, unlike other functions. --- dimelo/test/cases.py | 8 ++++---- dimelo/test/dimelo_test.py | 7 +++++++ 2 files changed, 11 insertions(+), 4 deletions(-) diff --git a/dimelo/test/cases.py b/dimelo/test/cases.py index ee02c90..a269008 100644 --- a/dimelo/test/cases.py +++ b/dimelo/test/cases.py @@ -34,7 +34,7 @@ "single_strand": False, "regions_5to3prime": False, "chunk_size": 1_000_000, - "cores": 2, + "cores": 1, }, # outputs dict function:values {}, # populated in subsequent cells @@ -55,7 +55,7 @@ "single_strand": False, "regions_5to3prime": False, "chunk_size": 100, - "cores": 4, + "cores": 2, }, # outputs dict function:values {}, # populated in subsequent cells @@ -76,7 +76,7 @@ "single_strand": True, "regions_5to3prime": True, "chunk_size": 10_000, - "cores": 1, + "cores": 3, }, # outputs dict function:values {}, # populated in subsequent cells @@ -118,7 +118,7 @@ "single_strand": False, "regions_5to3prime": True, "chunk_size": 100, - "cores": 3, + "cores": None, }, # outputs dict function:values {}, # populated in subsequent cells diff --git a/dimelo/test/dimelo_test.py b/dimelo/test/dimelo_test.py index 787aa83..73310cc 100644 --- a/dimelo/test/dimelo_test.py +++ b/dimelo/test/dimelo_test.py @@ -77,6 +77,13 @@ def test_unit__extract( ): kwargs_extract = filter_kwargs_for_func(dm.parse_bam.extract, kwargs) kwargs_extract["output_directory"] = cls.outDir + # extract can behave non-deterministically in terms of read output order + # if run on multiple cores. This is not an issue for sorted read loads, + # but means the file itself changes even though the meaningful contents don't + # given that parallelization here is purely within modkit anyway, the design + # choice was made to always single-core for extract testing + if "cores" in kwargs_extract: + del kwargs_extract["cores"] extract_h5, regions_processed = dm.parse_bam.extract( **kwargs_extract, ref_genome=cls.reference_genome, From 1da3d3c59d66c8560efd23acc6a126a6153c91f7 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 17 Jan 2025 16:27:54 -0800 Subject: [PATCH 16/21] DM-239 Adjusted generate_targets module to properly handle cores argument for extract. Updated test_matrix.pickle by re-running generate_targets. 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zMy{1J9E%u>Bx-IWtfE2~`lPCvvpxz#qDCjf3|%2IWx~KmC@=|9npUC{Sq6bu4P`<> z7R5j(6sDmW49ZMQMP0c+0?{3bS%s;-v1B<;!bA0k0H^#bgIF)XUka;8I3iT8QiBgO zwm^d|+=q62C~npgl5Lqe#0m!l28NhQ1C+5Gom$2NWL6=HCo1d+52~Vqn9t~#!3$zw3Q7JQ$$o|a8u#N9^GnKEZ`ItsW zJxWf%0k=2}Qsy#%K1K^epfA)na{!s!vSxU$g#q271$`4Nl2a^8A|82V7Cgw)iR@p) z+7G#zas!(Lde*mq!q~PzsrvT%stR6xb@!pFxQ16ehxZqZ3Ft&NhL6l5*n{Qd$;b{1 z*4>PSDY}Ye0HbRF5mp?4nt4W~R5M7rLP<%3=s65YX~USA)_>(EZtnh*6F=ea?Ea5; zT@-$2w|31%SD*c8{u31X`x8I9uXf4yzSi46Ot1g8lKuw8YJXcP2(0oOyi7dBZ}2j< z%5U&?&l!t$WY43-jL}>Tf^y zw_1(-IihL*!ut$=pdkKrA|-Xa8f^dFy%#GWzCBs4Kg;#EKlE`WsgQf{dbt2CBk-^IPOc4qv1!i#y#?EJ=ggU%SO2g+yygxO*Lm)5 zFZsb`r~G>>-~NFA-pYsn_>NZ(y;1)z?Yw&EP5Zvl_h9*T<*q-ya@mjhW%bQb-$TmQ zJ8pX6P5Sp{{d Date: Fri, 17 Jan 2025 16:33:08 -0800 Subject: [PATCH 17/21] Ruff format fixes --- dimelo/parse_bam.py | 6 +++--- dimelo/run_modkit.py | 4 ++-- dimelo/test/dimelo_test.py | 12 ++++++------ dimelo/utils.py | 2 +- 4 files changed, 12 insertions(+), 12 deletions(-) diff --git a/dimelo/parse_bam.py b/dimelo/parse_bam.py index 157ed59..2bb8a64 100644 --- a/dimelo/parse_bam.py +++ b/dimelo/parse_bam.py @@ -531,7 +531,7 @@ def verify_inputs( ) elif correct_bases / total_bases < 0.35: raise ValueError( - f"First {NUM_READS_TO_CHECK} reads have anomalously low alignment quality: only {100*correct_bases/total_bases}% of bases align.\nPlease verify that {input_file.name} is actually aligned to {ref_genome.name}." + f"First {NUM_READS_TO_CHECK} reads have anomalously low alignment quality: only {100 * correct_bases / total_bases}% of bases align.