lib prep using SPRIworks

[sgupta]

Hi,

We have been recently experiencing that sometimes library preps done using SPRIworks show up with a larger fragment size than expected on the Bioanalyzer. And if we go ahead and sequence those samples, the G signal intensity is very low for those samples which eventually leads to fewer G calls and a poor alignment rate.

I am wondering if anyone else has been experiencing something similar and may have more insight into the cause of this kind of phenotype.

Thanks for your help in advance.

[pmiguel]

I don't think SPRIworks controls the upper fragment size. Maximum amplicon length then would solely be be determined your input fragment size.

Are your input fragments larger than normal?

--
Phillip

[sgupta]

Hi pmiguel,

Thanks for your reply. We are seeing this phenotype predominately with whole cell extracts. Basically, the sample is split into 2 parts, one becomes the WCE, and the other goes through the ChIP. The IP looks fine after the library prep but the WCE's have a larger average fragment size.

We ran the original WCE (before library prep/ligation of adaptors) on bioanalyzer also and as expected, it looks like a smear.

Thanks again. Any inputs will be greatly appreciated.

[pmiguel]

You could do a size selection after SPRIworks to eliminate the larger amplicons.

What size do you see currently?

--
Phillip

[sgupta]

Hi Phillip,

That is actually the problem. We expected the fragment size to be between 200 and 400 (based on size selection). But we see larger fragment size. So, SPRIworks seems to not eliminate the larger fragment size. Basically the size selection on SPRIworks does not seem to work well for WCE's for some reason and we were hoping someone else might have experienced the same this.

Do you do library prep's for WCE using SPRIworks? If so, do you not see such a issue with WCE samples?

Thanks

[pmiguel]

Hi Phillip,

That is actually the problem. We expected the fragment size to be between 200 and 400 (based on size selection).

Do you mean your size selection or the SPRIwork's size selection?

But we see larger fragment size. So, SPRIworks seems to not eliminate the larger fragment size.

What is the size distribution of the fragments you put into your SPRIworks System?

Basically the size selection on SPRIworks does not seem to work well for WCE's for some reason and we were hoping someone else might have experienced the same this.

Do you do library prep's for WCE using SPRIworks? If so, do you not see such a issue with WCE samples?

Thanks

We do not have a SPRIworks System.

But I am familiar with SPRI and am unaware of any SPRI-only protocol that does an upper size selection. That would mean that if you put large DNA fragments in, you will get large DNA fragments out.

What size are your WCE fragments before you put them into your SPRIworks?

--
Phillip

[pmiguel]

Looks like I was wrong and the SPRIworks is supposed to do an upper size cut as well.

You will likely need to consult with SPRIworks tech support.

--
Phillip

[sgupta]

Hi Phillip,

Thanks for your efforts. I will speak with SPRIworks tech support again. In the past when we have spoken to them, they say that the size selection is not 100% accurate and 20% of higher size fragments "bleed over" and if one does not have enough DNA in the selected size range, the 20% "bleed over" stuff gets amplified. But I find it hard to believe that WCE has nothing or very little in that range because we ran it on bioanalyzer before library prep. Plus it is essentially the same sample that has been split into 2 parts (IP and WCE) as I mentioned in my 2nd post on this thread.

I wonder though, since you are familiar with the system, if you know how much of starting material is used for a WCE, for library prep on SPRIworks. If you do not know it is fine, I thought I ask.

Thanks again.

[rnal]

I think you can contact with the technical support of illumina, they say the large fragment(the large peak in agilent test) is the bead contaminate, not the dna fragment.

[pmiguel]

Okay, maybe you could post the electropherogram of the library from your bioanalyzer? Is there some reason you are steadfastly refusing to tell us the size range of your two libraries? You are making it very difficult to help you.

I don't actually know what the SPRIworks is doing exactly. But SPRI is a PEG cut, where molecules of a higher molecular weight are preferentially precipitated onto the surface of derivatized magnetic beads. So you can do one cut where you keep the stuff in solution and discard the precipitated stuff. That gets rid of the high molecular weight molecules. Then change the PEG concentration and precipitate fragments of the desired size on the beads and discard the lower molecular weight that remains in solution.

Solutes that effect the solubility of DNA in solution could conceivably shift your fragment size range under such a regimen. Does your WCE have a lot of salt in it? Maybe you could try desalting it prior to SPRIworks?

--
Phillip

[mgravina]

Having similar problems

Our laboratory has been having problems that sound suspiciously similar to yours. Here's the link to the thread:

http://seqanswers.com/forums/showthread.php?p=57156&posted=1#post57156

I see that you were having this problem back in July. Have you resolved it yet?

I am also in Cambridge, at MIT - we may be neighbors.

[sgupta]

Update

We have discontinued the use of SPRIworks for now. I think it is a issue with the bead purification/size selection process and the amount of sample in the 200 to 400 region after sonication. Collectively, it leads to a significant loss of sample.

It may affect ChIP samples only (or the cause-outcome relation is more apparent) because underlying characteristics of the sample type is different from others given sonication steps, antibody quality etc.

As a solution, we have moved to manual preps with size selection using pippin prep until we find a better solution. At this point, we do not have much confidence in SPRIworks prep and will not use it until Beckman resolves this issue.

[dottomarco]

Hi, we are now evaluating the SPRIworks System in order to do the library prep for our 454 and for HiSeq. I read your posts about the issues you faced with the system. We will be sequencing mostly exomes (TrueSeq/Nimblegen enriched), RNAseq and miRNA. Do you think the system works well with these types of samples or do you see this problems possibly affecting all the different types of libraries ? As you can immagine the main reason to use SPRIworks is to avoid labour intensive steps like size selection.. so if it is an issue with Beckman systems, I would not go for that. Does anyone know if they solved this issues? Any other feedback from SPRIworks users that can give some help regarding this choice would be greatly appreciated ! Thanks in advance to who ever could help