Multiplex Sample Prep: Three Primer Method question

[emucaki]

There is an older library prep method where samples were indexed using a 3 primer PCR method (forward and reverse primer + index primer). Illumina changed this, and I've seen people on SeqAnswers complain that it was inefficient. Unfortunately, those are the only primer types I have right now.

1) Do any labs have experience with this method, and have any tips dealing with these issues?

2) I assume that the 3 primers exist in equal amounts, but the protocol does not actually list concentrations (just volumes). Does anyone know if the primer concentrations should be equal for this amplification.

Thank you.

[kcchan]

Wow, that is a very old protocol. From what I remember, the three oligos are of equal concentration in the PCR reaction. The process was really tacked on and thus very inefficient since fragments that don't get the index sequence will not cluster. I believe that these libraries also require a primer that is different from TruSeq, but it may still be included in the standard sequencing primer mix. Nowadays all of the necessary sequences are added during the ligation step; PCR is optional and only used to amplify the yield. I would only use this method if you have no other alternative.

[beibeizhou]

three primers are not the same concentration.primer 1.0 and multiplex primer are 25uM,primer 2.0 is 0.5 uM

[emucaki]

Thank you for the responses.

Having the 2.0 primer at a lower concentration makes sense, as it creates the binding site for the indexing primer to amplify. However, since I'm getting conflicting results, I wanted to ask again if anyone could confirm the primer concentrations.

The "Illumina Customer Sequence Letter" lists TruSeq adapters with the index already present. Are those the ones that should be used instead of this older method? They do not seem to mention the phosphorylated 5' end as all the others require. Anyone know why this is?

[kcchan]

The sequences you're looking for with the older kit are in the section labeled "Oligonucleotide sequences for the Multiplexing Sample Prep Oligo Only Kit". The newer TruSeq adapters already have the index sequences in the forked adapter.

The adapters in the sequence letter are really only meant for QC and data filtering purposes. They don't disclose the modifications required to actually design some of the primers.

[emucaki]

We are performing a sequence capture step, so do you think this would improve amplification efficiency:
- amplify (PCR 1) with the forward and reverse primers normally
- capture sequence (with appropriate blocking primers, of course)
- amplify (PCR 2) this captured library DNA with the forward primer and the appropriate indexing primer (which hybridizes to a binding site left by the reverse primer overhang)

This way, we never have 3 primers in the PCR solution at once. The only downside is that I have to capture 1 sample per tube instead of pooling, which will help overall capture efficiency but increases work/costs involved.