\nPlease verify that {input_file.name} is actually aligned to {ref_genome.name}." ) return @@ -621,7 +621,7 @@ def check_bam_format( print( f""" WARNING: no modified appropriately-coded values found for {missing_bases} in the first {counter} reads. -Do you expect this file to contain these modifications? parse_bam is looking for {motifs} but for {missing_bases} found only found {[f'{base}+{mod_codes}' for base, mod_codes in mod_codes_found_dict.items()]}. +Do you expect this file to contain these modifications? parse_bam is looking for {motifs} but for {missing_bases} found only found {[f"{base}+{mod_codes}" for base, mod_codes in mod_codes_found_dict.items()]}. Consider passing only the motifs and mod codes (e.g. m,h,a) that you expect to be present in your file. You can use modkit adjust-mods --convert [OPTIONS] to update or consolidate mod codes. @@ -956,7 +956,7 @@ def read_by_base_txt_to_hdf5( iterator = tqdm( iterator, total=num_lines, - desc=f"Transferring {num_reads} from {input_txt.name} into {output_h5.name}, new size {old_size+num_reads}", + desc=f"Transferring {num_reads} from {input_txt.name} into {output_h5.name}, new size {old_size + num_reads}", bar_format="{bar}| {desc} {percentage:3.0f}% | {elapsed}<{remaining}", ) diff --git a/dimelo/run_modkit.py b/dimelo/run_modkit.py index aa8e707..298f0e9 100644 --- a/dimelo/run_modkit.py +++ b/dimelo/run_modkit.py @@ -28,8 +28,8 @@ print( f"PATH does not include the conda environment /bin folder. Adding {env_bin_path}." ) - os.environ["PATH"] = f'{env_bin_path}:{os.environ["PATH"]}' - print(f'PATH is now {os.environ["PATH"]}') + os.environ["PATH"] = f"{env_bin_path}:{os.environ['PATH']}" + print(f"PATH is now {os.environ['PATH']}") # Check modkit on first import: does it run; does it have the right version try: diff --git a/dimelo/test/dimelo_test.py b/dimelo/test/dimelo_test.py index 73310cc..d11dae9 100644 --- a/dimelo/test/dimelo_test.py +++ b/dimelo/test/dimelo_test.py @@ -270,9 +270,9 @@ def test_integration__extract_load_plot( assert np.allclose( actual[key], expected[key], atol=1e-5 ), f"""{test_case}: Arrays for {key} are not equal -mismatch at {np.where(value!=actual[key])} -mismatch values expected {value[np.where(value!=actual[key])]} vs actual {actual[key][np.where(value!=actual[key])]} -{value[np.where(value!=actual[key])[0]]} vs {actual[key][np.where(value!=actual[key])[0]]}. +mismatch at {np.where(value != actual[key])} +mismatch values expected {value[np.where(value != actual[key])]} vs actual {actual[key][np.where(value != actual[key])]} +{value[np.where(value != actual[key])[0]]} vs {actual[key][np.where(value != actual[key])[0]]}. """ elif isinstance(value, (str, int, bool)): assert ( @@ -439,9 +439,9 @@ def test_unit__read_vectors_from_hdf5( assert np.allclose( actual[key], expected[key], atol=1e-5 ), f"""{test_case}: Arrays for {key} are not equal -mismatch at {np.where(value!=actual[key])} -mismatch values expected {value[np.where(value!=actual[key])]} vs actual {actual[key][np.where(value!=actual[key])]} -{value[np.where(value!=actual[key])[0]]} vs {actual[key][np.where(value!=actual[key])[0]]}. +mismatch at {np.where(value != actual[key])} +mismatch values expected {value[np.where(value != actual[key])]} vs actual {actual[key][np.where(value != actual[key])]} +{value[np.where(value != actual[key])[0]]} vs {actual[key][np.where(value != actual[key])[0]]}. """ elif isinstance(value, (str, int, bool)): assert ( diff --git a/dimelo/utils.py b/dimelo/utils.py index aa85e55..66ed83d 100644 --- a/dimelo/utils.py +++ b/dimelo/utils.py @@ -84,7 +84,7 @@ def adjust_threshold( if thresh > 1: if not quiet: print( - f"Modification threshold of {thresh} assumed to be for