Hi everyone! I was using fastq-multx to demultiplex and met some problems.In the experiment the two samples were mixed in one sequence run, so I got R1.fastq and R2.fastq with mixed samples, with index and barcode designed as:
sample 1: forward barcode :TATAGCCT , reverse barcode CGAGTAAT sample 2: forward: TATAGCCT, reverse TCTCCGGA
First I use 8bp barcode to demultiplex, but the demultiplex result is bad. 99.99% count were unmatched....
and then I use the full length of index (33 nt, with 8 nt barcode) to demultiplex, and only 8%-10% reads can be demultiplex. ~90% reads were unmatched.
I checked the unmatched reads file, found some reads lack of barcode, some reads contain both forward and reverse barcode, some reads seems to have wrong barcode (e.g. forward barcode in R2 file).