Deciding WTF To Do (Nov. 2014)

This was a stressful time in my graduate school career. I felt torn by indecision about what the best wet lab method was to get the data I wanted- given the fact I had very little research funds. When I wrote a few grant proposals in Spring 2014, I had chosen to do the ezRAD method with pooling of 20 individuals from a site per library with one unique barcode per library. As the name suggests, this method is technically straightforward as it uses standard Illumina TruSeq preparation kits, with additional benefits of eliminating PCR-induced bias and not requiring sonication. I had enough money for one kit of 24 barcodes and 1 lane of Illumina HiSeq sequencing. Perfect! But in October, I met with a couple faculty members (one doing plant phylogeograhy using high throughput sequencing, the other a bioinformatics/population genomics guy) and they strongly discouraged against the pooling idea. While there was some support in the literature (Molecular Ecology 2013 Gautier) for the ability to get accurate allele frequencies from pooled data, numerous other papers (such as Molecular Ecology 2014 Anderson) cast doubt. By pooling individuals, I would limit the information I could glean from my sequencing data (ie observed heterozygosity or any form of haplotype analysis) and also be making an a priori assumption that the sites I collected were indeed separate populations.

Alright, so pooling was out. But if I could barely afford 24 barcodes, how could I possibly afford enough unique barcodes in order to sequence 96 individuals on a lane?? Fortunately, a curator at the Field Museum offered to share her lab’s Genotype-by-Sequencing (Elshire-2011-A Robust, Simple Gen) adaptors and barcodes for free. The only problem was they only had 48 barcodes, and my goal was to sequence at least 96 individuals per lane (each lane costs $1100-$1800). This act of scientific kindness led me down another path of obsessive pros and cons lists. Numerous grad students, postdocs, and professors (some I had never met and only stalked on the internet) kindly put up with my frantic emails as I tried to figure out wtf to do. Long story short, I decided to accept the 48 GBS barcodes and use a combinatorial index approach as in Double Digest RADSeq. Excessive pros/cons lists attached.

Cost of Pooling with Different Methods

Method:
  1. Make 40 libraries, with 2 for each population each containing 20 individuals. Sequence on 1 lane. Then resequence individuals from a subset (ie 8 pops) on another lane, will get more loci and better coverage. Can be pops that were not sequenced well previously or pops of interest.
    1. Use ApeKI and GBS
    2. Use ezRAD and REs of choice
    3. ddRAD
  2. Make 40 libraries, with 2 for each population each containing 20 individuals. Sequence on 1 lane. Resequence 8 pools that were poorly sequenced or of greater interest. OR sequence 20 each lane.
    1. Use ApeKI and GBS.
    2. Use ezRAD and REs of choice
    3. ddRAD
GBS (vs ezRAD)
Pros
Cons
$2000 cheaper
uneven sequencing of loci: may need to throw out loci. 2nd run def required
Advice on protocol
Not sensitive to methylation
blocked by some CpG methylation
PCR bias
~65,000 fragments
PCR cleanup vs Ampure beads
1a)40, then 96 GBS
Item
Estimated Cost
Sequencing
2200 (if joined with another group, otherwise 3600)
Adapters
~$200 for Y
RE
~$168
Ligase
80-200
PCR purification kit
$220-$524
Bionalyzer
$1088
Total
$3956-$5780
1b)40, 96 ezRAD
Item
Estimated Cost
Sequencing
3600
Adapters
2880
RE
236
PCR purification kit
0
Bionalyzer
$1088
Total
$7104

1c) and d) 40(48) to 40+96 ddRAD

Item
Estimated Cost
Sequencing
3600
Adapters
$4350
RE
$300
Ligase
80-$201
Ampure beads
$945
Pippin Prep
40-120
Bionalyzer
320-$1088
Total
9636-10,604
Not Pooling:
 
Method:
  1. Use GBS with ApeKI.
    1. 96 libraries of individuals using GBS with ApeKI on one lane. 5 from each of 19 populations or 6 from 16 pops. Look at sequencing, then do another lane with 96 individuals.
    2. 48 on two lanes (so as not to fiddle with adaptors).  ~7 from 13 populations.
  2. 96 libraries of individuals on one lane using ezRAD. 5 from 19 populations or 6 from 16.
    1. Adjust sequencing of additional individuals/pops
  3. 96 on one lane using ddRAD. Resequence additional on 2nd lane.
1a) 96 then 96 GBS
Item
Estimated Cost
Sequencing
1800-3600
Adapters
$200
RE
0-150
Ligase
80-$201
PCR Cleanup
330-524
Bionalyzer
$768-$1536
Total
$3178($33/96)-6211($32/192)
1b) 96 then 96 GBS (real)
Item
Estimated Cost
Sequencing
3600
Primers
$200
RE
$256
Ligase
$256
PCR Cleanup
$279-$389
Bionalyzer
$16
NEB Taq 2X Master Mix
$56
Total
$4663-4773 ($24.5/192)
2)96 then 96 ezRAD
Item
Estimated Cost
Sequencing
1800-3600
Adapters
$2880
RE
$150-300
Bionalyzer
$768-$1536 (12($96)-24($192)
qPCR
72-144
Total
5000($52/96)-7116($37/192)
3) 96 then 96 ddRAD
Item
Estimated Cost
Sequencing
1800-3600
Adapters
$4350
RE
$300
Ligase
80-$201
Ampure beads
$945
Pippin Prep
80-160
Bionalyzer
$768-$1536
Total
8323-11,092($86/96-$57/192)

