mgnify-pipelines-toolkit 1.2.2__py3-none-any.whl → 1.2.4__py3-none-any.whl

mgnify_pipelines_toolkit/analysis/amplicon/classify_var_regions.py

@@ -22,12 +22,12 @@ import os
 import logging
 import json
 import time
+import numpy as np
 
 from mgnify_pipelines_toolkit.constants.thresholds import (
     MIN_OVERLAP,
     MIN_SEQ_COUNT,
     MAX_ERROR_PROPORTION,
-    MAX_INTERNAL_PRIMER_PROPORTION,
 )
 from mgnify_pipelines_toolkit.constants.var_region_coordinates import (
     REGIONS_16S_BACTERIA,
@@ -62,8 +62,16 @@ def get_multiregion(raw_sequence_coords, regions):
 
     Returns:
         amplified_region: Amplified variable regions.
+        region_coverages: Coverage of all detected variable regions
 
     """
+
+    region_coverages = defaultdict(float)
+
+    for region, limits in regions.items():
+        overlap = calc_overlap(raw_sequence_coords, limits)
+        region_coverages[region] = overlap
+
     # check if any of the coords are inside the region
     matched_regions = [
         region
@@ -76,7 +84,7 @@ def get_multiregion(raw_sequence_coords, regions):
         amplified_region = matched_regions[0]
     else:
         amplified_region = ""
-    return amplified_region
+    return amplified_region, region_coverages
 
 
 def check_primer_position(raw_sequence_coords, regions):
@@ -90,7 +98,7 @@ def check_primer_position(raw_sequence_coords, regions):
 
     """
     result_flag = False
-    margin = 3  # allowed margin of error
+    margin = 10  # allowed margin of error
     for coord in raw_sequence_coords:
         for region in regions.values():
             if coord in range(region[0] + margin, region[1] - margin):
@@ -342,22 +350,30 @@ def retrieve_regions(
         per_read_info = (
             dict()
         )  # dictionary will contain read names for each variable region
+        all_region_coverages = defaultdict(lambda: defaultdict(list))
         for read in data:
+            # Example structure of `read`
+            # ('ERR14650515.1', 'SSU_rRNA_archaea', 'RF01959', 'hmm', '3', '525', '1', '518', '+', '-', '6', '0.55', '0.6', '363.6', '7.8e-107')
             regions = determine_cm(read[2])
             sequence_counter_total += 1
             limits = list(map(int, read[4:6]))
             domain = determine_domain(read[2])
             marker_gene = determine_marker_gene(domain)
             if not regions == "unsupported":
-                multiregion_matches.setdefault(read[2], []).append(
-                    get_multiregion(limits, regions)
-                )
+                matches, coverages = get_multiregion(limits, regions)
+
+                [
+                    all_region_coverages[domain][region].append(coverage)
+                    for region, coverage in coverages.items()
+                ]
+
+                multiregion_matches.setdefault(read[2], []).append(matches)
                 if check_primer_position(limits, regions):
                     primer_inside_vr += 1
                 sequence_counter_useful += 1
-                per_read_info.setdefault(
-                    marker_gene + "." + get_multiregion(limits, regions), []
-                ).append(read[0])
+                per_read_info.setdefault(marker_gene + "." + matches, []).append(
+                    read[0]
+                )
             else:
                 unsupported_matches += 1
 
@@ -385,18 +401,6 @@ def retrieve_regions(
             )
             continue
 
-        # filter out runs with too many sequences starting/ending inside variable regions
-        internal_seq_fract = primer_inside_vr / len(data)
-        if internal_seq_fract > MAX_INTERNAL_PRIMER_PROPORTION:
-            failed_run_counter += 1
-            logging.info("No output will be produced - too many internal mappings")
-            logging.info(
-                "Excluded due to high proportion of internal primers:\t{}\t{}\n".format(
-                    tblout_file, "{0:.2f}".format(internal_seq_fract)
-                )
-            )
-            continue
-
         normalised_matches[run_id] = dict()
         region_counter = defaultdict(int)
 
