Demultiplex fastq files (accepts gzip compression) acording to barcode sequences present on reads.
plexseq takes fastq formated reads and a barcode file to separate sequencing reads according to said barcodes.
Data may be provided in several ways:
1) single fastqfile (single end reads, -r option)
2) two fastq files (R1 R2 paired reads, -r and --r2 options)
3) interleaved fastqfile (R1 followed by R2, --ri option)
4) fastq data from STDIN (single or interleaved paired end, --s option)
A barcode file must be provided and data should be structured as follows:
tab separated file with at least two columns
| BARCODE_ID | BARCODE_SEQUENCE | BARCODE_SEQUENCE2(optional) |
|---|
Example:
| #ID | Seq | R2(optional) |
|---|---|---|
| A1F1 | CAGTCGAAT | TCACAGATG |
| A1F2 | TCAGTCGAA | TCACAGATG |
| A1F3 | CTCAGTCGA | TGCAGATGA |
| A1F4 | CAGAAAAGT | TTGTTCTCC |
| A1F5 | TCAGAAAAG | TGTTCTCCA |
| A1F6 | CTCAGAAAA | TGTTCTCCA |
| A1F7 | TCAGAAAAG | CTTGTTCTC |
| A1F8 | TCAGTCGAA | CTCACAGAT |
lines starting with # are ignored
plexseq works by looking the barcode sequence in specific parts of the sequence read. For now plexseq looks for barcode sequence 1 in the first 9 bases of the 5' end of read1. If a second barcode sequence is provided, plexseq will look for this sequence in the first 9 bases of the 5' end of read2 in the case of paired reads.
plexseq looks for exact matches between reads and barcodes.
This limited functionality is just for the moment. Future versions will automatically set the number of bases to look in each read based on the length of the barcode, and lookups with mismatches will be introduced.
Usage:
python demultiplex.py -r1 "read_file" -i "indexfile" -o "outdir" [--r2 "read2_file" --ri "interleaved_read_file"]
Or:
cat seqfile | python demultiplex.py -i "indexfile" -o "outdir" --s
For the moment plexseq can only separate sequences which exactly match barcode sequences
All barcode sequences have to be of the same length
Can not write gzip compressed files
Barcode sequences have to be located at the 5' end of reads
Only fastq files can be demultiplexed