Blood, Sweat, Mud, and Oyster Guts

WillapaMap

Thursday, July 26, 2014

Breakfast was at 4:30am, followed by gearing up for work in the tidal flats. This involves hip boots, a bright orange Pvc rain bib, my matching orange Carhartt rain jacket, and lots of layers. Unfortunately I don’t have the documentation of this fashionable outfit, but imagine a cross between Deadliest Catch and the Stay Puff Marshmallow Man and you’re close.
Jennifer Ruesink is part of a project called the Zostera Experimental Network (ZEN) , which is a collaboration of scientists around the world studying eelgrass (Zostera marina). I got to help out with one of their predation intensity experiments this morning, which involves super gluing live amphipods (tiny crustaceans about 6-10 mm long) to fishing line attached to poles and sticking them in an eelgrass bed to then check the next day. The number of creatures missing is a rough indicator of the level of predation, which can be compared to the level of herbivory (tested by, I’m not kidding, gluing organic kale to similar poles). While not always my first choice of an activity at 6:30a on a boat in the drizzly cold, the company was good and I got to pick Jennifer’s brain about feasible locations to sample from and the general ecology of the area. I also had my first taste of walking in tidal flat mud…
That afternoon I had an in depth conversation with Alan about his natural history observations of the bay and how the oceanography, such as river inputs and currents, may affect population structure. He also seems to know everyone who’s anyone that deals with oyster growing and management at the local and state levels. We mapped out a plan of where to put settlement substrates and where to collect adult oysters from so both the north and south parts of the bay are represented.
For the north part of the bay, the native oysters primarily live 30 feet underwater, and therefore can only be collected from trawling. Luckily for me, a trawl had been done that day to collect Pacific oysters to sell, and they were able to set aside a couple dozen native oysters that had also been picked up. These oysters had been cleaned on the outside and separated from each other, so excellent subjects for my first dissections! Up until then, I had never shucked an oyster, nor even seen an Olympia oyster myself, so this first group was slow going. For each oyster, I shucked off the top shell, rinsed the body with special purified water, and then dissected a small section of the adductor muscle to preserve in a buffer called RNALater (which keeps the DNA from degrading). I then noted the length/width of the shell, whether gonads were present, and the gender when I could tell.
oysterdiagram
For most native oysters, you need to look at the gametes (egg/sperm cells) under a microscope to tell if it is a boy or girl. Olympia oysters are different compared to most oysters in another interesting way. When the water temperature is just right, the males release their millions of sperm into the water for the females to filter out and use to fertilize their eggs. The young larvae are then brooded in the female’s mantle cavity for about 12 days until released into the water. This theoretically gives each larvae a much better chance of surviving the harsh ocean than the Pacific oyster larvae, which are floaters from the second a sperm fertilizes an egg.
OlyLife

PacOysterLife

  Friday, June 27, 2014

ParcelA
This day will forever remain in my memory as the day I almost died 500 meters from dry land…that’s obviously an exaggeration, but at the time it certainly felt that way. My goal was to collect adult Pacific oysters from a site that Jennifer and Alan termed “Parcel A”, look to see if there happened to be any natives hanging around, as well as pick some shell sticks they had set out earlier to look for Olympia oyster spat. Jennifer dropped me off on an oyster hummock with the boat around 7:30am, geared up in my orange Stay Puff outfit with my gloves and little collecting bag. The plan was for me to wait for the tide to go down so I could find the oyster stick, collect about 2 dozen Pacifics, and then walk back to shore and walk the little ways back to their house. I found a comfy spot amongst the razor sharp shells to sit, and then used an incredibly rough method to sample Pacific oysters at random from the surrounding area.
http://depts.washington.edu/jlrlab/oysters.phpLarge oyster hummocks
Oysters grow best on the shells of other oysters, and so in relatively undisturbed areas like this all of the oysters are in clumps, with the younger ones crowding on the older, larger ones. Some of these ladies were 16 cm long, and surprisingly heavy! An hour of traipsing around didn’t yield any native oysters, so I started back towards shore across what seemed to be a flat expanse of easily traversed damp brown. The second I stepped off the oyster reef, however, I sunk down to my knees. As thick mud is one of those non-Newtonian fluids where it actually solidifies under a shearing force, I tried to “sprint” forwards and tired quickly. This is where things got bad, like crying and calling for my momma bad. I made the mistake of sitting down and immediately sank so that mud and water went over my chest high rain bib and filled up my boots, making me even heavier. The Neverending Story came to mind more than a few times…

Admitting defeat, I trudged back to the safety of my oyster hummock, and decided the path of least resistance was to walk through the rising water around the rocks, climb over to the pier, and walk back to the house from there. Numerous people working around the docks must’ve seen this entire scene and I can only guess what they were thinking. Upon arriving at the house, Alan greeted me with a spray hose and congratulations- apparently plenty of people they’ve taken out to the tidal flats end up needing some kind of “rescuing”. He also recommended a bucket next time to help me push myself out when I get stuck (advice I have kept to avidly since).
The afternoon involved dissecting and measuring the Pacifics I had collected, and I discovered the joy of opening a “mudder”- a oyster shell that’s actually full of large polychaete worms and the same thick black mud that irked me so earlier that day. I also practiced collecting the tiny 0.5 mm spat off of the shell sticks. How I’m going to extract DNA from each of these tiny oyster babies is a challenge for back in Chicago.