@@ -432,14 +436,12 @@ def retrieve_regions(
             multiregion_matches[model] = new_value
 
         [multiregion_matches.pop(model) for model in models_to_remove]
-        print(multiregion_matches)
 
         run_status = "one"
         run_result = dict()
         total_useful_sequences = 0.0
         temp_seq_counter = dict()
         for model, model_regions in multiregion_matches.items():
-            print(model)
             result = normalise_results(model_regions)
             if result is None:
                 run_status = "ambiguous"
@@ -469,6 +471,16 @@ def retrieve_regions(
             logging.info("No output will be produced - the run is ambiguous.")
             continue
 
+    coverage_fw = open(f"{outfile_prefix}_all_coverages.txt", "w")
+
+    for domain, regions in all_region_coverages.items():
+        for region in regions:
+            if len(regions[region]) < MIN_SEQ_COUNT:
+                continue
+            region_coverage = float(np.mean(regions[region]))
+            if region_coverage > 0:
+                coverage_fw.write(f"{domain}:{region}: {region_coverage}\n")
+
     json_outfile = "{}.json".format(outfile_prefix)
     tsv_outfile = "{}.tsv".format(outfile_prefix)
     with open(json_outfile, "w") as f:

mgnify_pipelines_toolkit/analysis/amplicon/permute_primers.py

@@ -0,0 +1,87 @@
+import argparse
+from itertools import product
+from pathlib import Path
+
+from Bio import SeqIO
+
+
+def parse_args():
+
+    parser = argparse.ArgumentParser()
+    parser.add_argument(
+        "-i",
+        "--input_primers",
+        required=True,
+        type=str,
+        help="Input primers to generate permutations for due to IUPAC ambiguous codes",
+    )
+    parser.add_argument("-p", "--prefix", required=True, type=str, help="Output prefix")
+
+    args = parser.parse_args()
+
+    input_path = args.input_primers
+    prefix = args.prefix
+
+    return input_path, prefix
+
+
+def permute_seq(seq):
+
+    ambiguous_bases_dict = {
+        "R": ["A", "G"],
+        "Y": ["C", "T"],
+        "S": ["G", "C"],
+        "W": ["A", "T"],
+        "K": ["G", "T"],
+        "M": ["A", "C"],
+        "B": ["C", "G", "T"],
+        "D": ["A", "G", "T"],
+        "H": ["A", "C", "T"],
+        "V": ["A", "C", "G"],
+        "N": ["A", "C", "T", "G"],
+    }
+
+    seq_template = []
+
+    for base in seq:
+        if base in ["A", "C", "T", "G"]:
+            seq_template.append(base)
+        else:
+            seq_template.append(ambiguous_bases_dict[base])
+
+    seq_permutations = []
+    for combo in product(*seq_template):
+        seq_permutations.append("".join(combo))
+
+    return seq_permutations
+
+
+def make_primer_permutations(primers_dict, prefix):
+
+    with open(f"{prefix}_permuted_primers.fasta", "w") as fw:
+        for primer_name, seq in primers_dict.items():
+
+            primer_seq = seq.seq
+            fw.write(f">{primer_name}\n{primer_seq}\n")
+
+            if primer_name == "F_auto" or primer_name[-1] == "F":
+                strand = "F"
+            elif primer_name == "R_auto" or primer_name[-1] == "R":
+                strand = "R"
+
+            seq_permutations = permute_seq(primer_seq)
+
+            for counter, permuted_seq in enumerate(seq_permutations, 1):
+                variant_name = f"{primer_name}_variant_{counter}_{strand}"
+                fw.write(f">{variant_name}\n{permuted_seq}\n")
+
+
+def main():
+
+    input_path, prefix = parse_args()
+    primers_dict = SeqIO.to_dict(SeqIO.parse(Path(input_path), "fasta"))
+    make_primer_permutations(primers_dict, prefix)
+
+
+if __name__ == "__main__":
+    main()

