Erythranthe guttata Write-up

We conducted a Double Digest Restriction Associated DNA study on Mimulus guttatus (Erythranthe guttata). The first step was collecting samples, which we did on two field trips (see Lab 3 and Lab 4 posts).

Next, we extracted DNA from samples we collected, as well as samples previously collected by Alec Chiono (see Lab 8 post).

Next, we double digested our DNA using two restriction enzymes. (See Lab 11 post). These enzymes cut up the genome into many pieces.

Next, we ligated unique DNA barcodes onto each individual (see Lab 11 post).

Next, we used PCR (see Lab 12) in order to 1) add a second barcode and 2) to test if our library construction was successful. Our PCR was successful, as evidenced by the results of our gel run (photo to be uploaded by Dr. Paul).

After the test PCR, we did a larger reaction that was identical and will be used for the following test. (This was the last step that we were able to do as a class.) In a perfect world, we would do the following other tests:

The next step would be size selection. Size selection selects DNA of specific sizes. Specifically, we would target approximately 400-600 bp fragments. Size selection can be done in 3 different ways, 1) PPEN prep (we have one housed in the Suni Lab at USF — #Suni), or 2) gel extraction, or finally 3) use magnetic beads to isolate the DNA fragments.

After size selection, we would then normalize our DNA samples. This means to bring all of our DNA samples to approximately the same concentration. Having equal concentrations makes more likely that equal number of DNA fragments get sequenced.

The final step is to combine all of our size selected, normalized PCR products into one vessel. We would then run these samples on any Illumina sequencer (our class would use the in-house iSeq 1000 aka Wall-e). Sequencing would take approximately 16 hours, and if successful, would generate 10s of millions of reads. These data would be run through a bioinformatics pipeline (“Help, Dr. Z!”). Ultimately, we would align these sequence data with the published Mimulus guttatus genome and call SNPs (identify SNPs in dataset).

Finally, we would use these SNPs to infer population differentiation using a metric like Fst, and assess population genetic diversity, looking at things like number of alleles, allelic diversity, etc.

Based on what I know about Mimulus guttatus, I would expect populations that are geographically divergent to be genetically divergent. Within that geographic differentiation, I would expect that populations that are ecologically divergent to be more genetically divergent.

 

#molecularecologyforever

Lab 12 – Final PCR

Last week, we ran DD-RADSeq and Ligation on our Erythranthe guttata samples. This week, we began by running a test run of PCR to test for successful library construction.

  1. I began by labelling new PCR tubes to correspond to our samples #9-16 for this new PCR run.
  2. We created a Master Mix for the table, enough for 11 reactions, using the following recipe:
  • NEB One-Taq 2x Master Mix: 8uL/rxn (88 uL total)
  • Forward Primer (10mM): 0.4 uL/rxn (4.4 uL total)
  • Reverse Primer (10mM): 0.4 uL/rxn (4.4 uL total)
  • Pure H2O: 6.2uL/rxn (68.2 uL total)

3. We added 15 uL of the Master Mix and 1 uL of the library DNA template to each corresponding PCR tube.

4. We added these PCR tubes to the thermocycler BIORAD #1/2 –>DDRAD –>2_PCR1. This runs at 94 degrees Celsius for 2 min, then 20 cycles of (94 degrees C for 30 sec, 60 degrees C for 30 seconds, 68 degrees C for 45 sec). 4-10 degree C infinite hold.

Gel Run

  1. After PCR was finished, we ran the PCR products in a 1.5% agarose gel (0.75 g agarose in 50 mL 1XTAE) with a 100 bp ladder at 130V for 40 minutes.

