#!/usr/bin/env python """ Copyright 2021 Daryl Gohl Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. """ from Bio import SeqIO import regex import numpy as np import matplotlib.pyplot as plt import os import argparse __version__ = "1.0" def get_args(x): x.add_argument("-i", "--input_folder", type = str, default = None, metavar = '', help="Input folder containing raw FASTQ files [required].") x.add_argument("-r", "--reference_file", type = str, default = None, metavar = '', help="Input reference FASTA file [required].") x.add_argument("-l", "--length", type = str, default = '14', metavar = '', help="Barcode length (default: 14).") x.add_argument("-o", "--output_dir", type = str, default = '', metavar = '', help = "Output directory for barcode count file (default: same folder as input file)") x.add_argument("-m", "--mismatches_allowed", type = str, default = '2', metavar = '', help = "Number of mismatches to barcode reference sequences allowed (default 2)") args = x.parse_args() return args # Parses command line arguments argparser = argparse.ArgumentParser(description = "TaG-EM barcode counting script (v" + __version__ +")\n" + \ "by Daryl Gohl\n" + \ "This program takes in a FASTQ file and barcode reference FASTA file and outputs a plot and a .txt file of barcode counts.", add_help = True, epilog ='') args = get_args(argparser) args = vars(args) # possible input args folder = args['input_folder'] Ref_filename = args['reference_file'] mismatches = args['mismatches_allowed'] out_folder = args['output_dir'] bc_length = int(args['length']) if out_folder == '': out_dir = os.path.dirname(folder) else: if not os.path.exists(out_folder): os.makedirs(out_folder) out_dir = out_folder #Possible_Amplicons #CTTCCAACAACCGGAAGTGANNNNNNNNNNNNNNtggttacaaataaagc #XXCTTCCAACAACCGGAAGTGANNNNNNNNNNNNNNtggttacaaataaa #XXXXCTTCCAACAACCGGAAGTGANNNNNNNNNNNNNNtggttacaaata #XXXXXXCTTCCAACAACCGGAAGTGANNNNNNNNNNNNNNtggttacaaa R1_primer = "CTTCCAACAACCGGAAGTGA" os.chdir(folder) data_file_names = os.listdir() #Trim reads for i in data_file_names: if i[-3:] == ".gz": c_file = i[:-9] R1 = c_file + "_trimmed.fastq" execute = "cutadapt -g " + R1_primer + " " + i + " > " + R1 os.system(execute) elif i[-5:] == "fastq": c_file = i[:-6] R1 = c_file + "_trimmed.fastq" execute = "cutadapt -g " + R1_primer + " " + i + " > " + R1 os.system(execute) data_file_names = os.listdir() files = [] for i in data_file_names: if i[-14:] == "_trimmed.fastq": fx = os.path.join(folder, i) files.append(fx) #Count up barcodes in fasta file bc_ID_list = [] #Construct name bc_seq_list = [] #Barcode sequence for record in SeqIO.parse(Ref_filename, "fasta"): bc_ID = record.id #Collect standard IDs from reference file bc_seq = str(record.seq) #Collect standard barcode sequences from reference file bc_ID_list.append(bc_ID) bc_seq_list.append(bc_seq) #Make counts file save_name = os.path.join(out_dir,("Barcode_counts.txt")) save_file = open(save_name, "w") newtab = '\t' newline = '\n' save_file.write("Sample") save_file.write(newtab) for i, item in enumerate(bc_ID_list): save_file.write(item) save_file.write(newtab) save_file.write(newline) save_file.write("Name") save_file.write(newtab) for i, item in enumerate(bc_seq_list): save_file.write(item) save_file.write(newtab) save_file.write(newline) #Count number of records in the file for i in files: filename = i file = os.path.split(filename)[1] #Count up barcodes in fastq file bc_ID_list = [] #Construct name bc_seq_list = [] #Barcode sequence count_list = [] #Barcode counts bc_all_list = [] count_all = 0 count_bc = 0 for record in SeqIO.parse(Ref_filename, "fasta"): count_bc += 1 count = 0 bc_sub_list = [] bc_ID = record.id #Collect standard IDs from reference file bc_seq = str(record.seq) #Collect standard barcode sequences from reference file for i in SeqIO.parse(filename, "fastq"): if count_bc == 1: count_all +=1 query = r'(?:' + bc_seq +'){s<=' + mismatches + '}' #fuzzy matching - allow up to mismatches test = regex.findall(query, str(i.seq[:bc_length])) if test != []: count += 1 bc_sub_list.append(str(i.seq[:bc_length])) count_list.append(count) bc_all_list.append(bc_sub_list) bc_ID_list.append(bc_ID) bc_seq_list.append(bc_seq) print("done with " + bc_ID) #print("There were " + str(count) + " records in file " + filename) total_recs = count_all account = float(np.sum(count_list)) perc = account/total_recs*100 #Plot fig = plt.figure() ax = fig.add_subplot(111) ind = np.arange(len(count_list)) ax1 = ax.bar(ind,count_list, width=0.8, bottom=None) width=0.8 ax.set_ylabel('counts') ax.set_title('Barcode counts') ax.set_xticks(ind+width/2) xtickNames = ax.set_xticklabels(bc_ID_list) plt.setp(xtickNames, rotation=90, fontsize=8) fig_name = os.path.join(out_dir,(file[:-6] + "_barcode_counts.png")) plt.savefig(fig_name,bbox_inches='tight') #Write out barcode count file save_file.write(file) save_file.write(newtab) for i, item in enumerate(count_list): save_file.write(str(item)) save_file.write(newtab) save_file.write(newline) print("There were " + str(account) + "/" + str(total_recs) + "(" + str(perc) + "%) records accounted for.") save_file.close()