Multiplexing

[attila.szanto]

Thanks. The confusion came from the difference between the two illumina primers I mentioned (PE PCR Primer 2.0 and Multiplexing PCR Primer 2.0). The first one is for regular paired end seq and the other is for multiplexing. So, I had to design my oligos, so that I could do paired end seq and also include a barcode to filter out pcr artifacts. And I followed the logic of the standard illumina multiplexing oligos. However, instead of using 3 primers in the pcr, I add the barcode to the adapters. And just want to double check if the library ready for seq has the right sequences to bind to the flow cell and also to seq with Multiplexing Read 1, Read 2 and Index Read primers. I could find such a sequence only for PE but not for multiplexing.
Anyway, thanks for your help.

[cascoamarillo]

It's easy to check if you do a little of research. What I see is a standard Illumina RNA-seq library ready for multiplexing; with the read primer 1 (Rd1 SP) region, the index SP primer and the read primer 2 (Rd2 SP).

I'm not sure about the other primers you mention, couldn't find any match with what I have. Are they for DNA, RNA-seq, smallRNA...?

[attila.szanto]

Can anyone please check the following seq if it could work for a multiplexing PCR product?

AATGATACGGCGACCACCGAGATCT ACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNN AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC NNNNNN ATCTCGTATGCCGTCTTCTGCTTG
TTACTATGCCGCTGGTGGCTCTAGA TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA NNNNNN TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG NNNNNN TAGAGCATACGGCAGAAGACGAAC

Also, I would appreciate if someone could tell me why there is a difference between PE PCR Primer 2.0 and Multiplexing PCR Primer 2.0. I mean between the middle part:

CAAGCAGAAGACGGCATACGAGAT CGGTCTCGGCATTCCTGCTGAACC GCTCTTCCGATCT and the
----------------------------GTGACTGGAGTTCAGACGTGT GCTCTTCCGATCT