Final PCR

  1. To add Illumina flow cell annealing sequences, multiplexing indices, and sequencing primer annealing regions to all fragments and to increase concentrations of sequencing libraries, we perform a PCR amplification with a Fusion Polymerase kit.
  2. We prepared a Master Mix 2 for the table, enough for 11 reactions, following this recipe:
  • Fusion DNA polymerase: 0.31 uL/rxn (3.4 uL total)
  • 5X Fusion HF Buffer: 6.25 uL/rxn (68.8 uL total)
  • 10uM Forward Primer (PCR1_X): 1.56 uL/rxn (17.2 uL total)
  • 10uM Reverse Primer (PCR2_5): 1.56 uL/rxn (17.2 uL total)
  • 10mM dNTPs: 0.63 uL/rxn (6.9 uL total)
  • DMSO: 0.94 uL/rxn (10.3 uL total)
  • Pure H2O: 10.8 uL/rxn (118.3 uL total)

4. For each reaction, we combined 3 uL of the library template DNA to a new, labeled PCR tube.

5. We then added 25 uL of the Master Mix 2 to each tube.

6. We vortexed, and then spun down in the centrifuge.

7. We ran PCR2 on thermocycler BIORAD #2. Specifically, this cycle ran as:

Cycle Step Cycles Temp Time
Initial Denaturation 1 98 degrees C 30 sec
Denaturation

Annealing

Extension

20 98 degrees C

65 degrees C

72 degrees C

10 sec

30 sec

30 sec (b/c 1kb genome)

Final Extension 1 72 degrees C 5 min
Hold 1 4 degrees C infinite

 

The next steps would be to run this PCR product on a gel and then select for bp size.

Lab 11 – DD-RADSeq

This week in lab we performed double-digest restriction associated DNA sequencing

Double Digest of Erythranthe guttate samples:

  1. To double digest 100-1000ng of high quality genomic DNA with selected restriction enzymes and appropriate digestion buffers, we performed the following steps.
  2. I labeled 8 PCR tubes 1-8, and added 6uL of each of the following samples’ DNA, to store on ice.
    1. MONO 005
    2. DIRA 007
    3. INVR 001
    4. CHIM 003
    5. CHIM 002
    6. PRBM 005
    7. CHIM 001
    8. PRBM 004
  3. We prepared a Master Mix for our table. Due to the small volumes used and the viscous nature of the glycerol the enzymes are stored in, we made 130% excess (enough for 11 reactions) of the following recipe:
  • 0.9 uL CutSmart 10X buffer/rxn   (9.9 uL total)
  • 0.28 uL EcoRI-HF enzyme/rxn.   (3.1 uL total)
  • 0.12 uL MPSI enzyme/rxn.           (1.32 uL total)
  • 1.7 uL pure H2O/rxn                     (18.7 uL total)

4. We stored the Master Mix 1 on ice.

5. We added 3 uL of Master Mix 1 to each sample’s DNA, changing pipette tips with every sample.

6. I sealed, vortexes and centrifuged the samples, and then added them to thermocycler with a heated lid set to 50 degrees Celsius, to incubate at 37 degrees Celsius for 8 hours (ran “DD” on BIORAD #1/2 –> DDRAD –>DD).

 

Adapter Ligation:

For this next step in the lab, we used samples that had previously undergone the first 6 steps, and were ready for adapter ligation.

  1. We pulled out the working stock EcoRI and Mspl adapters made previously, to thaw.
  2. We added 1 uL of the working stock EcoRI adapter directly to the digested DNA. We used different adapters in each sample, as paired below:

Sample 9 – Eco 2

Sample 10 – Eco 3

Sample 11 – Eco 4

Sample 12 – Eco 5

Sample 13 – Eco 6

Sample 14 – Eco 7

Sample 15 – Eco 8

Sample 16 – Eco 9

3. We prepared a Master Mix 2 for our table. Due to the small volumes used and the viscous nature of the glycerol the enzymes are stored in, we made 130% excess (enough for 11 reactions) of the following recipe:

  • 0.4 uL CutSmart 10X buffer/rxn                      (4.4 uL total)
  • 1.3 uL ATP 10mM/rxn                                        (14.3 uL total)
  • 0.2 uL T4 Ligase enzyme/rxn                            (2.2 uL total)
  • 0.7 uL pure H2O/rxn                                             (7.7 uL total)
  • 1.0 uL working stock universal P2 Mspl adapter  (11 uL)

4. I added 3 uL of Master Mix 2 to the digested DNA samples.

5. I sealed the samples, vortexes, centrifuged and added to a thermocycler with a heated lid set to 50 degrees Celsius to incubate at 16 degrees celsius for 6 hours (ran “LIGATE” of BIORAD #1/2 –>DDRAD –>LIGATE).

