Demultiplexer - GenomicsCoreLeuven/GBSX GitHub Wiki

This program demultiplexes fastq or fastq.gz files obtained from sequencing with inline barcodes.
Like used in GBS, RAD, ... protocols.

These parameters are mandatory:

  • -f1 the name and path of the fastq or fastq.gz file to demultiplex
  • -i the name and path of the info file. This is a tab delimeted file without headings, with three columns: sample, sequence of the barcode, name of the enzyme

These parameters are optional:

  • -f2 the name of the second fastq or fastq.gz file (only with paired-end sequencing)

  • -o the name of the output directory (standard the directory of the call)

  • -lf use long file names (standard false) filename is standard the sample name, long file names is sample name _ barcode _ enzyme

  • -rad if the data is rad data or not (-rad true for RAD data, -rad false for GBS data) standard false (GBS)

  • -gzip the input and output are/must be gziped (.gz) (standard false: input and output are .fastq, if true this is .fastq.gz)

  • -m the allowed mismatches in the barcodes + enzymes (standard this value is 1)

  • -mb the allowed mismatches in the barcodes (overrides the option -m)

  • -me the allowed mismatches in the enzymes (overrides the option -m)

  • -minsl the minimum allowed length for the sequences (standard 0, rejected sequences are found in the stats for each sample in the rejected.count column. The sequences are found untrimmed in the undetermined file.)

  • -n keep sequences where N occurs as a "nucleotide" (standard true)

  • -ca the common adaptor used in the sequencing (standard (only first piece) AGATCGGAAGAGCG) currently only used for adaptor ligase see -al and when -rad is true) (minimum length is 10)

  • -s the posible distance of the start. This is the distance count from the start of the read to the first basepair of the barcode or enzyme (standard 0, maximum 20)

  • -cc Checks the complete read for the enzyme (if false, stops at the first possible enzyme cutsite) (use values true or false, standard is true). If used, the sequence after the enzyme site is compared to the adaptors, if the first basepairs of the sequence are compaired to the first basepairs of the adaptor

  • -kc Keep the enzyme cut-site remains (standard true) (example: enzyme ApeKI and restriction site G^CWGC: "ApeKI \tab CAGC,CTGC")

  • -ea Add enzymes from the given file (keeps the standard enzymes, and add the new) (enzyme file: no header, enzyme name tab cutsites (multiple cutsites are comma separeted)) (only use once, not use -er) (example: enzyme ApeKI and restriction site G^CWGC: "ApeKI \tab CAGC,CTGC")

  • -er Replace enzymes from the given file (do not keep the standard enzymes) (enzyme file: no header, enzyme name tab cutsites (multiple cutsites are comma separeted)) (only use once, not use -ea)

  • -al check for adaptor ligase: no (for no check) or a positive integer (starts at 0), for the number of mismatches (only checks 10 basepairs of the adaptor), standard 1

  • -scb Use self correcting barcodes (barcodes created by the barcodeGenerator) (standard false)

  • -malg the used algorithm to find mismatches and indels, possible algorithms:
    * hammings (Standard) Checks for mismatches (no indels)
    * knuth Faster than hammings, but can miss some locations
    * indelmis Checks for mismatches and indels, the barcode/enzyme/ adaptor with the least errors (mismatches or indels) is taken
    * misindel Checks for mismatches and indels, the mismatches are supperior to the indels (faster than indelmis, but errors can be higher)

  • -q the kind of quality scores used in the fastq file (including how phred scores are encoded):
    * Illumina1.8 (Standard)
    * Illumina1.5
    * Illumina1.3
    * Sanger
    * Solid

Possible Standard Enzymes for the info file: (NAN is no enzyme)

  • ApeKI
  • PstI
  • EcoT22I
  • PasI
  • HpaII
  • MspI
  • PstI-EcoT22I
  • PstI-MspI
  • PstI-TaqI
  • SbfI-MspI
  • AsiSI-MspI
  • BssHII-MspI
  • FseI-MspI
  • SalI-MspI
  • ApoI
  • BamHI
  • MseI
  • Sau3AI
  • RBSTA
  • RBSCG
  • NspI
  • NAN