Grace’s Notebook: DecaPod Season 1 Ep 11 – Crab Mtg

Today I edited and published DecaPod S1E11: Crab Meeting #5. I also have a plan for the format of the podcast.

S1 E11: Crab Meeting #5

After talking with Yaamini about my thoughts on how I should plan out the seasons for DecaPod, she mentioned that it could be based on the phase of the project, which makes perfect sense to me.

The seasons may vary by episode numbers, but I’ll separate the seasons by project phase.

Season 1: planning and troubleshooting RNA isolation, libraries Season 2: data analysis Season 3: results/ findings/ etc.

And I think I’m going to keep doing intermittent interviews on crab subjects with new people I meet along the way who are willing to do so.

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Yaamini’s Notebook: DML Analysis Part 5

Blasting for Uniprot codes (again)

Turns out I didn’t do my blastx correctly.

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Mistake 1: Not properly saving my blastx output…

I don’t know how I didn’t realize I never added in a line of code to actually SAVE my blastx output. How. What. Why.

Mistake 2: Blasting the entire genome against the Uniprot database, and not just the genes!

Again, how.

The solution is to re-blast! To help my own brain think clearly, I created a new folder for gene enrichment analyses. I also moved the database files and R Markdown file I created previously into this folder. Thankfully, I could use the same Uniprot database. I then downloaded the C. virginica transcrpt file.

Once all of my input files were assembled, it was time to blastx. I looked through the blastx help menu, but I couldn’t find the arguments I needed awash in the “oh shit I just did everything incorrectly” feeling. I posted this Github issue, and Sam helped identify the arguments I needed. My code looked like this:

  1. Path to blastx
  2. -query provides the file we want to blast 3.-db specifies database created in the previous step
  3. -outfmt specifies the type of output file. I will use 6, a tabular file
  4. -out will allow me to save the output as a new file
  5. -num_threads 4 uses 4 CPUs in the BLAST search
  6. -max_target_seqs 1 keeps only one aligned sequence (i.e. the best match)
/Users/Shared/Apps/ncbi-blast-2.2.29+/bin/blastx \ -query 2018-09-06-Virginica-transcripts.fna \ -db uniprot-filtered-reviewed.fasta \ -outfmt 6 \ -out 2018-09-06-Transcript-Uniprot-blastx.txt \ -num_threads 4 \ -max_target_seqs 1 

Hopefully that will finish running sometime this weekend! Once I have the blastx results, I can merge them with the overlap files and do some gene enrichment. I also need to send the transcript-Uniprot results to Mike Riffle in Genome Sciences so he can fix the background in the gene enrichment tool he built. Until that gets fixed, I can use DAVID to get some preliminary gene enrichment results for PCSGA. I can also review the methods in Emma’s geoduck paper so I understand the statistical analyses that accompany gene enrichment.

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Kaitlyn’s notebook: Prepping geoduck for outplanting

After dissecting oysters, Roberto and I headed to Pt.Whitney. We barely missed the 355 and caught the 445 instead. The lines quickly grew behind us and it was clearly rush hour. Once we arrived, we chatted a bit with Matt and Brent and started working.

Originally, the plan was to screen the geoduck and select half to go into a container for the night before transport. However, because of divider in the heath tray it would be nearly impossible to keep them separate while screening. Instead the geoduck were randomly hand selected out of the tray. They were placed in fine mesh bags that Matt provided since the bags Brent gave me were too large. The bags were the cut corners of larger bags so they were triangular in shaped. I used a sharpie to write the label on, and then vigorously scrubbed and rinsed the label with saltwater so the geoduck wouldn’t be exposed to any chemicals.

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25 geoduck were placed in each mesh bag and closed with a heavy duty zip tie woven between the mesh (except one that is very, very snug). The colored zip ties were added onto the mesh nets at the top, and the colors were selected according to the plot maps.

Colors for each group:

  • 3-H1-T: pink
  • 5-H2-T: green
  • 8-H3-B: orange
  • 2-H0-B: grey
  • 1-H0-T: yellow
  • 7-H3-T: dark blue
  • 4-H1-B: light blue
  • 6-H2-B: red

 

Closed nets were placed back in the trays. Any mortality we found while selecting geoduck was recorded (although we weren’t searching for mortality). Dead geoduck were usually a millimeter or less in length, so they clearly never grew as the live ones were around 5-8mm.

Number found dead in the order sampled:

  • 3-H1-T: 1
  • 5-H2-T: 3
  • 8-H3-B: 0
  • 2-H0-B: 6
  • 1-H0-T: 3
  • 7-H3-T: 3
  • 4-H1-B: 3
  • 6-H2-B: 2

The geoduck looked very healthy across all trays. Here is an example of some in group 7.

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However, the final tray on the bottom was very dirty with lots of algae/other accumulation compared to the rest of the tanks. It was difficult to find living geoduck towards the end, let alone dead ones. This is a photo about halfway through (it looks better on camera than in person).

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Because of this, we switched the trays around as randomly as you could with 4 trays. Here is before and after:

 

More photos of us prepping!

 

 

 

Kaitlyn’s notebook: Fidalgo Bay Dissections

Emily and Micah arrived with the oysters from Fidalgo Bay at 10:45. The oysters were either in bare beds or eelgrass beds. We got to measuring, weighing, and dissecting right away. Per the protocol, large oysters were dissected and small oysters were only massed and measured. Any dead oysters were saved but not included in the mass of the group or measured otherwise. The remaining shells are being kept in the freezer in 209 in labeled sandwhich bags with their corresponding, labeled PVC pipe. Following dissection, tissue was snap frozen in liquid nitrogen and put into the left -80C (shelf 1, column 1, row 5) in a box labeled “Laura’s oly samples 20180906 Fidalgo Bay”.

The oysters and bags were quite dirty with plant debris from the eelgrass beds and mud from the bare bed samples. We rinsed the oysters in weigh boats with tap water and a wash bottle before weighing.

Unfortunately, mortality was very high in both treatments. It was difficult to tell what were large oysters or small oysters. Many labelled large were less than 9mm and some labelled small were up to 15mm. This may have been due to mortality as well as many may not had had the chance to grow. We expected to use about 180 tubes today, but because of mortality only 50 were used. Only 2 bags had full survival while most had 4-5. More bags had 0 survivorship than 75% survival by far. Micah and Emily were able to do some guy and gill dissections though.

There were several polycheates in these bags. One dead oyster might have 4 worms upon separating the shells. Small oysters faired far worse than the large ones (15mm or more). More worms were found in the bag, not in shells, and many worms occupied the inside of the PVC pipe. Here are some images of the worms and everyone sampling.

An example of what we found in most bags:

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