6. After the run, the samples were stored in a freezer.

Lab 10 – Erythranthe guttata PCR set up

In this week’s lab, we did a PCR run on our samples, using the following methods.

  1. We partnered up and labeled 6 PCR tubes P1-P6 (for Pyrimidudes 1-6), noting which label corresponds to which samples. Ours were labeled:

P1 – HWH CATB

P2 – MAPL 002

P3 – SHOR 001

P4 – NPI-1

P5 – MONO 007

P6 – PRBW 006

  1. We micro-pipetted 1 microliter of our samples of extracted DNA into the appropriate PCR tube.
  2. We created a master mix enough for 16 PCR reactions, for our lab group to share. This master mix consisted of:
    1. ddH2O (214 microliters)
    2. 10x buffer (32 microliters)
    3. MgCl2 (32 microliters)
    4. BSA (16 microliters)
    5. dNTPs (3.2 microliters)
    6. Forward primer (3.2 microliters)
    7. Reverse primer (3.2 microliters)
    8. Taq (0.64 microliters)
  3. We vortexed the master to mix it up. Then we added 19 microliters of the master mix to each of the PCR tubes with the DNA in them.
  4. These PCR tubes were then added to the thermocycler to run PCR.

Lab 9 – Mimulus DNA Gel

This week in lab, we ran our plant DNA that we extracted last week from Erythranthe guttata samples through a gel electrophoresis process.

We aloquoted 2-microliter drops of loading dye onto a piece of parafilm. Then, we added 3-4 microliters of our extracted DNA samples.  The entire solution was added to the gel cells, along with the other samples and a ladder in the final cell.  These samples were run in the gel for the remaining lecture (about an hour).

Figure 1. Photo of gel electrophoresis run by my lab group. My samples are the first three cells on the left.

Lab 8 – Plant DNA Extraction

This week, we extracted DNA from Erythranthe guttata leaf samples collected in the field (see Lab 3 for collection methods). The following methods are modified from Alexander et al. DNA extraction protocol.

  1. I labeled three 2.0 milliliter tubes with my sample codes: NPI-1, PRBW 006, and MONO 007.
  2. I added three sterile, 3.2-mm stainless steel beads to each tube.
  3. I added a small amount of dried leaf tissue from the field collection vials to each tube, making sure to clean the forceps between tubes to prevent contamination.
  4. My lab group loaded our samples into a modified reciprocating saw rack and mounted the rack to the saw. We turned on the saw, which shook the samples back d forth on speed 3 for 40 seconds.
  5. We briefly spun the tubes in a centrifuge for 20 seconds at full speed to pull the plant material down to the bottom of the tube.
  6. I added 434 microliters of the preheated grind buffer to each tube.
  7. Then, I incubated the buffered grandame at 65 degrees Celsius for 10 minutes in a hot water bath, mixing the tubes by inverting them every 3 minutes.
  8. Next, I added 130 microliters of 3M pH 4.7 potassium acetate, inverted the tubes several times, and incubated on ice for 5 minutes.
  9. I loaded the tubes in a centrifuge and spun at maximum speed (14,000) for 20 minutes.
  10. In the meantime, I labeled new 1.5mL micro centrifuge tubes with the sample IDs. I transferred the supernatant from the centrifuged samples into the new tubes. I avoided transfer of the precipitate.
  11. Then, I added 1.5x the sample volume of binding buffer (700 microliters) to each new sample tube.
  12. I added 650 microliters of these mixture to Epoch spin column tubes. I centrifuged these tubes for 10 min at 14,000rpm. When finished, I disposed of the flow-through in a hazardous waste container.
  13. I added 650 more microliters of the mixture from step 11 into the Epoch spin column tube, and repeated the spin and disposal of waste.
  14. To wash the DNA bound to the silica membrane, I added 500 microliters of 70% EtOH to the column and centrifuged at 14,000 rpm for 8 minutes. Then I disposed of the flow-through.
  15. I repeated the washing step, adding more EtOH, spinning, and discarding the waste.
  16. Then I ran another centrifuge spin with the tubes for 5 min at 14,000 rpm to dry out any residual ethanol.
  17. I placed the columns in new, sterile, labeled 1.5-mL micro centrifuge tubes.
  18. We added 100 microliters of preheated (65 degrees Celsius) pure sterile H2O to each tube.  We let them stand for 5 minutes, then centrifuged for 2 min at 14,000 rpm.

