Broodstock Setup

Out with the old…

In a classic fieldwork moment, I had just finished setting up one system for my broodstock, when a newly published paper and a phone chat changed my mind and I had to almost completely redo it. Initially, I had decided to split my adults into 4 or 5 buckets per population of ~25 oysters each in order to maximize genetic diversity by minimizing the chance of one male fertilizing all females. I built a manifold for this system (new term I learned meaning “pipe branching into several openings”) and had 0.5 gal/hour drippers delivering 16degC seawater and algae to the buckets. The broodstock were taken out of their combined tank on Tues. May 31 and separated into a single 5 gal bucket per population. They were split up into ~25 per bucket on Wed. June 15, with 4 buckets for California and 5 buckets each for BC and OR. My cleaning schedule for buckets was to rinse them and wash with Vortexx 3 times a week, and change out the drippers every day I was at the hatchery with ones that had been sitting overnight in Vortexx.

Then on Friday June 17 I read this new paper out of George Waldbusser’s lab at OSU. They conducted a carbonate chemistry manipulation experiment with fertilized eggs extracted from brooding Olympia oysters. A quick reminder- male Olympia oysters release sperm into the water column which is then filtered out by females for fertilization. The females then keep the fertilized eggs in their mantle cavity for up to 12 days before releasing the now weakly swimming larvae. In Waldbusser et al. 2016,they found that larvae extracted ~36 hours after fertilization developed at a similar rate as larvae left in the brood chamber. As far as I know, this was one of the first experiments where Ostrea larvae had been raised outside of the brood. In order to identify a newly fertilized female, they looked for eggs that had been essentially spit out by an adult oyster. Chatting with Matthew Gray from the project was really helpful in figuring out how I can adapt my system to identify brooding females and possibly try to run some experiments of my own on newly fertilized larvae.

…in with the new

On Thursday June 23, I did away with most of the buckets and laid out oysters 2″ apart in black plastic 9 gal tubs (originally meant for cement mixing), with 40-50 oysters per tub and 3 tubs for BC and 2 tubs for CA and OR making 7 tubs total. This number was primarily dictated by space, or else I would’ve split them up into more trays. Some oysters were stuck together in a way that made it impossible to separate without killing them, and so were put in 3gal buckets with small 100 um banjo filters. Each tray has an airstone, either two 1/2 gal/hour drippers or a single 1 gal/hour dripper, and a banjo filter.

Cleaning these trays is a pain as they are too heavy and awkward to pick up and pour out into a bucket, so we use a hose to drain the tray into a bucket and keep the broodstock in a clam bag and separate bucket of seawater during cleaning and filtering for larvae. As we screened out the first batch of larvae on Thursday, we will be cleaning all trays/buckets every day to check for larvae. The official start of the experiment will be when all three populations are releasing enough larvae.

Back in the swing of things

Friday May 13 was my first day back out at Manchester. This summer I will be conducting an acidification stress experiment on Olympia oyster larvae from San Francisco Bay, CA, Coos Bay, OR, and Ladysmith Harbor, BC. Wild adults were collected from each site in December and January and shipped in coolers to a quarantine room at the Manchester Research Station so that the parents may experience the same over-wintering conditions together. They were kept in the same large tank in separate, triply labelled clam bags and fed a mix of live algae from the hatchery and algae paste.

Since arriving in Washington, I’ve mostly been getting my experiment system ready and conditioning my broodstock for spawning. There have been some logistical issues, as a rock scallop project also housed in the quarantine room hasn’t completed yet so we’ve had to adapt the OA system for the possibility of both experiments running at the same time. With many Home Depot/Harrington’s Plastics runs and hours of fitting PVC pipe together, it has felt like slow going. At the very least, if science doesn’t work out I think I’m now qualified to be an assistant plumber!

Attached below is a todo list document I made at one point to help keep track of all the things that needed to be accomplished.

Exp_5_27_16

One great improvement for this summer over last is the help of a part-time intern, Sean, who recently graduated from the University of Washington. As someone who has extensive experience building and running shellfish experiments through his work with the Friedman lab, I’m sure I couldn’t complete this project without his help.

 

Wet Lab posts on Benchling

A few weeks ago I made 2 more Genotype-by-Sequencing libraries for my Olympia Oyster population structure study, made up mostly of redos and some samples from the sister species Ostrea conchaphila. I tried out the free version of Benchling for it and really liked the interface and how it organizes protocols with your notes. I’ll continue using it for my wet lab notes and post the links to my entries here. All field work posts and analysis updates will still be posted here. The link to my Benchling is:

https://benchling.com/ksil91/

This link is on my About page as well.

FST and Admixture Ananlysis of 2bRAD Subset

I did a quick FST and Admixture analysis of the subset of 20 individuals from each population (19 from SS as one of them had inconsistent genotyping across replicates). You can view the results at this Jupyter Notebook on my github. These are very preliminary and will likely benefit from more individuals and by playing around more with the parameters in the mapping/genotyping. I plan to also look at nucleotide diversity.

 

 

Size & 2bRAD Read #’s for MBD BSseq Samples

18 of the adult Olympia oyster broodstock samples that were 2bRAD sequenced were also sent off for MBD-enriched and bisulfite treatment sequencing to get epigenetic information (see project wiki). We wanted to associate size data with these samples and check if the 2brad sequencing worked.

First, some correction of sample names in the 2brad demultiplexing:

When I was on the last step of the 2brad protocol I accidentally deleted some of the sample names in Library 3 from the “Library” sheet in the master sample sheet. I didn’t notice until making the barcode sample sheet and so denoted those on the barcode sheet by putting “w” next to the name. I went back in the sheet revision history to 11/21/15 at 3:22pm and associated the correct name with the well and barcode for these and checked all other samples.

