demultiplex paired end fastq file with mixed samples with barcode
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3 months ago
hellocita ▴ 30

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).

RNAseq fastq demultiplex • 306 views
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3 months ago
GenoMax 104k

You should be able demultiplex the data using demultiplexbyname.sh from BBMap suite. Use directions here: demuxbyname.sh output help

You can also use deML but this should be straight forward.

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thanks for your recommending!!! I then checked at the unmatched reads and found some R2.fastq reads and some R1.fastq reads contain both reverse and forward barcode. that seems to be why these reads were unmatched. Would this be caused by problems of sequencing? Do you think if I try demultiplexbyname.sh can improve the proformance?

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There can be only one version of the index sequence present in the output. If you expect TATAGCCT+CGAGTAAT combination then you should use that when you demultiplex the data.

If you experienced problems with sequencing that is a different issue. No tool is not going to help with those.