My goals for the remainder of July are to obtain DNA samples of high quality and purity for ddRAD / EpiRad library generation. This includes performing ethanol precipitation of the DNA samples I have.

For August, my goal is to generate the RAD libraries and submit them for sequencing. Maybe I will even get to start in on the bioinformatics.

In addition to the samples above, I collected the following samples of isolated symbionts using the NaOH treatment protocol of Zamoum et al. (2012).

Incubation 1:

0.5 hr incubation in 1.5 ml 2M NaOH at ambient temperature (28C). Samples vigorously shaken every ~15 min. Final incubation medium centrifuged at 2000 rpm for 2 min and fixed in 1.5 ml SS-DMSO

Samples:

Past1, Past2, Pdiv1, Pdiv2, Ppor

Incubation 2:

1.5 hr incubation in 1.4 ml 1M NaOH @ 28.5C. Samples shaken vigorously every ~15 min. Final incubation medium centrifuged at 2000 rpm for 2 min and fixed in 1.5 ml SS-DMSO.

Samples (note: these do not necessarily match individuals from previous incubation):

Past1, Past2, Pdiv1, Pdiv2, Ppor

It’s high time I laid out some goals for this spring and summer to keep me on track, so here they are:

May

1. Prep reagents and supplies for Carrie Bow trip, including SS-DMSO, tubes, and coolers. Need at least 750 ml SS-DMSO, shoot for 1 L. Need at least 30 tubes, shoot for 50. Bring some shears.

2. Think about methods for zooxanthellae separation while at Carrie Bow, especially this method. May need to bring some NaOH – check with Zach. Ultimately the goal is to get some separated host and symbiont DNA as reference material for RADseq. Will need proper centrifuge tubes – check with Zach.

3. Collect coral samples for Porites porites study while at Carrie Bow.

4. Collect isolated P. astreoides and P. porites host and symbiont cells/tissue while at Carrie Bow.

June

1. Extract DNA from samples collected in May, as well as re-extract any samples from November with low quantity/quality.

2. Purify DNA for all samples destined for RADseq.

3. Confirm with Adam the timing of RADseq work.

4. Start REU project on symbiont genotyping. Narrow down specific samples / question. Currently favoring idea of evaluating symbiont identity in the three branching Porites related to the Hall Award proposal. This could be a separate paper.

July/August

1. Wrap up REU project

2. Perform RADseq and submit samples for sequencing.

Today I continued my assessment of DNA quality for P. astreoides samples extracted earlier this winter by running them on a gel (using this protocol). Here is the gel:

Again, quite a range of quality. I now have looked at nearly all samples and here is my assessment (this data can also be found in this spreadsheet):

Today I determined DNA yield for several samples that I had not yet tested with the Qubit. Here are the results (along with previous Nanodrop results), which can also be found in this spreadsheet:

I also evaluated DNA quality on a gel for several samples. Here is the gel:

As can be seen here, some samples look pretty good, some look ok, and some look like crap. Why such a range? I have no idea, they were all preserved and extracted the same way. Past95 and past19 in particular are particularly bad. These samples should be re-extracted.

Today I completed the final round of DNA extractions from my Belize Porites samples collected in November using the Qiagen DNeasy Blood and Tissue kit. Based on my experience in previous extraction efforts (see previous post), I was generous with tissue quantity. And again, in many cases the tissue was so loosely attached to the skeleton that it was easy to either scoop or scrape off with little skeletal material. As before, I used a single elution volume of 50 ul, which was passed though the column twice. I only evaluated yield this time on the Nanodrop. All samples were in the range of 100 – 400 ng/ul. Based on comparative Qubit results from last time, these Nanodrop values may overestimate yield by approximately double. Also note that most samples had significant absorbance around 230 nm, suggesting the presence of salts, carbohydrates, or phenolic compounds.

Note here that some of the samples in this batch were Porites porites (ppor) as opposed to Porites astreoides (past). Also included in this batch were three samples collected from the top, middle, and bottom of a single P. astreoides colony (w, for within).

Here is the Nanodrop report:

I also ran these samples on a gel. I followed this gel protocol with one modification: I used 1 ul of all samples mixed with 4 ul AE buffer and 1 ul loading dye. There appears to be high molecular weight DNA in all visible samples.

