TITLE: Coral Resilience Lab StepOnePlus™ qPCR protocol for Cladocopium and Durusdinium Symbiodiniaceae detection 
AUTHORS: Mariana  Rocha De Souza, Shayle Matsuda, Jenna Dilworth, Crawford   Drury, Ross Cunning

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The purpose of this protocol is to quantify </span><span style = "font-style:italic;">Cladocopium</span><span> and </span><span style = "font-style:italic;">Durusdinium</span><span> Symbiodiniaceae cell ratio densities (symbiont to host cell ratios) from individual coral DNA samples using the StepOnePlus™ Real-Time PCR System. It allows for a quick assessment of the symbiont community composition and ratio using targeted primers. </span></div></div>

[STEPS]
?. [Mastermix preparation]
Primer and Probe DilutionsPrimers usually come in lyophilized form. We recommend diluting them to a 100µM stock, then diluting the stock to get the desired concentration for the reactions. For the reacions, Forward primers should be 1μM and Reverse primers shold be 1.5 μM. Probes should be diluted to 2μM (Table 1).Stock should be kept at -20 ºC and when in use,  kept at 4 ºC (avoid freezing and thawing). Mastermix should be kept at 4 ºC at all times. Probes for the detection of Symbiodiniaceae Cladocopium (former clade C) and Durusdinium (former clade D) were labeled by Cunning et al. (2013) with different fluorescent dyes (VIC and FAM respectively), allowing those reactions to me multiplexed. Primers and probes for two coral hosts (Montipora capitata and Pocillopora damicornis) have been developed by Cunning et al. (2017) and Cunning & Baker (2013) respectively.   Table 1. Primers and probes for symbionts and coral host.   ABCDE1TargetNameWorking ConcentrationSequence2Symbiodiniaceae3Cladocopium (C)CActFForward1μM5′-CCAGGTGCGATGTCGATATTC- 3′4CActRReverse1.5 μM5′-TGGTCATTCGCTCACCAATG- 3'5CActProbeProbe2 μM5′-VIC-AGGATCTCTATGCCAACG-MGB’ 3′67Durusdinium (D)DActFForward1μM5′-GGCATGGGGTAAGCACTTCTT- 3′8DActRReverse1.5 μM5′-GATCCTTGAACTAGCCTTGGAAAC- 3′9DActProbeProbe2 μM5′-6FAM-CAAGAACGATACCGCC-MGB- 3′1011Coral host12Montipora capitataPaxC-ForForward1μM5’-GTGCAGGTGAGATTGAGTCTTATAACA-3'13PdAct-RevReverse1.5 μM5’-CGGTTGAGCTTCGCTAAACAG-3'14PaxC-ProbeProbe2 μM5’-FAM-CAGTTCTTCCAACAATG-MGB-3'1516Pocillopora damicornisPdAct-ForForward1μM5′-GAGAAGCTCTGCTATGTTGCCA- 317PdAct-RevReverse1.5 μM5′-TCCACGGAATCGCTCGTT- 3′18PdActProbeProbe2 μM5′-NED-AGACTGCTGCCTCAAC-MGB- 3′19Mastermix preparationNote: Always run duplicates or triplicates of each sampleThe Micro Amp Fast plate has 96 wells, but in our machine, the 4 corners of the plate don’t provide a good read, so in a 96 well plate, we only use 92 wells. You might need to test that for the machine you are using. Print a 96 well plate template and write the sample ID and the reaction it will be running (S for Symbiodiniaceae and H for coral host) on the printed wells .To minimize the time the mastermix will sit on the bench at room temperature, separate the DNA samples you will use in the order they will be plated before you start preparing the mastermix. Leave them in order in a microcentrifuge rack in the freezer. It is always recommended to do the whole protocol with the reagents and the samples on ice. CalculationEach well should have 9 ul of your qPCR mastermix and 1 ul of DNA sample. Prepare two separate mastermixes, one for the host and one for the Symbiodiniaceae .qPCR Symbiodiniaceae C and D mastermix (amount per well)5 μl GTMM0.5 μl Symb C primer (F), 0.5 μl Symb C primer (R), 0.5 μl, 0.5ul Symb C  probe0.5 μl Symb D primer (F), 0.5 μl Symb D primer  (R), 0.5 μl Symb D probe1 μl Hi clone ddH20qPCR Coral Host master mix (amount per well)5 μl GTMM0.5 μl Host primer (F), 0.5 μl Host primer (R), 0.5 μl Host probe 2.5 μl Hi clone ddH20Remember to have 3 Controls:Positive control: coral with Symb C and Symb D (a sample that has been confirmed to have both symbionts you are testing)Negative control 1:  Hi clone ddH20 (will test for external source of DNA)Negative control 2:  control TE buffer (extraction control, replace with the reagent your DNA samples had been eluted with)Calculate the quantity of mastermix