After yesterday’s attempt at quantification revealed insufficient dilution of the libraries, I repeated the qPCRs using 1:100000 dilutions of each of the libraries. Used the KAPA Illumina Quantification Kit (KAPA Biosystems) according to the manufacturer’s protocol.

Made 1:100000 dilutions of each library were made with NanoPure H2O.

Ran all samples, including standards, in triplicate on the Roberts Lab Opticon2 (BioRad).

Plate set up and master mix can be found here: 20151117_qPCR_plate_layout_Oly_RAD.JPG

Results:

qPCR Data File (Opticon2): Sam_20151117_100745.tad

qPCR Data (Google Sheet): 20151117_RAD_qPCR_data

Overall, the new dilutions worked well, with all the library samples coming up between Ct 9 – 15, which is well within the range of the standard curve.

Manually adjusted the baseline threshold to be above any background fluorescence (see images below).

All samples, except Oly RAD 30, exhibit two peaks in the melt curve indicating contaminating primer dimers. Additionally, the peak heights appear to be roughly equivalent. Can we use this fact to effectively “halve” the concentration of our sample to make a rough estimate of library-only PCR products?

Here are the calculated library concentrations, based on the KAPA Biosystems formulas

Amplification plots of standard curve samples:

Melt curve plots of standard curve samples. Shows expected “shoulder” to the left of the primary peak:

Amplification plots of RAD library samples:

Melt curve plots of RAD library samples. Peak on the right corresponds to primer dimer. Peak heights between primer dimer and desired PCR product are nearly equivalent for each respective sample, suggesting that each product is contributing equally to the fluorescence generated in the reactions:

Melt curve plot of Oly RAD library 30. Notice there’s only a single peak due to the lack of primer dimers in this sample:

The final step before sequencing these 2bRAD libraries is to quantify them. Used the KAPA Illumina Quantification Kit (KAPA Biosystems) according to the manufacturer’s protocol.

Made 1:4 dilutions of each library to use as template.

Ran all samples, including standards, in triplicate on the Roberts Lab Opticon2 (BioRad).

Plate set up and master mix can be found here: 20151116_qPCR_plate_layout_Oly_RAD.JPG

Results:

qPCR Data File (TAD): Sam_20151116_144718.tad

The take home messages from this qPCR are this:

• The amplification plots that are pushed up against the left side of the graph (essentially at ~ cycle 1) are all of the libraries. A 1:4 dilution was insufficient to have the libraries amplify within the range of the standard curve.
• All libraries except one (Oly RAD Library 30) have detectable levels of primer dimer. This confounds library quantification (because both the intended PCR product and the primer dimers contribute to the fluorescence accumulation), as well as potentially interfering with the subsequent Illumina sequencing (primer dimers will be sequenced and contain no insert sequence).

Will repeat the qPCR with more appropriately diluted libraries.

See the info below for more deets on this run.

Default analysis settings need to be adjusted to account for how early the standard curve comes up. Otherwise, the Opticon software sets the baseline incorrectly:

The KAPA Quantification Kit indicates that the baseline calculations need to be extended to cycles 1 through 3. This allows the software to set the baseline threshold correctly:

Melt curve analysis of the standard curve shows the expected profile – slight hump leading into the peak:

Melt curve analysis of the libraries. Dual peaks indicate primer dimer contamination:

Melt curve analysis of Oly RAD Library 30. Shows the desired single peak, suggesting library is free of primer dimers: