Sean’s Notebook: Olympia Oyster genome…

Sean’s Notebook: Olympia Oyster genome assembly in Platanus.

I’ve been trying to incorporate the new PacBio data we recently got and we’ve settled on a combination Platanus/Redundans approach for assembling a new genome from scratch.

I’ve finished the first portion surprisingly quickly. Hooray for Hyak I guess?

Some stats on the final gap closed Platanus assembly:

stats for /Users/Sean/Documents/RobertsLab/Oly__gapClosed.fa
sum = 4121175, n = 28721, ave = 143.49, largest = 1971
N50 = 133, n = 10380
N60 = 122, n = 13618
N70 = 114, n = 17127
N80 = 109, n = 20829
N90 = 105, n = 24686
N100 = 100, n = 28721
N_count = 32133
Gaps = 975

Still a lot of gaps. Hopefully the Redundans run with the PacBio stuff will help with that.

Data: here

Gap filled Assembly:

Now on to Redundans.

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Sean’s Notebook: Bismark mapping efficiency…

Sean’s Notebook: Bismark mapping efficiency with Hard trimmed C. virginica sample.

Yesterday Mackenzie Gavery came by and offered some suggestions to increase mapping rates for our Virginica BS-Seq data using Bismark. Her two suggestions were using the –non_directional flag to account for the PBATness of the data, which had a huge effect, and hard trim the first 16 bases in our samples, because they look weird.

I tried everything on a single sample for speed and finished it this morning.

The weirdness

Untrimmed:
untrimmed

Default Trimmed:
trimmed

Hard Trimming of the first 16 bases:
hardtrimmed

That cleans stuff up for sure. Unfortunately it didn’t have much of an effect on mapping rate, bringing us up from 28% to 28.3%. Was worth a shot though!

Final Alignment report
======================
Sequences analysed in total:	12197930
Number of alignments with a unique best hit from the different alignments:	3456338
Mapping efficiency:	28.3%
Sequences with no alignments under any condition:	5760842
Sequences did not map uniquely:	2980750
Sequences which were discarded because genomic sequence could not be extracted:0

Number of sequences with unique best (first) alignment came from the bowtie output:
CT/CT:	181719	((converted) top strand)
CT/GA:	166362	((converted) bottom strand)
GA/CT:	1588675	(complementary to (converted) top strand)
GA/GA:	1519582	(complementary to (converted) bottom strand)

Final Cytosine Methylation Report
=================================
Total number of C's analysed:	61813350

Total methylated C's in CpG context:	12572131
Total methylated C's in CHG context:	4005979
Total methylated C's in CHH context:	12350257
Total methylated C's in Unknown context:	0

Total unmethylated C's in CpG context:	2271987
Total unmethylated C's in CHG context:	12442077
Total unmethylated C's in CHH context:	18170919
Total unmethylated C's in Unknown context:	5

C methylated in CpG context:	84.7%
C methylated in CHG context:	24.4%
C methylated in CHH context:	40.5%
C methylated in Unknown context (CN or CHN):	0.0%

Bismark output files located: here

now time to run the rest of them!

Yaamini’s Notebook: Another Skyline Fail

The DIA pipeline has been quite a #strugglebus

ride-this-virtual-struggle-bus-and-watch-your-lif-1-11214-1380570808-0_big

It’s unfortunate that the strugglebus website no longer works :/ Let’s go through our current struggle:

  • I showed Emma my error checking results
  • She said my error rate was pretty high (and I agreed)
  • She looked at my blib and Laura’s blib with people from her lab (a.k.a. the Skyline creators)
  • They found asterisks at the end of some of the blib sequences that could affect Skyline’s ability to peak peaks
  • Another problem may be that brecan reports the same peptide multiple times, and Skyline doesn’t like that

Overall, this means that my current Skyline output isn’t valid anymore. I can’t work on MSstats or target identification using Skyline data, but I can still look through the literature for proteins of interest. Emma and her team are working on fixing Skyline for our files, so I just have to wait! After everything’s fixed, I will need to check error rates again for the same peptides.

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Katie’s Notebook: Getting Started!