range 0-255. {thresh}/255={thresh/255} will be sent to modkit." + f"Modification threshold of {thresh} assumed to be for range 0-255. {thresh}/255={thresh / 255} will be sent to modkit." ) thresh_scaled = thresh / 255 else: From bd94e785673c52bcea041bf6ef167795db1d1c2a Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 21 Feb 2025 10:13:41 -0800 Subject: [PATCH 18/21] Fixed small mistakes from merge --- dimelo/export.py | 2 +- dimelo/load_processed.py | 23 ++++++++++++++--------- 2 files changed, 15 insertions(+), 10 deletions(-) diff --git a/dimelo/export.py b/dimelo/export.py index 8ccf34b..e2c973c 100644 --- a/dimelo/export.py +++ b/dimelo/export.py @@ -131,7 +131,7 @@ def pileup_to_bigwig( for row in tqdm( tabix.fetch(contig), desc=f"Writing {contig}.", - total=contig_lines[contig], + total=lines_by_contig[contig], leave=False, ): keep_basemod, genomic_coord, modified_in_row, valid_in_row = ( diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index 043a3f2..ad4c79b 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -18,6 +18,7 @@ #### Loader wrappers #### ################################################################################################################ + def regions_to_list( function_handle, regions, @@ -79,7 +80,7 @@ def regions_to_list( else 1, # if parallelization is within region **kwargs, ) - + if cores_to_run > 1: # Use executor.map without lambda results = list( tqdm( @@ -88,17 +89,21 @@ def regions_to_list( desc=f"Processing regions in parallel across {cores_to_run}", ) ) - else: - # Single-threaded fallback - results = [ - process_region( - region_string=region, function_handle=function_handle, cores=1, **kwargs - ) - for region in tqdm(region_strings, desc="Processing regions") - ] + else: + # Single-threaded fallback + results = [ + process_region( + region_string=region, + function_handle=function_handle, + cores=1, + **kwargs, + ) + for region in tqdm(region_strings, desc="Processing regions") + ] return results + def process_region(region_string, function_handle, **kwargs): """ process_region simply exists to convert position arguments into keyword arguments to make executor.map work From 060c4bf265f34225c740040456c2d7e1a26d5647 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 21 Feb 2025 10:57:16 -0800 Subject: [PATCH 19/21] DM-242 environment spec loosening. Python and modkit can both be newer versions but must meet minimum requirements. --- environment.yml | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/environment.yml b/environment.yml index 5837208..5e17ccf 100644 --- a/environment.yml +++ b/environment.yml @@ -4,5 +4,5 @@ channels: - nanoporetech - defaults dependencies: - - python=3.11 - - nanoporetech::modkit==0.2.4 \ No newline at end of file + - python>=3.11 + - nanoporetech::modkit>=0.2.4 \ No newline at end of file From f0d638737cd709d5818dbe8c0ac4b066264c98b2 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Fri, 21 Feb 2025 12:45:39 -0800 Subject: [PATCH 20/21] DM-243 Version checking changed to min rather than exact; minor logic changes. Fixed import order --- dimelo/load_processed.py | 4 +-- dimelo/run_modkit.py | 60 ++++++++++++++++++++++++++-------------- 2 files changed, 41 insertions(+), 23 deletions(-) diff --git a/dimelo/load_processed.py b/dimelo/load_processed.py index ad4c79b..bc59a82 100644 --- a/dimelo/load_processed.py +++ b/dimelo/load_processed.py @@ -202,7 +202,7 @@ def pileup_counts_from_bedmethyl( concurrent.futures.as_completed(futures), total=len(futures), disable=quiet, - desc=f"Loading genomic chunks, up to {chunk_size/1000}kb per chunk", + desc=f"Loading genomic chunks, up to {chunk_size / 1000}kb per chunk", ): try: future.result() @@ -320,7 +320,7 @@ def