NEBNext Multiplex Oligos for Illumina: library preparation - (Dec/06/2013 )
I have a question about library preparation using NEBNext multiplex oligo for Illumina (index primers set 1). During PCR enrichment of adaptor ligated DNA, both the universal PCR primer and the index primer are added. I have noted that the complimentary regions (to the NEBNext adaptor for Illumina) of both these primers is identical (5'... GCTCTTCCGATC*T 3') and for both is at the 3' end. Therefore during PCR 3 types of products will form; the target DNA with the universal primer at both ends, the target DNA with the index primer at both ends and the target DNA with the universal primer at one end and the index primer at the other. This doesn't seem logical to me as some product will not have the addition of the index. I think therefore I must have made a mistake but I cannot understand where. Can anyone help me in letting me know how the index primer and the universal primer bind to the adaptor ligated DNA and thus what product will be formed. Thank you.
The annealing temperature for the two primers is quite high (65) which may minimize incorrect binding. But even if incorrect binding occurs, and you do get a mix such as you describe, bridge formation on the flow cell will not occur with those AA or BB fragments -- only with the AB fragments. So, at worst you will incorrectly quantify the library preparation when loading it on the flow cell. The situation is even more extreme with Nextera libraries, where the 19 bp adjacent to the desired sequencing region is identical, and reverse complement. Other sample prep kits and index primer sequences avoid priming so close to the Read 1 and Read 2 starts, and prime instead to regions further 5' on the adapters, probably to avoid this issue.