Library QC

[BigAlPal]

Hi everyone,

I'm a newbie to the Miseq and was wondering if anybody could take a look at the attached electropherogram and tell me if you think it is a good library? It was prepared using the Nextera XT kit and the sample is from the CAN plate (just after cleanup, not normalized) and was ran on a Tapestation 2200.

Kappa qPCR results showed the library at 13.333 nM and Qbit showed sample at 2.15 ng/ul.

Thank you for your help!
Big Alpal

[ychang]

it is not a perfect library because the fragment is too large, the ratio of DNA/transponase is very sensitive to the insert fragment, I will use a little more enzyme when I do next time. however, if you still want to seq it, make sure load more template according to the NEXTera manual. BTW, when you use KAPA kit, how long is the extension step? I am wondering it need at least 80s since the fragment is so long.

Hope it will be helpful! good luck!

[BigAlPal]

Thank you for your reply ychang. The extension on qPCR was 45 seconds. I went ahead and tried to sequence the library using Nextera denature and dilution protocol and got no clustering. Illumina is not sure if it's the library or the instrument and is sending a Phix control and sequencing reagents to check.

[mbeale]

Hi BigAlPal,

We've done TapeStation analysis of Nextera XT libraries and we had similar results, with a broad library size range and largish fragments. We found that the DK1 screen tapes are inadequate for the libraries because of this, since they cut off at 1Kb, as shown in your image. We switched to using the newer genomic DNA screen tapes, and those allow you to fully image the library and calculate the mean/median fragment size.

Like you, we also had overly large fragment sizes, but Illumina seemed to think it wasn't a big problem and advised us to adjust for it in our quantitation when we clustered on the MiSeq. For example, after normalisation the Nextera XT kit says to add 24ul of PAL to the HT1 - if you increase the ratio of PAL to HT1 (e.g. add 30ul) you allow for a larger fragment size reducing your molarity. You may need to experiment a bit - our libraries had a median fragment size of about 900bp, and our clustering was disappointing with 24ul and 30ul of PAL. Having said that, we still got useable sequences, just not as much depth as hoped for.

Good luck!

[asr1]

Hi

We had this exact same problem. We wanted to use it as a QC check before running our libraries. Although we found we were getting excellent sequencing results, we were confused as to why the Tapestation was giving us weird results.

But after a lot of investigating, many many frustrating emails, and purchasing many Agilent kits, we discovered Nextera XT is actually incompatible with the Tapestation and that the two had never been checked for compatibility by Agilent. The old Nextera kits were compatible.

We found XT was incompatible both before and after normalization with both the HS D1K screen tapes and the gDNA screen tapes. As well it doesn't work with the Bioanalyzer, which some people from Agilent came out and tested for us. In the end we finally agreed that we would return the Tapestation for a refund.

So basically your electropherogram is not actually showing your insert size, it has simply not worked.

Hope this saves you some time!

[BigAlPal]

Thanks, mbeale. That's good to know. I actually was able to get a good sequencing run. I now have some actual experience! I'll definitely be checking out the genomic tapes.

Appreciate it!
BigAlpal

[sebl]

But after a lot of investigating, many many frustrating emails, and purchasing many Agilent kits, we discovered Nextera XT is actually incompatible with the Tapestation and that the two had never been checked for compatibility by Agilent.

Is there any explanation for that? How are you supposed to QC the libraries otherwise?

[nucacidhunter]

DNA TapeScreens quantify and size the fragments. They have a size and concentration range for accurate measurements as specified by manufacturer. The isuuse with Nextera libraries is that they peak at 1.5 kb and range can be up to 2 kb. D1000 tapes upper marker is 1.5 kb fragment, so fragments larger than that will not be accurately sized and also concentration will be underestimated because large library fragments will blend with upper marker which its concentration is used for quantification.

For QC and quantification purposes they are adequate, because all one need to do is to get average size up to 1kb (these fragments would form clusters and larger ones are less likely to cluster in presence of smaller fragments) and use data from qPCR to calculate molar concentration. Note that KAPA qPCR kit with 45 sec extension also mostly quantifies fraction of library with fragment shorter than 1 kb. In this way longer fragments which are not contributing to clustering process are ignored both in average size calculation and qPCR.