Successfully made it to my first day at the Roberts Lab yesterday! I attended the lab meeting and logged into all the sites and platforms that I will need access to this summer. I left for the day with some readings to catch myself up on basic information pertaining to Olympic and Pacific Oysters. I also did a lot of browsing of Yaamini’s and Laura’s notebooks to learn about their current projects!

Yaamini’s Notebook: Protein Extractions Round 2, Part 2

More cowbell? More samples.

I started thinking about my next round of protein extractions a few weeks ago, but now I have a better plan. I’m going to focus solely on the first outplant. I also noticed that the second ouptlant Case Inlet organisms had the lowest sample size. There were only five in the bare treatment, and one in the eelgrass treatment. For this reason, using only the first outplant would give me a larger sample size and allow me to understand the differences between all sites and eelgrass conditions.

I’m going to extract proteins in two blocks of 25 samples each:

Table 1. Proteins to extract. The bolded samples are those I will extract in the first block.

Site Condition 1 2 3 4 5
PG B O25 O83 O54 O51 O52
PG E O31 O71 O78 O56 O30
FB B O36 O70 O43 O40 O35
FB E O64 O46 O32 O24 O49
WB B O129 O126 O135 O121 O122
WB E O140 O145 O147 O131 O144
SK B O111 O120 O99 O96 O113
SK E O103 O101 O106 O102 O91
CI B O11 O13 O16 O21 O22
CI E O01 O08 O10 O06 O04

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Yaamini’s Notebook: Protein Extractions Round 2, Part 2

More cowbell? More samples.

I started thinking about my next round of protein extractions a few weeks ago, but now I have a better plan. I’m going to focus solely on the first outplant. I also noticed that the second ouptlant Case Inlet organisms had the lowest sample size. There were only five in the bare treatment, and one in the eelgrass treatment. For this reason, using only the first outplant would give me a larger sample size and allow me to understand the differences between all sites and eelgrass conditions.

I’m going to extract proteins in two blocks of 25 samples each:

Table 1. Proteins to extract. The bolded samples are those I will extract in the first block.

Site Condition 1 2 3 4 5
PG B O25 O83 O54 O51 O52
PG E O31 O71 O78 O56 O30
FB B O36 O70 O43 O40 O35
FB E O64 O46 O32 O24 O49
WB B O129 O126 O135 O121 O122
WB E O140 O145 O147 O131 O144
SK B O111 O120 O99 O96 O113
SK E O103 O101 O106 O102 O91
CI B O11 O13 O16 O21 O22
CI E O01 O08 O10 O06 O04

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Yaamini’s Notebook: Heat Shock Practice

It’s always good to practice your methods of torture.

On May 12, I figured out my methods for the heat shock experiment I wnat to conduct with the C. gigas we brought back from Manchester. The reason why I want to conduct a heat shock is because I want to see if different stressors methylate different regions of the genome, and if a combination of stressors will create a unique methylation pattern.

The first thing I did was create a hot water bath with saltwater from our flow-through system. Although the water bath has a temperature reading on it, I also added a thermometer to double check. The temperature I aimed for was 40 ºC.

img_7380

After checking that the temperature was at 40ºC, I grabbed four oysters from our set-up…

img_7378

…and plopped them in the water bath!

img_7382

I started the heat shock treatment at 4:17 p.m., and let it run for an hour. At 5:17 p.m., I killed the heat and took the oysters out. The temperature on the water bath and on the thermometer read around 40ºC.

img_7384img_7385

I checked mortality everyday since then, and they’re still alive! Looks like one hour at 40ºC should work for a heat shock.

Things I need to do before Monday:

  • Find a heater for the 100 L tanks
  • Figure out how many oysters to shock
  • Find a time to start heating the water before the day of the shock

The bigger question, however, is whether or not I’m going to do a heat shock at all! Because I have 45 oysters for each OA condition, and not all of those oysters will be fertile when I’m ready to spawn and the sex ratio of oysters can’t be predicted, I don’t want to use oysters I could have spawned with. I may apply a heat shock to some of the oysters that were kept on the ambient line continously, and then cross those with oysters from the OA treatment. Either way, I’m going to expose spat to both OA and heat shock conditions, so I’m still going to assess the effect of multiple stressors on the next generation.

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