according to the number of samples you are running and make each mix in a 1.5 μl centrifuge tube, one for the Symbiodiniaceae and one for the coral host.If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20CalculationEach well should have 9 ul of your qPCR mastermix and 1 ul of DNA sample. Prepare two separate mastermixes, one for the host and one for the Symbiodiniaceae .qPCR Symbiodiniaceae C and D mastermix (amount per well)5 μl GTMM0.5 μl Symb C primer (F), 0.5 μl Symb C primer (R), 0.5 μl, 0.5ul Symb C  probe0.5 μl Symb D primer (F), 0.5 μl Symb D primer  (R), 0.5 μl Symb D probe1 μl Hi clone ddH20qPCR Coral Host master mix (amount per well)5 μl GTMM0.5 μl Host primer (F), 0.5 μl Host primer (R), 0.5 μl Host probe 2.5 μl Hi clone ddH20Remember to have 3 Controls:Positive control: coral with Symb C and Symb D (a sample that has been confirmed to have both symbionts you are testing)Negative control 1:  Hi clone ddH20 (will test for external source of DNA)Negative control 2:  control TE buffer (extraction control, replace with the reagent your DNA samples had been eluted with)Calculate the quantity of mastermix according to the number of samples you are running and make each mix in a 1.5 μl centrifuge tube, one for the Symbiodiniaceae and one for the coral host.If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20qPCR Coral Host master mix (amount per well)5 μl GTMM0.5 μl Host primer (F), 0.5 μl Host primer (R), 0.5 μl Host probe 2.5 μl Hi clone ddH20Remember to have 3 Controls:Positive control: coral with Symb C and Symb D (a sample that has been confirmed to have both symbionts you are testing)Negative control 1:  Hi clone ddH20 (will test for external source of DNA)Negative control 2:  control TE buffer (extraction control, replace with the reagent your DNA samples had been eluted with)Calculate the quantity of mastermix according to the number of samples you are running and make each mix in a 1.5 μl centrifuge tube, one for the Symbiodiniaceae and one for the coral host.If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20Remember to have 3 Controls:Positive control: coral with Symb C and Symb D (a sample that has been confirmed to have both symbionts you are testing)Negative control 1:  Hi clone ddH20 (will test for external source of DNA)Negative control 2:  control TE buffer (extraction control, replace with the reagent your DNA samples had been eluted with)Calculate the quantity of mastermix according to the number of samples you are running and make each mix in a 1.5 μl centrifuge tube, one for the Symbiodiniaceae and one for the coral host.If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20Positive control: coral with Symb C and Symb D (a sample that has been confirmed to have both symbionts you are testing)Negative control 1:  Hi clone ddH20 (will test for external source of DNA)Negative control 2:  control TE buffer (extraction control, replace with the reagent your DNA samples had been eluted with)Calculate the quantity of mastermix according to the number of samples you are running and make each mix in a 1.5 μl centrifuge tube, one for the Symbiodiniaceae and one for the coral host.If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20If doing a full plate, here are the calculations (92 samples):  qPCR Symbiodiniaceae C and D (46 samples)230 μl GTMM23 μl Symb C primer (F), 23 μl Symb C primer (R), 23 μl Symb C  probe23 μl Symb D primer (F), 23 μl Symb D primer  (R), 23 μl Symb D probe46 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20qPCR Coral Host master mix (46 samples)230 μl GTMM23 μl Host primer (F), 23 μl Host primer (R), 23 μl Host probe 115 μl Hi clone ddH20
ABCDE1TargetNameWorking ConcentrationSequence2Symbiodiniaceae3Cladocopium (C)CActFForward1μM5′-CCAGGTGCGATGTCGATATTC- 3′4CActRReverse1.5 μM5′-TGGTCATTCGCTCACCAATG- 3'5CActProbeProbe2 μM5′-VIC-AGGATCTCTATGCCAACG-MGB’ 3′67Durusdinium (D)DActFForward1μM5′-GGCATGGGGTAAGCACTTCTT- 3′8DActRReverse1.5 μM5′-GATCCTTGAACTAGCCTTGGAAAC- 3′9DActProbeProbe2 μM5′-6FAM-CAAGAACGATACCGCC-MGB- 3′1011Coral host12Montipora capitataPaxC-ForForward1μM5’-GTGCAGGTGAGATTGAGTCTTATAACA-3'13PdAct-RevReverse1.5 μM5’-CGGTTGAGCTTCGCTAAACAG-3'14PaxC-ProbeProbe2 μM5’-FAM-CAGTTCTTCCAACAATG-MGB-3'1516Pocillopora damicornisPdAct-ForForward1μM5′-GAGAAGCTCTGCTATGTTGCCA- 317PdAct-RevReverse1.5 μM5′-TCCACGGAATCGCTCGTT- 3′18PdActProbeProbe2 μM5′-NED-AGACTGCTGCCTCAAC-MGB- 3′19