I have been moving forward with extraction of DNA from P. astreoides samples using the Qiagen DNeasy kit. On Monday/Tuesday I did 16 samples, then another 10 samples (actually 9; one was divided in two). For the first set of 16, I did an overnight lysis (6pm to 9am) with no homogenization. Most of the tissue was very loose and easily scraped off the skeleton or scooped from the bottom of the tube. My initial results (see previous post) suggested that larger quantities of tissue were more successful, so I was fairly generous with tissue quantity. I did two 200 ul elutions into two separate tubes. I measured DNA quantity in the first elution with the Qubit HS assay. Here are the results (again, for the first elution only):

For the next set of 10 samples, I did a few things differently. Again, I tried to be generous with tissue quantity. For a few samples, the tissue volume of the pellet was nearly 100 ul. I ranked the tissue quantity on a scale of 1 to 10, 1 being the lowest. I also ranked the sample color after the lysis period to get a second qualitative measure of tissue quantity. I homogenized three of the most dense samples lightly with a plastic pestle (86, 90, 83). The lysis period was from 4:30pm to 9:30am. The lysis did not appear complete, so for the last hour of lysis, I added an additional 180 ul of buffer and 40 ul of proteinase K, and disrupted a few of the most dense samples by pipetting. Sam suggested elution with 50 ul instead of 200 ul, and reusing the original eluate for the second elution. I quantitated the DNA with both Nanodrop and Qubit for comparison. Here are the results:

The Nanodrop quantity estimates were approximately double those from the Qubit. Qubit values were used for estimation of total yield in the last column. Also, from my qualitative estimate of tissue quantity, it appears that higher quantities tended to be better. If you plot the data, there appears to be an asymptote, so I may have reached optimum tissue quantities.

Yesterday I did a second trial run with the same P. astreoides samples used in the previous run (sample #s 1, 10, 97, 100; see previous post). This time, however, I used the Qiagen DNeasy Blood and Tissue Kit. I used approximately the same amount of tissue used in the previous runs, perhaps slightly more. All tissue samples were largely detached (sloughed off) from the skeleton as before, so for most samples I simply scooped loose tissue from the tubes. I did not do any homogenization. However, I did rinse the tissues three times in PBS, compared to only once in the previous run with the EZNA Mollusc Kit. Another thing that I did differently was a shorter 2.5 hr lysis at 56ºC, instead of a 24 hr lysis at 37ºC with the mollusc kit. I chose this lysis duration mainly out of convenience since I was unable to come back the following day. I also vortexed the samples periodically during lysis. At the end of the lysis period, I noted the following with respect to the appearance of the samples: samples 1 and 10, no visible skeletal material, some bits of intact tissue; sample 97, some skeletal material present, very little visible tissue; sample 100, no skeletal material, many small bits of tissue. So from these I estimated that the only sample that was fully lysed was #97. However, I think this was also the sample with the least amount of tissue.

The good news is that this time there was significantly more DNA in most of the samples. Here is the nanodrop report (the second half of the samples with a -2 are the second elution).

My sense is that sample 97 had low DNA concentration due to a low tissue quantity. As for sample 100, I think there may have been too much tissue and incomplete lysis. But I would not say there was huge variance in the amount of sample used, so the large differences in DNA quantity are a bit perplexing. In any case, the results are encouraging and I will move forward with DNeasy for all samples, perhaps with a longer lysis duration and some light homogenization.

I wanted to try out the Omega EZNA Mollusc DNA extraction kit on some of my samples, so I processed four of them with this method. I used Porites astreoides samples 1, 10, 97, and 100. I collected a small amount of tissue that had sloughed off the skeleton from the samples preserved in salt saturated DMSO at room temp. The tissue was fairly loose, so opted not to homogenize it in any way. For the proteinase K incubation, I opted for 37º overnight. Otherwise, I performed the extraction as per the manufacturer’s instructions, without any of the optional steps.

The results were somewhat disappointing; only one sample had a concentration above 5 ng/ul. Sample #10 had a concentration of 9.3 ng/ul; the rest were below 4 ng/ul. Next time I will try more starting material, and do some homogenization. The samples are stored at -20ºC.

On Friday 12/4 I homogenized 6 P. astreoides samples that were part of a short-term (5 day) transplant experiment to look at gene expression. Collection and transplant details of these samples can be found in the previous post. These samples (Past 92, 93, and 94 from 11/20/15 and 11/25/15) are the only ones from my November Belize trip that were preserved in RNAlater. I prioritized dealing with these specimens since RNA preserved in RNAlater has a limited lifetime if not frozen. Half of these samples (the ones collected on 11/20) spent half their time at 4C, but then were kept at room temperature for about a week with the ones collected on 11/25. For each sample, I scraped the top tissue layer off the coral skeleton with a box cutter in a mortar chilled with dry ice, then ground the samples in the mortar with a pestle. I placed the homogenate in 15ml tubes kept chilled on dry ice. I put all samples in the -80°C freezer.