Ronit’s Notebook: Identifying Unknown Oyster Sample from Marinelli Shellfish Company (C. gigas vs C. sikamea)

The Marinelli Shellfish Company had an issue with one of their bags of oysters being labelled as Kumamoto oysters (C. sikamea) AND as Pacific Oysters (C. gigas). Obviously, confusion abounded, and ultimately we were tasked with figuring out what the true identity of these mystery oysters were. To do so, DNA was isolated from mantle tissue from the unknown oysters, a sample set of known C. gigas, and a sample set of known C. sikamea. 4 PCR primers were targetting the cytochrome oxidase gene: universal forward and reverse primers (HC02198, LCO1490); reverse primer specific to C. gigas (COCgi269r); and a reverse primer specific to C. sikamea (COCsi546r). Note: this was a multiplex PCR.

Cycling parameters were as follows:

95°C for 10 mins; 30 cycles of 95°C (1 min), 51°C (1 min), 72°C (1 min); 72°C (10 min).

PCR reactions were run on a gel and results are visualized below:

IMG_0493.jpg

The first set of 4 samples (offset by ladders) are the unknown samples; the second set of 4 samples are C. gigas; and the third set of 4 samples are C. sikamea.

Using the GeneRuler DNA Ladder as a guide,

Screen Shot 2019-12-17 at 8.04.15 PM.png

First, we can see that there is a band of approximately 700bp in all samples, indicating that the universal forward and reverse primers did their job (positive primer). Next, we expect to see a band of approximately 260-270 bp in the known C. gigas samples, which we do! Similarly, we also expect to see a band of 550 bp in the known C. sikamea samples, which we also do. (Note: it looks like there is a faint band of 270bp in the C. sikamea samples. Could be a sign of contamination with C. gigas samples?).

In the unknown samples, a prominent band of 270bp is clearly visible, which is what we should see in C. gigas samples, Thus, it seems that these mystery samples are in fact C. gigas. Case closed!

Ronit’s Notebook: Finalized qPCR Plots

I worked on figuring out some of the stats for my qPCR data with Shelly on Thursday. I decided to go with an ANOVA/Tukey’s Honest Significance Difference Test, with the data being normalized using a log transform. Final qPCR plots are attached below–capital letters indicate differences in treatment, asterisks denote differences between individual groups, and capital letters next to ploidy key indicate differences between diploids and triploids. ATP SynthetaseCOX1DNMT1HATHATHaP2HIF1AHSC70HSP90MBD2MeCP2SOD.png

Ronit’s Notebook: Adjusted qPCR Data, COX1 Relative Mitochondrial Abundance Plot

To account for N/A Cq values, I substituted in a Cq value of 45 wherever the Cq value was nonexistent. This allows for some initial analysis of the data to see which genes might warrant further work. Below are the adjusted qPCR plots. I also generated the COX1 Cq plot (not normalized to actin) to examine relative mitochondrial abundance between stressed and non-stressed diploids and triploids.

atpsynthetasecox1dnmt1hathif1ahsc70hsp90mbd2mecp2sod

Ronit’s Notebook: qPCRs and Data Analysis

I ran SOD and ATP Synthetase qPCRs last week. Currently, I’m working on getting box plots worked out with the R script–the main issue I’m trying to sort out is how to account for the N/A values for some of the samples as the current plots just ignore it. I’m also adding a statistical analysis portion to the script so that we can start visualizing which treatments are statistically different.

Link to ATP Synthetase qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-23%2014-56-33_BR006896.pcrd

Link to SOD qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2019-01-09%2015-08-24_BR006896.pcrd

Ronit’s Notebook: Generating Plots for qPCR Data

Today, I made a GitHub account and generated box plots for all my qPCR data using R. Attached below are pictures of the box plots:

hat_plothathap2_plothif1a_plotdnmt1_plothsc70_plothsp90_plotmbd2_plotmecp2_plot

Ronit’s Notebook: HSC70, MBD2, and MeCP2 qPCR

I ran a qPCR assay on my desiccated + elevated temp. samples with HSC70, MBD2, and MeCP2 as my gene targets. For protocol used, please refer to previous notebook entries.

Link to Excel spreadsheet of Cq values: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing

Link to HSC70 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-19%2016-24-55_BR006896.pcrd

Link to MBD2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2013-17-34_BR006896.pcrd

Link to MeCP2 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-20%2014-58-17_BR006896.pcrd

Ronit’s Notebook: DNMT1 and Actin qPCR

In the last two qPCR assays, no fluorescence was detected. Turns out we were using Promega master mix which is specifically for probe-based assays…as such, it doesn’t have any fluorescent dye in it, explaining the lack of fluorescence detection in any of the assays. I switched over to using a different master mix that should solve the issue.

I reran the DNMT1 primer assays and will run an actin qPCR to determine if it might be a possible normalizing gene. For protocol used, please refer to previous lab book entries.

Link to Excel spreadsheet of Cq values for both actin and DNMT1: https://docs.google.com/spreadsheets/d/1AhdJLEcXt6vnNBcGVLnc926KlZ5J-Nt8JMpiCdrUvaU/edit?usp=sharing

Link to DNMT1 qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-12%2015-24-28_BR006896.pcrd

Link to actin qPCR data: http://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/admin_2018-12-14%2016-16-16_BR006896.pcrd

Plate map:
All replicates are in adjacent wells. The order of the samples in the well plate by rows is: D01, D02, D03, D04, D05, D06, D07, D08, D09, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, T01, T02, T03, T04, T05, T06, T07, T08, T09, T10, T11, T12, T13, T14, T15, T16, T17, T18, T19, T20

Example:
Well—–Sample
A01——D01
A02——D01
A03——D02
A04——D02