import pkg_resources import tempfile import magic import subprocess import tempfile import logging import re import io import gzip log = logging.getLogger(__name__ ) def read_fasta(sequence): entries = 0 bases = [] label = None for line in sequence: if line.startswith(">"): label = line entries += 1 else: bases.append(line) if entries > 1: log.debug("FASTA file contains multiple entries") raise ValueError("FASTA file contains multiple entries") return label, bases def qc_fasta(arg_sequence, check_with_clustalw=True): log.debug("Starting qc_fasta") schema_resource = pkg_resources.resource_stream(__name__, "validation/formats") with tempfile.NamedTemporaryFile() as tmp: tmp.write(schema_resource.read()) tmp.flush() val = magic.Magic(magic_file=tmp.name, uncompress=False, mime=True) gz = "" if arg_sequence.name.endswith(".gz"): sequence = gzip.GzipFile(fileobj=arg_sequence, mode='rb') gz = ".gz" else: sequence = arg_sequence sequence = io.TextIOWrapper(sequence) r = sequence.read(4096) sequence.seek(0) seqlabel = r[1:r.index("\n")] seq_type = val.from_buffer(r).lower() if seq_type == "text/fasta": # ensure that contains only one entry submitlabel, submitseq = read_fasta(sequence) sequence.seek(0) sequence.detach() if not check_with_clustalw: return ("sequence.fasta"+gz, seqlabel) with tempfile.NamedTemporaryFile() as tmp1: with tempfile.NamedTemporaryFile() as tmp2: refstring = pkg_resources.resource_string(__name__, "SARS-CoV-2-reference.fasta") tmp1.write(refstring) tmp1.flush() tmp2.write(submitlabel.encode("utf8")) tmp2.write(("".join(submitseq)).encode("utf8")) tmp2.flush() subbp = 0 refbp = 0 similarity = 0 try: cmd = ["minimap2", "-c", tmp1.name, tmp2.name] logging.info("QC checking similarity to reference") logging.info(" ".join(cmd)) result = subprocess.run(cmd, stdout=subprocess.PIPE) result.check_returncode() res = result.stdout.decode("utf-8") mm = res.split("\t") if len(mm) >= 10: # divide Number of matching bases in the mapping / Target sequence length similarity = (float(mm[9]) / float(mm[6])) * 100.0 else: similarity = 0 except Exception as e: logging.warn("QC against reference sequence using 'minimap2': %s", e, exc_info=e) if similarity < 70.0: raise ValueError("QC fail: alignment to reference was less than 70%% (was %2.2f%%)" % (similarity)) return ("sequence.fasta"+gz, seqlabel) elif seq_type == "text/fastq": sequence.seek(0) sequence.detach() return ("reads.fastq"+gz, seqlabel) else: raise ValueError("Sequence file does not look like a DNA FASTA or FASTQ")