This script is demultiplexing fastq.gz file based on the header barcode using multi processors. The script is subsampling the fastq file, and return the abundance of the headers that will be used if, you do not have a barcode file.

In some cases, sequencing centers will put the barcode/indexes sequences in the fastq sequence labels/headers before handing it off.

In some cases, sequence barcodes are not provided in a separate file, or a dual barcoding strategy may have been applied during sequencing. From the headers, the script will generate a list of barcodes that will be used to demultiplex the file.

It is important to know the exact length of the barcode:

##Dependencies Biopython: [sudo] pip install biopython

##Example of headers:

    Dual indexed
        @M01132:152:000000000-AUA7D:1:1102:16025:1335 1:N:0:ACGCAAC+CCGATTG
        GGTGATATTGTTTGTTATCGTTTAATATTGCGCTATATTTTAAAAAAGCTATATTTATTCCCGTATATACTCGGCGATTGCTAAATTCACAATTATATTTTTTGTTTATCATTCAATTCAGATAAAAAACAACGATAAATTGATTCTAAAAAAGAAATGAGGTTATAAAGACATTAAGAAAACAGGCAATAAAATATAGCGATCGAAACACGTTAACAAAATGAGTCTCATTATCAGAGTAGGACAACAGG
        +
        AAAA>FFFFDBFGFFGGGGGFGEHBBGGHDG?GGAFHFFFGFGDFHEEHHHHFHHHHHHHHHGGEHHHHFFHGFGE>EHGBGHHHHHHGGHHHGHHHHHHGHGGGHCEGHHHHHGHHHHHHEHGFHHHCGEHECFHGGHGHHHHHHHFHDGB@?FG<FGEHHHFHHGHFHGHHHHHHHHHHHHHHEEHHHHGHHHGGHHGGGEECGGFGGGGFGGGGGGEFGFGGFFFGFGGGGGGFBFFFFF/BBFFFFF

    Single indexed
        @MISEQ03:64:000000000-A2H3D:1:1101:14358:1530 1:N:0:TCCACAGGAGT
        TNCAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGAAACTGACACTGAGGGGCGAAAGCGGGGGGGGCAAACG
        +
        ?#5<????DDDDDDDDEEEEFFHHHHHHHHHHHHHHDCCHHFGDEHEH>CCE5AEEHHHHHHHHHHHHHHHHHFFFFHHHHHHEEADEEEEEEEEEEEEEEEEEEEEEEE?BEEEEEEEEEEEAEEEE0?A:?EE)8;)0ACEEECECCECAACEE?>)8CCC?CCA8?88ACC*A*::A??:0?C?.?0:?8884>'.''..'0?8C?C**0:0::?ECEE?############################
       

##Usage

usage: Demultiplex_by_headers2.0.py [-h] -i IFILE [-t THREAD] [-m MIN_READS]
                                    -l LENGTH [-L LIST]

optional arguments:
  -h, --help            show this help message and exit
  -i IFILE, --ifile IFILE
                        Input file
  -t THREAD, --thread THREAD
                        Input Number of threads
  -m MIN_READS, --min_reads MIN_READS
                        Minimum reads per barcode [OPTIONAL]
  -l LENGTH, --length LENGTH
                        Length of the barcode
  -L LIST, --list LIST  List of barcodes[OPTIONAL]

##Example:

    Dual indexed
        @M01132:152:000000000-AUA7D:1:1102:16025:1335 1:N:0:ACGCAAC+CCGATTG
        GGTGATATTGTTTGTTATCGTTTAATATTGCGCTATATTTTAAAAAAGCTATATTTATTCCCGTATATACTCGGCGATTGCTAAATTCACAATTATATTTTTTGTTTATCATTCAATTCAGATAAAAAACAACGATAAATTGATTCTAAAAAAGAAATGAGGTTATAAAGACATTAAGAAAACAGGCAATAAAATATAGCGATCGAAACACGTTAACAAAATGAGTCTCATTATCAGAGTAGGACAACAGG
        +
        AAAA>FFFFDBFGFFGGGGGFGEHBBGGHDG?GGAFHFFFGFGDFHEEHHHHFHHHHHHHHHGGEHHHHFFHGFGE>EHGBGHHHHHHGGHHHGHHHHHHGHGGGHCEGHHHHHGHHHHHHEHGFHHHCGEHECFHGGHGHHHHHHHFHDGB@?FG<FGEHHHFHHGHFHGHHHHHHHHHHHHHHEEHHHHGHHHGGHHGGGEECGGFGGGGFGGGGGGEFGFGGFFFGFGGGGGGFBFFFFF/BBFFFFF

    List barcode:
    The file containing the list of indexes should be identical to the header in the fastq file. 

    ACGCAAC+CCGATTG
    
    Command line:
        If you have a list of barcodes:
            python Demultiplex_by_headers.py -i file.fastq.gz -t 8 -l 15 -L index.list
        
        If you do not have a list of barcodes:
            python Demultiplex_by_headers.py -i file.fastq.gz -t 8 -l 15 -m 1000