mgnify_pipelines_toolkit/analysis/amplicon/primer_val_classification.py

@@ -19,6 +19,7 @@ from collections import defaultdict
 import re
 
 from Bio import SeqIO
+from Bio.Seq import Seq
 import pandas as pd
 
 from mgnify_pipelines_toolkit.constants.var_region_coordinates import (
@@ -49,18 +50,26 @@ def parse_args():
         help="Path to concatenated primers fasta file",
     )
     parser.add_argument("-s", "--sample", required=True, type=str, help="Sample ID")
+    parser.add_argument(
+        "--se",
+        action=argparse.BooleanOptionalAction,
+        help="Flag for if run is single-end",
+    )
     args = parser.parse_args()
 
     input = args.input
     fasta = args.fasta
     sample = args.sample
+    single_end = args.se
 
-    return input, fasta, sample
+    return input, fasta, sample, single_end
 
 
 def get_amp_region(beg, end, strand, model):
     prev_region = ""
 
+    margin = -10
+
     for region, region_coords in model.items():
 
         region_beg = region_coords[0]
@@ -68,10 +77,10 @@ def get_amp_region(beg, end, strand, model):
         end_diff = region_beg - end
 
         if strand == STRAND_FWD:
-            if beg_diff > 0 and end_diff > 0:
+            if beg_diff >= margin and end_diff >= margin:
                 return region
         else:
-            if beg_diff > 0 and end_diff > 0:
+            if beg_diff >= margin and end_diff >= margin:
                 return prev_region
 
         prev_region = region
@@ -80,10 +89,16 @@ def get_amp_region(beg, end, strand, model):
 
 
 def main():
-    input, fasta, sample = parse_args()
+
+    input, fasta, sample, single_end = parse_args()
     res_dict = defaultdict(list)
     fasta_dict = SeqIO.to_dict(SeqIO.parse(fasta, "fasta"))
 
+    fwd_primers_fw = open("./fwd_primers.fasta", "w")
+    rev_primers_fw = open("./rev_primers.fasta", "w")
+
+    matched_primers_list = []
+
     with open(input, "r") as fr:
         for line in fr:
             line = line.strip()
@@ -95,6 +110,10 @@ def main():
             beg = float(line_lst[5])
             end = float(line_lst[6])
 
+            cleaned_primer_name = "_".join(primer_name.split("_")[0:-3])
+            if cleaned_primer_name in matched_primers_list:
+                continue
+
             if rfam == "RF00177":
                 gene = "16S"
                 model = REGIONS_16S_BACTERIA
@@ -104,8 +123,12 @@ def main():
             elif rfam == "RF01960":
                 gene = "18S"
                 model = REGIONS_18S
-            else:
-                continue
+            else:  # For cases when it's a std primer but for some reason hasn't matched the model
+                if cleaned_primer_name == "F_auto" or cleaned_primer_name == "R_auto":
+                    continue
+                gene = "Unknown"
+                amp_region = "Unknown"
+                model = ""
 
             res_dict["Run"].append(sample)
             res_dict["AssertionEvidence"].append("ECO_0000363")
@@ -113,23 +136,39 @@ def main():
 
             strand = ""
 
-            if "F" in primer_name:
+            if primer_name[-1] == "F":
                 strand = STRAND_FWD
-            elif "R" in primer_name:
+            elif primer_name[-1] == "R":
                 strand = STRAND_REV
+            else:
+                print(f"Not sure what strand this is, exiting: {primer_name}")
+
+            if model:
+                amp_region = get_amp_region(beg, end, strand, model)
 
-            amp_region = get_amp_region(beg, end, strand, model)
-            primer_seq = str(fasta_dict[primer_name].seq)
+            primer_seq = str(fasta_dict[cleaned_primer_name].seq)
 
             res_dict["Gene"].append(gene)
             res_dict["VariableRegion"].append(amp_region)
-            res_dict["PrimerName"].append(primer_name)
+            res_dict["PrimerName"].append(cleaned_primer_name)
             res_dict["PrimerStrand"].append(strand)
             res_dict["PrimerSeq"].append(primer_seq)
 
