[pmiguel]

Even less likely. Have a look at the flow cell sequences and how the bridge PCR works. Any 5' distal sequence would really be problematic.

I'm going to try to amplify a standard PE library with the TruSeq primers. If they amplify, we know that the primers are not full length. If they don't, maybe they are.

Yes, I agree with your logic. Interested to see your result.

One final piece of information: I was able to get directly from Illumina (and a very good source) that the PCR primer cocktail does NOT contain a mix of all 12 indexes and that the sequences are not full length. They were not able to provide any more details than that.

Okay. Good to hear that. Probably some modification or something innocuous causing them to run as if they were much longer molecules.

--
Phillip

[csquared]

I finally had a chance to play around with the TruSeq primers and how they amplify the TruSeq adaptors as well as standard PE adaptors.

The summary version is that the TruSeq primers are highly unlikely to be the full length of the adaptors and very very likely to be the same sequences that Roche released for use in the Nimblegen capture protocols some time ago.

A standard Illumina PE library that has undergone PCR amplification to create a complete and sequenceable library amplifies very well with the TruSeq PCR primer cocktail. However, an unamplified standard PE library does not amplify at all, suggesting that the TruSeq primers are located 5' of the adaptor sequences and also 5' of the sequence differences between the TruSeq and PE adaptors.

In other words, you can make TruSeq adaptors however you like and then use the Nimblegen PCR primers to amplify the libraries and get results equivalent to the full TruSeq reagents for those looking for the equivalent to a TruSeq oligo only kit.

Message me if anyone wants the bioanalysis traces for the different conditions.
.

[pmiguel]

Interesting and good to know.

Okay, the TruSeq PCR primers (PPC) do not extend the full length of the adapters. But I still wonder what cause them to migrate as if they were 80+ nt long. The obvious hypotheses include:

(1) They really are that long. But your results show that they prime from the 3' end of the TruSeq adapters. So any extra bases would need to be on the 3' end and extend 3' beyond the end of the adapters.
I think you discount this possibility earlier in this thread due to issues with compatibility with the flow cell oligos. But I was not clear as to what your objection was.

(2) Something other than nucleotides (actual oligo length) produces the aberrant migration rates. This could be a lot of things. Something positively charged or bulky would fit the bill. Ideas?

--
Phillip

[csquared]

(1) They really are that long. But your results show that they prime from the 3' end of the TruSeq adapters. So any extra bases would need to be on the 3' end and extend 3' beyond the end of the adapters.
I think you discount this possibility earlier in this thread due to issues with compatibility with the flow cell oligos. But I was not clear as to what your objection was.

My concern is that anything that is added to the complete fragment during PCR that results in the addition of sequences beyond the sequences that hybridize to the flowcell (P5 and P7 sequences) would possibly interfere with the cluster generation and therefore unlikely to be added with no purpose. There is a short spacer to get the P5 and P7 sequences away from the surface of the flow cell, but I don't think an additional sequence would be added during PCR.

Plus, final TruSeq library QC always shows about a 120bp increase in size over the input DNA and no difference in final fragment size pre and post amplification, further indicating that the pre and post PCR fragment size is the same and the primers don't add anything extra to the TruSeq fragments.

[ECO]

I only do amplification-free library prep with TruseqV2 reagents, and homemade reagents/adapters, and can confirm that they cluster just great.

To the OP's question, it's totally straightforward to just cut the Truseq rxns directly in half. The only small challenge you may encounter is eluting the ER reaction in 10ul, and taking ~8.75 on to the AT/ligation step, but it's very doable.

[mchotalia]

To ECO,

When you say that you cut the TruSeq reactions in half, would you make any changes to the AMPure purification after ER and ligation?

Thanks

[zwirko]

Does anyone have experience with picogram-scale input for library construction? If not, ideas, suggestions, or general musings?

[hawaii454-0]

I have managed to get some okay-looking TruSeq libraries from some tests on E coli which were sheared at around 150bp and the starting material was 50ng, 25ng, and 10ng. The 50ng library (obviously) looks the best. The adjustments I made to the protocol were in the ratios of ampure beads to sample in the clean ups through the protocol. Prior to the adapters being added, the ratio was 1.6:1 and afterwards it was 1.2:1. This seemed to work well for the size of the fragments I sheared. The next step will be checking the Agilent concentration with the ones on the Qubit and after qPCR.