?. [Mastermix preparation]
Plating mastermix IMPORTANT: Vortex each mix thoroughly before plating.Add 9 μl of mastermix to the bottom of each well (remember to check which ones are Symbiont wells and which ones are Host wells). Change pipettes between mastermixes.Flick each DNA sample before use, for better mixingAdd 1 μl of DNA sample to the top side of each well. Change pipette between each DNA sample. As 1 μl is such a small volume, adding to the side makes it easy to track which samples have been loaded already. The droplets will be visible on the side. Plus, adding to the side instead of directly into the mix in the bottom of the well helps prevent bubbles.  Seal the plate with qPCR sealing tape and use roller to ensure it is well sealed.Put plate in centrifuge to spin DNA into mastermix at 10,000 g for about 10s.Check that the liquids are at the bottom of each well, that all wells have  same amount of liquid, and that there are no bubbles. If there are bubbles, flick the wells and centrifuge again.
?. [Mastermix preparation]
Running the samples in the Step One Plus machinePut samples in thermocycler (StepOnePlus machine), and check that the samples are placed in the same orientationyou loaded them.Thermocycler settings have previously been saved with the name TaqMan_Std (Figure 1).Check that the settings are correct and press "Save"Change volume to your sample volume (10ul)Select "Run" and wait for reaction to start to make sure it is running
?. [Mastermix preparation]
Collecting results and visualizing results on computerWhen reaction is finished, insert a USB thumbdrive into the Step One Plus Machine and select "Save Results", once the file has been saved, remove the USB thumbdrive. Results can also be taken directly from the computer connected to the Step One Machine by selecting "Collect experimental results" on the computer. Open your Taq file on the computer (computer should have StepOne software installed). Click on "Setup" on the left hand column. In "Define Targets"  (your C and D and host) and your samples. Click “Add New Target”  into "Define Targets" to get a new row. For reporter, select the fluorescent dye for each reaction (Table 2). Choose also the color you would like to display each reporter.  Table 2. Target and fluorescent dyes Now on "Define  Samples", on the right side,  list all your sample names (Figure 2).      Now click on the tab "Assign Targets and Samples". Here you will assign targets and samples to each well (Figure 3). Highlight the well with your mouse and then check the box next to the targets and the sample names. You can highlight all the wells and assign targets at once, and do the sample names individually.   Now click on "Analysis" on the left hand column, then click "Analyze" on the top right corner. On left, click "Multicomponent Plot". Here we are looking for the number of cycles before amplification. We want to tell the machine what our baseline is. We want it to be large, but remember, it needs to be the same for all of your runs. On the top right, click on "Analysis settings" (Figure 4). Enter the threshold (usually 0.01) and the baseline for both C and D (usually 6-23). Then hit "Apply Analysis Settings" (we tend not to use the "auto" settings and instead edit them ourselves).   Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.Table 2. Target and fluorescent dyesTargetFluorescent dyeCladocopium (C)VICDurusdinium (D)FAMMontipora capitataFAMPocillopora damicornisNEDNow on "Define  Samples", on the right side,  list all your sample names (Figure 2).      Now click on the tab "Assign Targets and Samples". Here you will assign targets and samples to each well (Figure 3). Highlight the well with your mouse and then check the box next to the targets and the sample names. You can highlight all the wells and assign targets at once, and do the sample names individually.   