# qPCR – Oly RAD-Seq Library Quantification

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

 Library Library Stock Conc. (nM) Stock Halved (nM) Oly RAD 02 46.70 23.35 Oly RAD 03 79.35 39.67 Oly RAD 04 61.35 30.67 Oly RAD 06 30.61 15.30 Oly RAD 07 477.05 238.53 Oly RAD 08 46.32 23.16 Oly RAD 14 224.91 112.46 Oly RAD 17 24.56 12.28 Oly RAD 23 49.56 24.78 Oly RAD 30 11.19 NA

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:

# qPCR – Oly RAD-Seq Library Quantification

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:

# Uninterruptible Power Supplies (UPS)

A new UPS we installed this week for our qPCR machine (Opticon2 – BioRad) to handle power surges and power outages doesn’t seem to be working properly. With the qPCR machine (and computer and NanoDrop1000) plugged into the “battery” outlets on the UPS, this is what happens when the Opticon goes through a heating cycle:

The UPS becomes overloaded when the Opticon is in a heating cycle.

And, sometimes, that results in triggering a fault, shutting everything off in the middle of a qPCR run:

Fault message indicating unit overload.

This is supremely lame because having a battery backup is a great way to prevent the qPCR machine from shutting off when a power outage occurs!

I switched the Opticon (and computer and NanoDrop1000) to the outlets that are solely for surge protection. Check out what happens when I run the qPCR machine now:

Opticon plugged in to surge protection outlet while in heating cycle. Notice that output load is 0%.

So, I guess we’ll settle for at least having the surge protection aspect of things.

While handling this UPS issue, I realized that the two Synology servers we have possess a built-in UPS monitor. So, I connected the USB cables to/from each of the UPS that each server is plugged into and enabled UPS shutdown in the Synology Diskstation Management (DSM):

Eagle

Owl

Now, both Synology units will enter Safe Mode when the UPS they’re connected to reaches a low battery status. This will help minimize data loss/corruption during the next extended power outage we experience.

# Opticon2 Calibration

Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.

Ordered 5nmol of 6-FAM T10 Calibration Standard from Biosearch Technologies and resuspended it in 50μL of 1x dilution buffer (10mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl2) to make a 100μM solution. Buffer and dye were stored @ -20C after use.

Buffer calculations: Total Volume = 15mL

• 1.5mL of 100mM Tris-HCl
• 150μL of 5M NaCl
• 750μL of 100mM MgCl2

Made a working dilution of the 6-FAM dye of 300nM in 5mL of 1x dilution buffer (15uL of 100uM dye in 5mL of buffer).

Ran the calibration protocol on the Opticon2 (BioRad) using 50μL of dye in all wells when required by the calibration protocol.

Results:

### EMPTY PLATE MEASUREMENTS

Empty Plate – Channel 1 voltage measurements

Empty Plate – Channel 2 voltage measurements

Empty Plate – Ratio of Channel 1 to Channel 2 voltage measurements.

The empty plate measurements above show the expected low voltage measurements, but also show a  ~5-fold difference in min/max voltages in each channel. Additionally, the voltage ratios (the third image above) show a wavy pattern, but a smooth, even level from well-to-well is what would be expected if the Opticon was in measuring things properly.

DYE PLATE MEASUREMENTS

Dye Measurements – Channel 1 voltage measurements

Dye Measurements – Channel 2 voltage measurements

Dye Measurements – Channel 1 to Channel 2 voltage measurement ratios.

The voltages measured in each channel show the expected increase in voltages relative to the empty plate (> 10x voltage than empty plate). However, the spread between the min/max voltages in both channels is ~4-fold. Additionally, the ratio between the two channels still shows the wavy pattern across all the wells instead of the expected even ratio from well-to-well that should result from the calibration.

It appears the calibration has not resolved the issue.

To verify that calibration has failed, I ran two sets of qPCR “protocols” that simply read the dye plate to measure fluorescence across the plate in two plate orientations.

Original Orientation Data File (TAD): 20150630_162622_calibration_test.tad
180 degree rotation Data File (TAD): 20150630_162622_calibration_test_180.tad

Dye Fluorescence – Original Orientation

Dye Fluorescence – Original Orientation with Fluorescence Graph

Dye Fluorescence – 180 Degree Rotation

Dye Fluorescence – 180 Degree Rotation with Fluorescence Graph

First thing to notice is that there’s clearly uneven fluorescence detection across the plate. Viewing the images that also contain the fluorescence graphs reveals a spread of ~8-fold between the highest and lowest fluorescence detection.

The second thing to notice is that, despite rotating the plate 180 degrees, the rotation has no effect on the fluorescence detected in each block location.

Both of these taken together provide strong evidence that there’s an issue with the machine.

# qPCR – Re-run Jake’s O.lurida DNased RNA Samples NC1, SC1, SC2, SC4 from 20150514

The following DNased RNA samples showed inconsistencies between qPCR reps (one rep showed amplification, the other rep did not) on 20150514:

• NC1
• SC1
• SC2
• SC4

Reran these four samples to obtain a definitive answer as to whether or not they have residual gDNA in them prior to using them to make cDNA.

Used Oly_Actin primers (SR IDs: 1504, 1505)

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

• 95C – 2.5mins
• 40 cycles of:
• 95C – 10s
• 60C – 20s
• Melt curve

Master mix calcs: 20150521_qPCR_Oly_DNased_RNA

Plate layout: 20150521_qPCR_plate_Jake_Oly_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150521_145749.tad
qPCR Report (Google Sheet): 20150521_qPCR_Report_Jake_Oly_DNased_RNA

No amplification in any of the RNA samples, nor the NTCs. Will make cDNA.

# qPCR – Jake’s O.lurida ctenidia DNased RNA (1hr Heat Shock Samples)

Ran qPCR on DNased RNA from earlier today to assess whether there was any residual gDNA after the DNase treatment with Oly_Actin_F/R primers (SR IDs: 1505, 1504).

Used 1μL from all templates.

All samples were run in duplicate.

Positive control was HL1 O.lurida DNA isolated by Jake on 20150323.

Cycling params:

• 95C – 2.5mins
• 40 cycles of:
• 95C – 10s
• 60C – 20s
• Melt curve

Master mix calcs are here (used same calcs from the other day): 20150512_qPCR_Oly_RNA

Plate layout: 20150514_qPCR_plate_Jake_Oly_1hr_HS_DNased_RNA

Results:

qPCR Data File (Opticon): Sam_20150514_170332.tad

qPCR Report (Google Spreadsheeet): 20150514_qPCR_Report_Jake_Oly_DNased_1hr_HS_RNA

Positive control samples are the only samples that produced amplification (cycle ~20). Will proceed to making cDNA.

# ISO Creation – OpticonMonitor3 Disc Cloning

Since many newer computers are coming without optical disc drives (including my laptop, which I want to install this software on), I created an .iso disc image of the OpticonMonitor3 (BioRad) installation disc.

Using OS X Disk Utility:

1. File > New Disk Image

2. Dropdown > CD/DVD Master

This creates a Mac-specific .cdr image of the installation CD. Converted to a universal .iso disc image with the following command line:

\$hdiutil makehybrid -iso -joliet -o [filename].iso [filename].cdr

Replaced [filename] with OpticonMonitor3.

Moved the newly created OpticonMonitor3.iso file to our server (Eagle/Backup/Software/Windows).

Now the .iso file should be able to be mounted and installed on any Windows computer without the need for a physical installation CD.