The sort function demultiplexes the pooled sequence data into barcodes corresponding to each of the tag combinations that were used in the pool. The usage of sort is given below.
usage: Begum sort [-h] [-p PrimerFile] [-p1 FwdPrimer] [-p2 RevPrimer] -t
TagFile -s SampleInformationFile -l PoolInformationFile
[-m] [-pm PrimerMismatches] [-tm TagMismatches]
[-mo MinOverlap] [-mm OverlapErrRate] [-d OutDirectory]
[-o OutPrefix]
optional arguments:
-h, --help show this help message and exit
-p PrimerFile, --primers PrimerFile
File with forward and reverse primer sequence (Format:
ForwardPrimer ReversePrimer)
-p1 FwdPrimer, --fwdPrimer FwdPrimer
Sequence of forward primer
-p2 RevPrimer, --revPrimer RevPrimer
Sequence of reverse primer
-t TagFile, --tags TagFile
File with tag name and sequence (Format: TagName
FwdTagSequence)
-s SampleInformationFile, --sampleInfo SampleInformationFile
File with tag combo and pool for each sample (Format:
Sample FwdTagName RevTagName PoolName)
-l PoolInformationFile, --pool PoolInformationFile
File with pool information (Format: Poolname
Read1Fastq [Read2Fastq])
-m, --allowMultiplePrimers
Allow more one occurrance of the primer sequence in
read. (Default False)
-pm PrimerMismatches, --primerMismatches PrimerMismatches
Number of mismatches in primer. (Default 0)
-tm TagMismatches, --tagMismatches TagMismatches
Number of allowed mismatches in tags. (Default 0)
-mo MinOverlap, --merge_overlap MinOverlap
Merge read1 and read2 if overlapping by given number
of bases or more (>=5) __NOT IMPLEMENTED YET__
-mm OverlapErrRate, --merge_errors OverlapErrRate
Rate of mismatches allowed in overlap between reads:
range [0,0.2] __NOT IMPLEMENTED YET__
-d OutDirectory, --output_directory OutDirectory
Output directory. (Default: .)
-o OutPrefix, --output_prefix OutPrefix
Prefix for output files. (Default : '')
The sort function requires 4 files, which are now described in detail.
The primer file contains just one line, with two dna sequences on it, seperated by whitespace. The first sequence is the forward primer, and the second sequence is the reverse primer. Instead of specifying the primers in a file, one can specify the primers on the command line directly, using teh -p1 and -p2 arguments. Primer sequences need to be specified for begum to function properly. Primers sequences are allowed to have IUPAC ambiguity in them.
The tag file contains a list of all the tag sequences used in the pooling of multiple samples in one pool. The format of the tag file is a two column file, with each line representing a tag sequence. The first column is the tag name whereas the second column is the tag sequence. Note that, unlike the primer sequence, the tag sequences are NOT allowed to have IUPAC ambiguity codes in them.
The pool information file contains information on the fastq sequence files for each of the pools in a study. The file contains 2 or 3 columns, with each line containing information on a single pool. The first column is the name of the pool (case sensitive), the second column is the name of the single end fastq file (Read 1 if paired end) and the third column contains the name of the second read in a paired end sequencing experiment (absent if single end). Note that all the pools must have the same type of sequencing, i.e., either all the pools must use single end sequencing (or collapsed using AdapterRemoval/cutadapt), or paired end sequencing.
The sample information file contains information on the various replicates for each sample. This is a 4 column file, with the first column being the sample name, the second column being the name of the forward tag, the third column being the name of the reverse tag, and the last column being the pool that this sample-tag combination is present in. It is important to note that the order of the samples is NOT important, and that the replicate number for a sample is assigned automatically. Further, the name of the samples, pools and tags are case sensitive and must be exactly the same across differnt files.