[ssing]

So, I ended up size-selecting my libraries, and then I used Bioanalyzer to look at the curve -- and it was exactly the same. Again, the desired peak at 300 bp and the higher MW peak centered around 1400 bp. So, I took Philip's suggestion (seen on other posts) to heat up the library to 95 degrees, cool slowly and then ran on an agarose gel (we don't have a Bioanalyzer in house...), and finally, I only saw one band at exactly the desired size. Not sure why my bubble products/concatenamers/daisy chains (whatever they are) have such a different pattern from others seen on this forum, but there you go. I guess the denaturing during quantification via qPCR and during the actual sequencing should mean these products will have no downstream effects.

Submitting for sequencing tomorrow, so hopefully I'll have good results to report back to you in a month or so!

[ssing]

Just wanted to follow up and say that I got good quality sequences at good yields on the HiSeq. Our sequencing facility head said he had seen similar bioanalyzer profiles with the TruSeq kit -- why is unclear -- but the results are fine, nonetheless.

Thanks for all your help!

[KB*]

also have HMW tail

Hi ssing,
Are you still around? I have samples looking similar to yours (check the image). I am preparing libraries on ChIPed DNA and all my IPs and IgG IPs have this tail. Only input does not have it. Input was amplified in a different PCR machine and had 3 less cycles. But the initial concentration of my input DNA was 2.5-5 times more than that of IP samples.

I did not experiment with the "stock" libraries, but I diluted them to ~100-300 pg/uL (100 ul total). I tried separation on the magnet; re-size-select; spin - nothing changed. Just like in your case..... well may be my libraries were too diluted for second run with Ampure beads??

You said you denatured the library at 95 oC and then cooled slowly. What do you mean "slowly"? I would like to try that as well. As I understand that was just a test. Or did you denatured all of your libraries before submission? I need to pool the libraries together before submission. If you did denaturation for your libraries, shall I denature them before pooling? I guess the concentration stays the same?

The sequencing facility proposed to do a trail on MiSeq first and then to sequence on NovaSeq. Huh.. that is the huge overpay They gave the quote for all the samples. I guess if I do miSeq trail I only shall do one sample although not sure it is possible...