Barcode mismatch R1 and R2

[vl80]

Hi,

We are demultiplexing some in house barcodes, 5 nt in length as mentioned in a previous post for 2 x 150 Miseq runs
When this is done, there is a very high frequency of mismatch between R1 and R2 in barcode splitter. The barcode file used is the same. Is there any need to use the complment, or the reverse for the splitting of R2?

Thanks a lot

[vl80]

Just adding to the above question, has anyone had any experiences with barcode/index (inhouse) mistmatch between R1 and R2 and the percentage of such mistmatched reads?We are really stuck with the issue as nearly 90% of our reads show a mistmatch between R1 and R2. To give more details, the barcodes are at the end of the amplicons (both 5' and 3') and are 5 nt in length.

[GenoMax]

How can there be a mismatch as long as the experiment was done right? I assume you have R2 barcodes that look completely different than expected i.e. this is not a case of one base pair mismatch out of expected 5 bp? Are you using a custom script to do the demultiplexing?