TruSeq / SPRIworks?

[jlove]

Is anyone else using the SPRIworks system with Illumina's TruSeq kits? We are noticing some unfortunate changes. We started using SPRIworks with the old adapters and didn't have much trouble. Now that we are using TruSeq, suddenly the adapter dimer situation is much worse. Also, some libraries are running higher in size than they should be after PCR - for instance, a 200-400 bp size selection is resulting in some libraries with an avg size of 500 bp.

It seems like the size selection is inconsistent.

I'd love to hear any feedback or experiences from other groups.

[ashchin]

Hi ,
If you overload the pcr reaction you get fragments greater than 600bp along with 200 to 400 bp.

[HESmith]

The TruSeq adapters are substantially different from the earlier ones, with a much larger region of unpaired sequence, that causes the libraries to migrate anomalously during gel electrophoresis. Illumina recommends using Sybr Gold staining to mitigate the problem.

[jlove]

Hi ,
If you overload the pcr reaction you get fragments greater than 600bp along with 200 to 400 bp.

Ashchin - do you mean overload the PCR with template? I don't *think* that's happening -this is ChIP-Seq and thus I am using very little input. It actually doesn't seem to happen as much with greater input, such as with genomic DNA sequencing.

Thanks for the suggestion, I'd like to hear more!

[jlove]

The TruSeq adapters are substantially different from the earlier ones, with a much larger region of unpaired sequence, that causes the libraries to migrate anomalously during gel electrophoresis. Illumina recommends using Sybr Gold staining to mitigate the problem.

Thanks HESmith - I am actually using SPRIworks, which does the size selection using beads, so SYBR staining isn't an option here... but the question is, why are they migrating anomalously? I am under the impression that they are slipping through the size selection because they are just under the cutoff size of 200-400 bp. Do you think they are also clumping together and running larger? This might make sense, if so, since they are showing up in the clusters in larger numbers than the bioA leads me to believe when I look at the 125 bp peak.

[vtosha]

Do you run size selection after adapter ligation? As I can suppose some fragments could ligate to each other in spend of adapter ligation, and so we get chimera libraries with length approximately twice than we wait. Try to take more adapters.

[jlove]

Do you run size selection after adapter ligation? As I can suppose some fragments could ligate to each other in spend of adapter ligation, and so we get chimera libraries with length approximately twice than we wait. Try to take more adapters.

vtosha- Thank you for your reply. We do run the size selection after ligation, so I don't know if this is happening or not. But it's worth looking into, thank you.

[genbio64]

@jlove, did you figure out the SPRI works problem?

[jlove]

Hi GenBio64- We stopped using SPRIworks altogether, and I think the reason for our troubles had to do with the fact that we were doing ChIP-Seq (with tiny amounts of input), which does not size select very well on beads. It works fine with larger inputs, generally, but I don't have any experience with it using TruSeq, since it's been so long since I tried using it. I just do them manually.

[pmiguel]

Ampure beads work well with ng levels of DNA. However the rest of the SPRIworks reagents are likely calibrated for much higher levels of inserts. Might have worked to dilute the adapters 10x to maintain a reasonable molar ratio of insert:adapters.

--
Phillip

[jlove]

Phillip- you're right, I do ChIP manually with bead purifications and it works very well. The reaction volumes (and bead amounts) were not compatible with the small inputs in the SPRI cartridges.