Lab 7 – Phylogenetic Inference

I finished the previous lab by conducting a BLAST search of the COI gene from 24 ray-finned fishes (Actinopterygii) and one shark (Chondrichthyes). I created alignment of these 25 COI genes, and my two samples EZ02 and EZ03.

I started this lab by cleaning up the alignment, and deleting any extra base pairs from each end. Next, I used the program jModelTest2 to find the best model for these sequences. I exported my alignment from Geneious in a relaxed Phyllip format (.phy), and loaded it into jModelTest2.

From this alignment, I computed the likelihood scores, keeping all defaults for this calculation. I then used two different methods to find the best model. I first tried the Akaike Information Criterion (AIC) method. The best resulting method from this analysis was TVM+G. Then I ran the Bayesian Information Criterion (BIC) method. The best model based on BIC was TPM2uf+G.

Next, I ran a Bayesian inference to create a tree using MrBayes in Geneious. I used ‘HKY85’ for the substitution model, and gamma for the rate variation. I selected the shark gene for the outgroup. I first used a chain length of 10,000, but later repeated the methods with a chain length of 3,000,000. I used an unconstrained branch length and shape parameter of 10.

Next, I used maximum likelihood to infer a phylogenetic tree of my aligned data set. I did this with the RAxML plugin for Geneious. I used the rapid bootstrap with rapid hill climbing method. I used the resulting file to create a consensus tree with a support threshold of 50%.

Then, I tried a different program for maximum likelihood, called PHYML.

My final tree is shown in Figure 1.

Figure 1. Final Phylogeny

Figure 2. Phylogengy Trace

Figure 3. Parameter Estimates

Lab 6 – Intro to Geneious

This week, we worked with the program Geneious to sequence our fish DNA.  Unfortunately, the fish samples that I PCR’d did not produce a useable barcode sequence.  For this lab, I used barcodes from a previous student.  The samples were EZ02 and EZ03.  When she collected the sushi, the student was told that EZ02 was red snapper and EZ03 was yellowtail.  I used Geneious to assemble, edit and BLAST these DNA sequences, and found the results of the species barcodes.  EZ02 was not red snapper as advertised, it was Oreochromis niloticus, or Nile tilapia.  The other sample, EZ03 was Seriola quinqueradiata, or yellowtail, so this one was correctly labeled.

 

The EZ02 alignment I built had only one polymorphism, in column 663.  The EZ03 alignment had seven polymorphisms, found in columns 5, 6, 7, 11, 12, 15, and 201.

Lab 5: Mimulus Encounters Part 2

This week, the molecular ecology lab took a field trip to Marin County to visit more wild populations of our focal plant species, Mimulus guttatus(=Erythranthe guttata).

 

On Tuesday, September 24, 2019 at 1:00PM, the class left in two passenger vans, and headed to Red Rock Spring, Marin County.  The site is located at a gravel pull-out on the east side of Highway 1, just South of Stinson Beach.