+            if strand == STRAND_FWD:
+                fwd_primers_fw.write(f">{cleaned_primer_name}\n{primer_seq}\n")
+            elif strand == STRAND_REV:
+                if single_end:
+                    primer_seq = Seq(primer_seq).reverse_complement()
+                rev_primers_fw.write(f">{cleaned_primer_name}\n{primer_seq}\n")
+
+            matched_primers_list.append(cleaned_primer_name)
+
     res_df = pd.DataFrame.from_dict(res_dict)
     res_df.to_csv(f"./{sample}_primer_validation.tsv", sep="\t", index=False)
 
+    fwd_primers_fw.close()
+    rev_primers_fw.close()
+
 
 if __name__ == "__main__":
     main()

{mgnify_pipelines_toolkit-1.2.2.dist-info → mgnify_pipelines_toolkit-1.2.4.dist-info}/METADATA

@@ -1,6 +1,6 @@
 Metadata-Version: 2.4
 Name: mgnify_pipelines_toolkit
-Version: 1.2.2
+Version: 1.2.4
 Summary: Collection of scripts and tools for MGnify pipelines
 Author-email: MGnify team <metagenomics-help@ebi.ac.uk>
 License: Apache Software License 2.0

{mgnify_pipelines_toolkit-1.2.2.dist-info → mgnify_pipelines_toolkit-1.2.4.dist-info}/RECORD