pileup_vectors_from_bedmethyl( concurrent.futures.as_completed(futures), total=len(futures), disable=quiet, - desc=f"Loading genomic chunks, up to {chunk_size/1000}kb per chunk", + desc=f"Loading genomic chunks, up to {chunk_size / 1000}kb per chunk", ): try: future.result() diff --git a/dimelo/run_modkit.py b/dimelo/run_modkit.py index 298f0e9..4b96254 100644 --- a/dimelo/run_modkit.py +++ b/dimelo/run_modkit.py @@ -11,40 +11,58 @@ from pathlib import Path from typing import Optional, cast +from packaging import version from tqdm.auto import tqdm # This should be updated in tandem with the environment.yml nanoporetech::modkit version -EXPECTED_MODKIT_VERSION = "0.2.4" +MIN_MODKIT_VERSION = "0.2.4" """ Import checks """ -# Add conda env bin folder to path if it is not already present -# On some systems, the directory containing executables for the active environment isn't automatically on the path -# If this is the case, add that directory to the path so modkit can run -current_interpreter = sys.executable -env_bin_path = os.path.dirname(current_interpreter) -if env_bin_path not in os.environ["PATH"]: - print( - f"PATH does not include the conda environment /bin folder. Adding {env_bin_path}." - ) - os.environ["PATH"] = f"{env_bin_path}:{os.environ['PATH']}" - print(f"PATH is now {os.environ['PATH']}") - # Check modkit on first import: does it run; does it have the right version try: result = subprocess.run(["modkit", "--version"], stdout=subprocess.PIPE, text=True) - modkit_version = result.stdout - if modkit_version.split()[1] == EXPECTED_MODKIT_VERSION: - print(f"modkit found with expected version {EXPECTED_MODKIT_VERSION}") + modkit_version = result.stdout.strip().split()[-1] + if version.parse(modkit_version) >= version.parse(MIN_MODKIT_VERSION): + print( + f"modkit found with version {modkit_version}, which meets the minimum requirement ({MIN_MODKIT_VERSION})." + ) else: print( - f"modkit found with unexpected version {modkit_version.split()[1]}. Versions other than {EXPECTED_MODKIT_VERSION} may exhibit unexpected behavior. It is recommended that you use v{EXPECTED_MODKIT_VERSION}" + f"WARNING: modkit found with version {modkit_version}, but version {MIN_MODKIT_VERSION} or later is required. " + f"Consider updating modkit to avoid unexpected behavior." ) -except subprocess.CalledProcessError: - print( - 'Executable not found for modkit. Install dimelo using "conda env create -f environment.yml" or install modkit manually to your conda environment using "conda install nanoporetech::modkit==0.2.4". Without modkit you cannot run parse_bam functions.' - ) +except (FileNotFoundError, subprocess.CalledProcessError): + # Add conda env bin folder to path if it is not already present + # On some systems, the directory containing executables for the active environment isn't automatically on the path + # If this is the case, add that directory to the path so modkit can run + current_interpreter = sys.executable + env_bin_path = os.path.dirname(current_interpreter) + if env_bin_path not in os.environ["PATH"]: + print( + f"PATH does not include the conda environment /bin folder. Adding {env_bin_path}." + ) + os.environ["PATH"] = f"{env_bin_path}:{os.environ['PATH']}" + print(f"PATH is now {os.environ['PATH']}") + try: + result = subprocess.run( + ["modkit", "--version"], stdout=subprocess.PIPE, text=True + ) + modkit_version = result.stdout.strip().split()[-1] + if version.parse(modkit_version) >= version.parse(MIN_MODKIT_VERSION): + print( + f"modkit found with version {modkit_version}, which meets the minimum requirement ({MIN_MODKIT_VERSION})." + ) + else: + print( + f"WARNING: modkit found with version {modkit_version}, but version {MIN_MODKIT_VERSION} or later is required. " + f"Consider updating modkit to avoid unexpected behavior." + ) + except (FileNotFoundError, subprocess.CalledProcessError) as e: + raise RuntimeError( + 'Unable to execute modkit. Install dimelo using "conda env create -f environment.yml" or install modkit manually to your conda environment using "conda install nanoporetech::modkit==0.2.4". Without modkit you cannot run parse_bam functions.' + ) from e def run_with_progress_bars( From ec1c3f60817915a47d0e747f8b82806fe3389317 Mon Sep 17 00:00:00 2001 From: Oberon Dixon-Luinenburg Date: Mon, 24 Feb 2025 14:22:06 -0800 Subject: [PATCH 21/21] Adjusted version range. Something seems to have changed too much as of 0.4.0 and needs to be examined. For now, pin it to a tight range. --- dimelo/run_modkit.py | 36 +++++++++++++++++------------------- environment.yml | 2 +- 2 files changed, 18 insertions(+), 20 deletions(-) diff --git a/dimelo/run_modkit.py b/dimelo/run_modkit.py index 4b96254..df36d57 100644 --- a/dimelo/run_modkit.py +++ b/dimelo/run_modkit.py @@ -16,24 +16,17 @@ # This should be updated in tandem with the environment.yml nanoporetech::modkit version MIN_MODKIT_VERSION = "0.2.4" +MAX_MODKIT_VERSION = "0.2.6" """ Import checks """ # Check modkit on first import: does it run; does it have the right version +modkit_version = "NA" try: result = subprocess.run(["modkit", "--version"], stdout=subprocess.PIPE, text=True) modkit_version = result.stdout.strip().split()[-1] - if version.parse(modkit_version) >= version.parse(MIN_MODKIT_VERSION): - print( - f"modkit found with version {modkit_version}, which meets the minimum requirement ({MIN_MODKIT_VERSION})." - ) - else: - print( - f"WARNING: modkit found with version {modkit_version}, but version {MIN_MODKIT_VERSION} or later is required. " - f"Consider updating modkit to avoid unexpected behavior." - ) -except (FileNotFoundError, subprocess.CalledProcessError): +except Exception: # Add conda env bin folder to path if it is not already present # On some systems, the directory containing executables for the active environment isn't automatically on the path # If this is the case, add that directory to the path so modkit can run @@ -50,19 +43,24 @@ ["modkit", "--version"], stdout=subprocess.PIPE, text=True ) modkit_version = result.stdout.strip().split()[-1] - if version.parse(modkit_version) >= version.parse(MIN_MODKIT_VERSION): - print( - f"modkit found with version {modkit_version}, which meets the minimum requirement ({MIN_MODKIT_VERSION})." - ) - else: - print( - f"WARNING: modkit found with version {modkit_version}, but version {MIN_MODKIT_VERSION} or later is required. " - f"Consider updating modkit to avoid unexpected behavior." - ) except (FileNotFoundError, subprocess.CalledProcessError) as e: raise RuntimeError( 'Unable to execute modkit. Install dimelo using "conda env create -f environment.yml" or install modkit manually to your conda environment using "conda install nanoporetech::modkit==0.2.4". Without modkit you cannot run parse_bam functions.' ) from e +finally: + if ( + version.parse(MAX_MODKIT_VERSION) + >= version.parse(modkit_version) + >= version.parse(MIN_MODKIT_VERSION) + ): + print( + f"modkit found with version {modkit_version}, which meets the requirement ({MIN_MODKIT_VERSION}-{MAX_MODKIT_VERSION})." + ) + else: + print( + f"WARNING: modkit found with version {modkit_version}, but version {MIN_MODKIT_VERSION} or later is required. " + f"Consider updating modkit to avoid unexpected behavior." + ) def run_with_progress_bars( diff --git a/environment.yml b/environment.yml index 5e17ccf..296354b 100644 --- a/environment.yml +++ b/environment.yml @@ -5,4 +5,4 @@ channels: - defaults dependencies: - python>=3.11 - - nanoporetech::modkit>=0.2.4 \ No newline at end of file + - "nanoporetech::modkit>=0.2.4,<=0.2.6" \ No newline at end of file