Now click on "Analysis" on the left hand column, then click "Analyze" on the top right corner. On left, click "Multicomponent Plot". Here we are looking for the number of cycles before amplification. We want to tell the machine what our baseline is. We want it to be large, but remember, it needs to be the same for all of your runs. On the top right, click on "Analysis settings" (Figure 4). Enter the threshold (usually 0.01) and the baseline for both C and D (usually 6-23). Then hit "Apply Analysis Settings" (we tend not to use the "auto" settings and instead edit them ourselves).   Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop. Now click on the tab "Assign Targets and Samples". Here you will assign targets and samples to each well (Figure 3). Highlight the well with your mouse and then check the box next to the targets and the sample names. You can highlight all the wells and assign targets at once, and do the sample names individually.   Now click on "Analysis" on the left hand column, then click "Analyze" on the top right corner. On left, click "Multicomponent Plot". Here we are looking for the number of cycles before amplification. We want to tell the machine what our baseline is. We want it to be large, but remember, it needs to be the same for all of your runs. On the top right, click on "Analysis settings" (Figure 4). Enter the threshold (usually 0.01) and the baseline for both C and D (usually 6-23). Then hit "Apply Analysis Settings" (we tend not to use the "auto" settings and instead edit them ourselves).   Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.Now click on "Analysis" on the left hand column, then click "Analyze" on the top right corner. On left, click "Multicomponent Plot". Here we are looking for the number of cycles before amplification. We want to tell the machine what our baseline is. We want it to be large, but remember, it needs to be the same for all of your runs. On the top right, click on "Analysis settings" (Figure 4). Enter the threshold (usually 0.01) and the baseline for both C and D (usually 6-23). Then hit "Apply Analysis Settings" (we tend not to use the "auto" settings and instead edit them ourselves).   Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.On left, click "Multicomponent Plot". Here we are looking for the number of cycles before amplification. We want to tell the machine what our baseline is. We want it to be large, but remember, it needs to be the same for all of your runs. On the top right, click on "Analysis settings" (Figure 4). Enter the threshold (usually 0.01) and the baseline for both C and D (usually 6-23). Then hit "Apply Analysis Settings" (we tend not to use the "auto" settings and instead edit them ourselves).   Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.Now the screen should show you the amplification plot (if you don’t see the lines, make sure the “Show baseline” and “Show threshold” buttons are clicked at bottom). Also, select "Well" or Target into the Plot Color. Note, you can drag to make the wells larger so you can read the numbers as you would in an Excel sheet. You can also highlight specific wells, or rows (Figure 5).   Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.Now we want to export the data as an Excel sheet. Under "File", select "Export". Select "Results" as the data to export. Name your file, and be sure it is exported as an Excel file.Also save the StepOne settings and set up (in case you find a mistake, you won't have to re-do everything), by going into "saving as". Give a name for the settings and this run. Then you can copy it to your USB and save to your laptop.
TargetFluorescent dyeCladocopium (C)VICDurusdinium (D)FAMMontipora capitataFAMPocillopora damicornisNED
?. [Mastermix preparation]
BioinformaticsYou need to run your data through an R script to see what the values mean. Ross Cunning has developed calculations (Cunning et al. 2012, 2017) and an R script (you can see it in his GitHub). A modified version is below. The command line below can be copied to clipboard by clicking on the button at the bottom of the command line window. Once copied it can be pasted directly into R.