demultiplex paired end fastq file with mixed samples with barcode

1

Hi everyone! I was using fastq-multx to demultiplex. And the demultiplex result is bad. 99.99% count were unmatched....

In the experiment the two samples were mixed in one sequence run, so I got R1.fastq and R2.fastq with mixed samples, with barcode designed as:
sample 1: forward barcode :TATAGCCT , reverse barcode CGAGTAAT
sample 2: forward: TATAGCCT, reverse TCTCCGGA

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. In head of the reads that unmatched, the forward barcode appear once the unmatched fastq R2 file, and twice in unmatched R1 file. And reverse barcode also appear in some R1 read, while not in some R2 read.


RNAseq


fastq


demultiplex

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