[GenoMax]

Did you also remove the corresponding Q-score value? If you used trimmomatic/bbduk they would do this but if you used some other method then you may have an extra Q-score value remaining on line 4 for each fastq record.

I am not sure why you are having a problem but if I use the two reads you posted above I am able to demultiplex them fine (after taking the N's out).

[smitra]

I haven't tried trimmomatic/bbduk yet. But this barcode splitter is playing strange with me. Yes with N's out they demultiplex but never the whole data. every time some reads remained which is completely unexplainable. If I do again with those unmatched read again some bit match. which is very strange.

But at present i am just doing the N's out for the whole data and seems with two stage I am getting success. Thanks for helping.

[luc]

You might want to try Sabre instead:

https://github.com/najoshi/sabre

Works also on paired-end data.

The FASTX toolkit is very old by now.

[smitra]

Thank you Luc. I will give it a try.

[sjuzhet]

This might be a basic question, but I thought I'd hop in this topic to ask - half of my reads were read in the opposite direction, so the barcode is reverse complemented at the end of the read. Is there a way to get Fastx Toolkit to incorporate reverse complement barcodes at the end of the line as well, or do I have to do this manually?

[GenoMax]

This might be a basic question, but I thought I'd hop in this topic to ask - half of my reads were read in the opposite direction, so the barcode is reverse complemented at the end of the read. Is there a way to get Fastx Toolkit to incorporate reverse complement barcodes at the end of the line as well, or do I have to do this manually?

Fastx_barcode_splitter has --eol (end of line/read) option to check reads at the 3' end. Provide the barcodes appropriately in the barcode file. You will need to run --bol and --eol sequentially (with individual barcode files) since those two options can't be combined.

[sjuzhet]

Fastx_barcode_splitter has --eol (end of line/read) option to check reads at the 3' end. Provide the barcodes appropriately in the barcode file. You will need to run --bol and --eol sequentially (with individual barcode files) since those two options can't be combined.

Yeah, that's what I did. I was hoping to get a report on the number of reads that didn't match to either bol or eol, but I guess I can just calculate it.