  • HC4_12w -> HC4_11
  • HC4_13w -> HC4_10
  • HC4_15w -> HC4_15
  • HC4_1w -> HC4_7
  • HC4_5w -> HC4_8
  • HC5_1w -> HC5_1
  • SS3_15w -> SS3_20
  • SS3_16w -> SS3_21
  • SS4_1Aw -> SS4_7
  • SS4_1Bw -> SS4_1
  • SS4_3w -> SS4_16
  • SS4_7w -> SS4_9
  • SS4_9 -> SS4_3

Size

When the broodstock oysters were dissected at Manchester last summer, I weighed them in the shell and took pictures of them first with a ruler so later they could be measured (dissections done on 8/13/15 and 8/17/15). I took the pictures for families HC1,  HC2, HC3, SS2, SS3, and SS5 and opened them in ImageJ. For each picture, I set the scale by drawing a line on 1 cm of the ruler to get the number of pixels per mm 3 times and getting the average pixels/mm. To get shell width, I drew a line between the two widest dorsal-ventral points of the shell (with the hinge always on bottom. I did this 2x for each oyster and took the average of the measurements.

Screenshot from 2016-02-12 16:28:19

I also measured shell area by drawing a line around the oyster shell 2x and taking the average. These obviously have more spread in the values.

Screenshot from 2016-02-12 16:33:26

Read Numbers

 

 

2bradbsanalysisb-0

2bradbsanalysisb-1

2bradbsanalysisb-2

Sample Weight (grams in shell) Length (mm) Raw 2bRAD reads
hc1_2B 2.2 17.41 1521
hc1_4B 1.9 20.43 2294290
hc2_15B 2.2 25.33 2498
hc2_17 1.1 19.38 (seq 4x)best = 2964986
hc3_1 2.2 26.79 1863108
hc3_5 1.9 19.5 1915
hc3_7 1.4 18.43 2438612
hc3_10 1.2 19.80 4091281
hc3_11 2.1 20.54 2901608
ss2_9B 1.5 17.2 749
ss2_14B 2.2 21.02 29745
ss2_18B 5.3 35.76 3016
ss3_3B 2.1 22.71 2076769
ss3_14B 2.4 24.71 2661152
ss3_15B 2.6 26.78 2816364
ss3_16B 4.2 39.57 2070520
ss3_20 2.3 26.96 1656456
ss5_18 5.4 27.34 2358172

Wednesday 11/25/15

Ran gels of samples B4-B10 from 2bRAD Library 1 of the PCR done on 11/24/15. Some of the PCRs either did not seem to work or might’ve washed out of the gel. I still cut out bands around 170bp for these, but will look at the quantification to see if anything is recovered. Gel slices were left in 4degC.

Samples that did not work (likely need redos): F5 8(HC1_4B), E5 (HC1_9), B5(HC2_13), B7 (HC3_9), B8(HC3_17, library 4).

 

Tuesday 11/24/15

Finished gel extraction of samples B3-C4 from 2bRAD Library 1 started on 11/23/15 following the protocol described in that post.

Did the PCR of the rest of Library 1 (B5-C12) with the new Taq. There is no H9, however (due to how I labelled on the spreadsheet).

First made more Lib 1 and Lib 2 primer stock:

  • 10 uL stock + 90 uL NFW = 10 uM

PCR master mix. Added 3.75 of each 1 uM barcode (HT and BC) to wells.

1x
65
10 mM (each) dNTPS
1.5
97.5
10 uM ILL-Lib1
1.5
97.5
10 uM ILL-Lib2
1.5
97.5
5X Q5 buffer
15
975
Q5 Taq polymerase
.75
48.75
20.25 + barcode = 27.75
648

 

Monday 11/23/15

Took the tubes with gel slices from Sunday 11/22/15 and centrifuged them at high speed for 1 minute to bring gel in contact with water. Put them in the -80degC freezer for 1.5 hours (Meyer protocol recommends at least 1 hour). Afterwards, centrifuged at maximum speed in the refrigerated centrifuge at 4degC for 15 minutes (Meyer protocol recommends 10-20 minutes). Then pressed gel slice against tube wall and took out between 30-50uL per sample and added then to a PCR plate corresponding to their well positions during the previous 2bRAD steps. I found that if the gel slices were left out of the cold for too long that they were more likely to break up during this step and less supernatant was recovered- in the future will work in batches of 16 and leave the rest in the fridge. Leftover gel slices were put back in the fridge in case they needed to be gel extracted using a kit due to low DNA recovery.

Ran out the gel of wells B3-C4 on a regular gel and cut out the band at around 170bp. Still need to do B4 from the PCR on 11/22/15. Left gel in 40 uL overnight at 4degC.

Gel_2bRAD_Lib1_B3_C4

Gel of samples B-C4 of 2bRAD Library 1

Did the Ligation 2bRAD step on libraries 2 and 3- decided to use 4 as a backup for failed individuals and put that plate in the freezer.

First made new adapters by adding the oligos into 2 separate tubes and leaving at room temp for 10 minutes:

Adaptor 1
150x 
5ILL-NR
7.5
Anti-ILL
7.5
NFW
735
Adaptor 2
150x
3ILL -NR
7.5 uL
Anti-ILL
7.5 uL
NFW
735 uL
Ligation master mix.
1x
149x
2 uM Adaptor 1
5 uL
745
2 uM Adaptor 2
5 uL
745
T4 ligase
1 uL
149
T4 ligase buffer with 10 mM ATP
4 uL
596
NFW
22
3278
10 mM ATP
1
149
Total
38
5662

Added 38 uL to each well.