@@ -1,9 +1,10 @@
 mgnify_pipelines_toolkit/__init__.py,sha256=47DEQpj8HBSa-_TImW-5JCeuQeRkm5NMpJWZG3hSuFU,0
 mgnify_pipelines_toolkit/analysis/__init__.py,sha256=47DEQpj8HBSa-_TImW-5JCeuQeRkm5NMpJWZG3hSuFU,0
-mgnify_pipelines_toolkit/analysis/amplicon/classify_var_regions.py,sha256=W0ob9z_8sjrB1Ck48Ac-_5Vw2kyoRFalcxhrR6KSXpI,20196
+mgnify_pipelines_toolkit/analysis/amplicon/classify_var_regions.py,sha256=nUvboLz08RyqRE8Thfh8NRlmgJk0kVdXcSvgmAfKip0,20649
 mgnify_pipelines_toolkit/analysis/amplicon/make_asv_count_table.py,sha256=ohguvrMSg7GuiiZ5aHj1DvCnfThKFUG4s13LUSMM0mo,8892
 mgnify_pipelines_toolkit/analysis/amplicon/mapseq_to_asv_table.py,sha256=BLqhflblUegCvuQic16PrFXfIXlFWmGkmWJyl4wJoLQ,5040
-mgnify_pipelines_toolkit/analysis/amplicon/primer_val_classification.py,sha256=Bmc4Yu8inpT6AVTG1zwxp9F9mknIDLY33-UuFdaZuq0,3756
+mgnify_pipelines_toolkit/analysis/amplicon/permute_primers.py,sha256=1aGOJX9tC7M1rnd0U2PeJ681sUo02wxk7_ycJqeVt6s,2216
+mgnify_pipelines_toolkit/analysis/amplicon/primer_val_classification.py,sha256=I9JfkM_o6Wp4VINOMO6ff9mHqghdJw1kDIfiF37JtLo,5185
 mgnify_pipelines_toolkit/analysis/amplicon/remove_ambiguous_reads.py,sha256=Wu4tRtuRkgd3hoeuwPl_E5ghxIW7e_1vrcvFGWv_U4A,3173
 mgnify_pipelines_toolkit/analysis/amplicon/rev_comp_se_primers.py,sha256=yLpzkRJXAeXRUNgz60zopEwHcdprM2UDjquE-GkrFys,1722
 mgnify_pipelines_toolkit/analysis/amplicon/study_summary_generator.py,sha256=epVClL10QcllL8yu7YGjx0rXNVHL2GxHi-Ek0MOjsjo,13859
@@ -42,9 +43,9 @@ mgnify_pipelines_toolkit/schemas/schemas.py,sha256=pyDZvCuWbwccQF0D7c5BN1vv36wQd
 mgnify_pipelines_toolkit/utils/__init__.py,sha256=47DEQpj8HBSa-_TImW-5JCeuQeRkm5NMpJWZG3hSuFU,0
 mgnify_pipelines_toolkit/utils/fasta_to_delimited.py,sha256=lgYIR1S4crURY7C7nFtgE6QMV4u4zCNsUrVkcRnsEEo,3996
 mgnify_pipelines_toolkit/utils/get_mpt_version.py,sha256=aS9bWrC9CP7tpxoEVg6eEYt18-pmjG7fJl5Mchz4YOU,798
-mgnify_pipelines_toolkit-1.2.2.dist-info/licenses/LICENSE,sha256=xx0jnfkXJvxRnG63LTGOxlggYnIysveWIZ6H3PNdCrQ,11357
-mgnify_pipelines_toolkit-1.2.2.dist-info/METADATA,sha256=MRDDDp1xhJhoIc-rU1IKWASfleAvFXsJLyhaoG8FLUg,5775
-mgnify_pipelines_toolkit-1.2.2.dist-info/WHEEL,sha256=_zCd3N1l69ArxyTb8rzEoP9TpbYXkqRFSNOD5OuxnTs,91
-mgnify_pipelines_toolkit-1.2.2.dist-info/entry_points.txt,sha256=d7r4_VUS1hWNMnTJOy8u2kTRSFcy-sDN5NLRUXz-IhU,3041
-mgnify_pipelines_toolkit-1.2.2.dist-info/top_level.txt,sha256=xA_wC7C01V3VwuDnqwRM2QYeJJ45WtvF6LVav4tYxuE,25
-mgnify_pipelines_toolkit-1.2.2.dist-info/RECORD,,
+mgnify_pipelines_toolkit-1.2.4.dist-info/licenses/LICENSE,sha256=xx0jnfkXJvxRnG63LTGOxlggYnIysveWIZ6H3PNdCrQ,11357
+mgnify_pipelines_toolkit-1.2.4.dist-info/METADATA,sha256=UXCHFcEcjuPMZvUgtzITSY_iIG-j_nfGVBMGCWjBjjA,5775
+mgnify_pipelines_toolkit-1.2.4.dist-info/WHEEL,sha256=_zCd3N1l69ArxyTb8rzEoP9TpbYXkqRFSNOD5OuxnTs,91
+mgnify_pipelines_toolkit-1.2.4.dist-info/entry_points.txt,sha256=sHDxlHizt_iZPtkNp0EDuohDGvC4O12B57JtpUmHwYk,3123
+mgnify_pipelines_toolkit-1.2.4.dist-info/top_level.txt,sha256=xA_wC7C01V3VwuDnqwRM2QYeJJ45WtvF6LVav4tYxuE,25
+mgnify_pipelines_toolkit-1.2.4.dist-info/RECORD,,

{mgnify_pipelines_toolkit-1.2.2.dist-info → mgnify_pipelines_toolkit-1.2.4.dist-info}/entry_points.txt

@@ -23,6 +23,7 @@ make_asv_count_table = mgnify_pipelines_toolkit.analysis.amplicon.make_asv_count
 mapseq2biom = mgnify_pipelines_toolkit.analysis.shared.mapseq2biom:main
 mapseq_to_asv_table = mgnify_pipelines_toolkit.analysis.amplicon.mapseq_to_asv_table:main
 markergene_study_summary = mgnify_pipelines_toolkit.analysis.shared.markergene_study_summary:main
+permute_primers = mgnify_pipelines_toolkit.analysis.amplicon.permute_primers:main
 primer_val_classification = mgnify_pipelines_toolkit.analysis.amplicon.primer_val_classification:main
 process_dbcan_cazys = mgnify_pipelines_toolkit.analysis.assembly.process_dbcan_result_cazys:main
 process_dbcan_clusters = mgnify_pipelines_toolkit.analysis.assembly.process_dbcan_result_clusters:main