 

After getting lost on a Google-maps detour, we found the gravel parking lot and joined the rest of the class. We observed a natural spring, flowing out of PVC pipes from the hillside, and the population of Mimulus guttatus growing in the spring-fed puddle.  The population had many more flowers on display than we had seen at the populations from the Lab 3 field trip, two weeks prior.  All of the flowers appeared to have closed stigmas, suggesting that they had been pollinated.  The habitat was a steep, disturbed hillside that had been carved away to make room for Highway 1.  The population, although fed, seemingly perennially, by the stream, was in full sun, and we can vouch for the heat stress.

 

After leaving this population, the vans left and headed south along Highway 1, eventually turning east and arriving at the Redwood Creek Trailhead.  The trail crosses Redwood Creek, a perennial creek that flows through Muir Woods, all the way to Muir Beach.  The class followed the trail until we got to an old wooden bridge that crosses the creek.  We veered to the right, down the horse bypass trail.  Once we got to the creek, we followed it upstream, searching for Mimulus guttatus.  The creek is surrounded by a dense riparian corridor, a continuous canopy, and characterized by a gravelly, sandy substrate in the creek bed.

 

By now, the class recognized the familiar habitat, and expected to see our monkey flower growing near the waters’ edge.  After ducking under vegetation, crossing log bridges, and only a few submerged shoes, I spotted a population of Mimulus on a dry gravel bar along the edge of the creek.  The individuals were numerous, small, vegetative rosettes.  About 100 ft further upstream, the class found another populations of small Mimulus rosettes.  These populations had not yet flowered.  We considered the likelihood of these plants flowering before winter storms drown them out or wash them away.  We also pondered the movements of pollinators, and how that might affect gene flow between the populations we’ve seen in the field.

 

Eventually we made our way back along the creek, and headed back to campus.

 

Note: Sorry about the absence of photos, my phone was dead before arriving at the first population.

Lab Entry 4

Week 4 lab was an extension of the Sushi Lab started in Week 2.

 

Gel Electrophoresis

  1. We removed our PCR tubes with our samples of fish tissue from ice and given time to thaw.
  2. We laid out 15 dots of about 1 microliter of loading dye on a sheet of parafilm.
  3. We recorded the names and order of samples on a piece of paper.
  4. Each lab member pipetted 3 microliters of each PCR product onto a single dot of loading dye, changing pipette tips in between each sample.
  5. We loaded each dot of loading dye mixed with PCR product into a cell in the gel setup, including a control in the first cell and a ladder in the 15th
  6. We fastened the lid to the gel and turned on the machine to run at 130 volts for 30 minutes.

 

Clean-Up of PCR products for sequencing – ExoSAP

  1. We determined the number of PCR clean-ups for our table, to prepare sufficient master mix. This included one for each sample, and a few extra, for a total of 18.
  2. The master mix recipe was prepared as follows:

Master Mix:                                       Per Rxn              Total Rxn: 18

H2O                                                    10.59 uL    _____190.6_______

10x buffer (Sap 10x)                      1.25 uL      _____22.5______

SAP                                                    0.44 uL      ______7.9________

Exo                                                     0.22 uL      ______4.0______

 

Master Mix Total                     12.5 uL               _______225_______

 

PCR   Product                                       7.5 ul

Total Cleaned-up Volume        20.0 uL

 

3.  We put each reagent in the ice bucket on the table.

4.  We pipetted 7.5 uL of each PCR product into a clean, labeled 0.2 uL PCR tube.

5.  We made the ExoSap master mix, keeping the reagents on ice while it was made.

6.  We pipetted 12.5 uL into each PCR product tube.

7.  We placed the tubes in a thermocycler and started the EXOSAP program.

8.  After the program was done (~ 45 minutes later), Dr. Paul placed the PCR tubes in a labeled tube rack and placed them in the freezer.

 

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