question on making libraries from pcr products

[seqgirl123]

Does anyone make libraries for Solexa from their PCR products? What is the best way to start off trying to do this?

If I have 3 different amplicons, all are 800bp size, and I pool all three together, what is the best way to start making a library out of them for solexa sequencing?

If I have another 3 amplicons that are different sizes, like 200bp, 300bp, and 400bp, and I pool them together, how would I go about making a library out of the pool?

thanks for any help!

[greigite]

Does anyone make libraries for Solexa from their PCR products? What is the best way to start off trying to do this?

If I have 3 different amplicons, all are 800bp size, and I pool all three together, what is the best way to start making a library out of them for solexa sequencing?

If I have another 3 amplicons that are different sizes, like 200bp, 300bp, and 400bp, and I pool them together, how would I go about making a library out of the pool?

thanks for any help!

I have prepped pools of amplicons of different sizes for 454 libraries, not quite what you want to do but the initial steps should be similar. I found that covaris shearing worked best for fragments of 1-2 kb length. For your smaller amplicons, the issue with pooling is that the smaller fragments are going to be preferentially amplified in the post adapter ligation pcr. Is there a reason you need to pool them, or could you just tag each set with a barcoded adapter and mix them post-amplification?

[seqgirl123]

Ok I have some questions:

For #1, since the amplicons are all the same size (800bp), they need to be end repaired, ligated, and sheared first to a lower size for solexa sequencing? I've heard large library sizes cannnot be sequenced on Solexa and that most people use 200bp to obtain good solexa sequences, but an 800bp library may be too large. Is this all true? And if the amplicons are all 800bp and a 200bp library is made from them, there would be no PCR bias after adapter-ligation?

For question #2, the amplicons are of different sizes so yes there could be bias towards the smaller fragments (200bp) that will be amplified more than the larger fragments (400bp). But is it possible to pool the three different amplicons together, end repair and ligate the pool, shear the pool to 200bp, and then proceed through normal library construction? This would eliminate any bias for the smaller fragments because all amplicons are sheared to the same size? Also, ligation step of the pool would eliminate any bias towards small end fragments that amplification could also have a bias towards? Does shearing the 200bp amplicons in the pool produce fragments smaller than 200bp?


Are there protocols for these pooled PCR libraries? Would like to stay away from adapter barcodes and use basic solexa library construction at this time.

[krobison]

A recent review covers PCR along with other (Molecular Inversion Probe, hybridization) methods for targeted sequencing.

You might also look at this paper to see how they handled the situation:
Tewhey, R. et al. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat. Biotechnol. 27, 1025–1031 (2009).

Method for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology.
Harismendy O, Frazer K.
Biotechniques. 2009 Mar;46(3):229-31.
PMID: 19317667