Another question about Bridge amplification/insert size

[chenbati]

I have another question about the bridge amplification / optimal insert size which is similar to the one asked by BTS about a week ago.

I know that the Illumina recommended insert size is around 150-300 bp (for single-read). And in the previous thread people mentioned that longer inserts worked well too. My question is that how short the insert can be? I only plan to use an isert with a size of about 60bp. Is this doable? Anybody has any experience?

Any input will be appreciated.

[GW_OK]

I think a 60bp insert would be removed through the library making clean-up processes. Why so short?

[chenbati]

I think a 60bp insert would be removed through the library making clean-up processes. Why so short?

Thanks for your reply!

It is a complicated story. In my experiment, I will synthesize a complex ssDNA library (each sequence will be around 150mer) with adapters and sequencing primers designed in. So I will generate clusters using the ssDNA library directly and won't have any library preparation step.

I won't use the sequencer to do sequencing, instead, I will use it to count the number of clusters generated on the flowcell.

BTW, It should be OK if I start with ssDNA for cluster generation, right? cause for regular libraries, we have to denature them first. And based on what I understand, one sequence is enough for cluster formation for single-read sequencing.

[GW_OK]

Well, if you're generating the ssDNA with everything already on there, it ought to work.

I gotta ask: Why are you generating flowcells of synthetic DNA just to count them? Would qPCR not work?

[chenbati]

Well, if you're generating the ssDNA with everything already on there, it ought to work.

I gotta ask: Why are you generating flowcells of synthetic DNA just to count them? Would qPCR not work?

qPCR won't work if you are interested in 50k different sequences at the same time.

I wish I didn't have to do the sequencing - expensive, and not sure if it will work as the sequencer is not designed for this.

[kmcarr]

I only plan to use an isert with a size of about 60bp. Is this doable?

As I have occasionally observed primer dimers in my data that is evidence that you can amplify clusters with 0bp inserts. This was confirmed by an Illumina scientist as well. They wondered what the lower limit of insert length was for bridged amplification but upon observing primer dimers they concluded that the primers themselves provided enough length and flexibility for the bridged PCR.

[chenbati]

As I have occasionally observed primer dimers in my data that is evidence that you can amplify clusters with 0bp inserts. This was confirmed by an Illumina scientist as well. They wondered what the lower limit of insert length was for bridged amplification but upon observing primer dimers they concluded that the primers themselves provided enough length and flexibility for the bridged PCR.

This is great to know! I then feel more comfortable starting my experiments. Thank you, kmcarr!

[dkenned1]

Have you considered using a nanodrop to quantify your DNA concentration?

[dorix]

Hi,
I have libraries of insert size of about 600bp. I'm quantifing them by qPCR. Those are amplification free libraries, so I gess I may have same fragments wihout adapters and so on. Unfortunatelly comparing molarity from bioanalyzer and from qPCR are incosistent: qPCR shows two times lower amount. I'm wondering which is more likely: I have samples with not fully constructed fragments or amplification efficiency is that much lower with longer fragments. I'm affraid of overclustering, using results from qPCR, however I do not want to loose money by less density.

Does anyone has similar expirience?

[jdk787]

I would trust qPCR over the BA since the Bioanalyzer doesn't differentiate between ligated and non-ligated material.

[dorix]

jdk787, thank you for your resposne. Have you got any expirience with lower concentration from qPCR due to too short elongation time and how does it corresponds to amplifiaction on machine?

[nucacidhunter]

If qPCR suggest 2x less concentrated Library then you ligation based library prep is successful as most kits ligation efficiency is 20-30%.

Using shorter time for qPCR cycling steps would reduce amplification efficiency due to incomplete denaturation, extension or inefficient primer binding resulting in lower signal and quantity.

[dorix]

The final answer for my problem is even more complicated, as I found out that my actual library size is different, as T-shaped adapters cause higher migration of libraries, as it is shown in TruSeq PCR-free manual... So real concentration is sth between and I have to asume library size or I should check it by testing amplification product size.

I hope it will help someone in the future.

[GW_OK]

One thing we have found to be helpful is to clean up and run the qPCR products out on our Tapestation. This way you know exactly what size of product you have in your analysis.