Yaamini’s Notebook: Virginica MBDSeq Day 4

Rinse and repeat

Today I got really good at pipetting supernatant from tubes as they were on the magnetic rack, then adding liquid to the beads and placing the tubes on the rotating mixer. Maybe it’s because that’s all I really did………..

Step 0: Prepare for the day

  • Made 10 mL of the 1X Bind/Wash buffer using the same ratio as yesterday.
    • I used the same bottle of buffer I used yesterday and mixed everything. Then I realized there probably wasn’t a full 2 mL in that bottle since the pipet tip had lots of bubbles. I discarded the buffer I made, and remade it using 5X Bind/Wash Buffer from a new bottle. I labelled this bottle with the date and the volume I took from it.
  • Labelled six sets of 10 1.7 mL centrifuge tubes!
    • Sample Number + Wash 0 (ex. 31 W0)
    • Sample Number + Wash A (ex. 31 WA)
    • Sample Number + Wash B (ex. 31 WB)
    • Sample Number + Elution 1 (ex. 31 E1)
    • Sample Number + Elution 2 (ex. 31 E2)
    • Sample Number + Elution 3 (ex. 31 E3)
  • Got ice
    • Sidenote: the new ice machine is so fancy

Step 1: Remove non-captured DNA

  • Removed tubes from the rotating mixer at 4ºC
  • For each tube:
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “W0” tube, and placed “W0” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “WA” tube, and placed “WA” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
    • Repeated entire process once more
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “WB” tube, and placed “WB” tube on ice
    • Added 200 µL 1X Bind/Wash buffer to “B” tube
    • Repeated entire process once more, EXCEPT I added 400 µL of High Salt Elution Buffer to the “B” tube after pipetting the supernatant from the “B” tube and into the “WB” tube on the second round. I got the High Salt Elution Buffer from the 4ºC fridge

Step 2: Elute captured DNA

  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E1” tube, and placed “E1” tube on ice
      • During this step, I spilled some of sample 106 E1 as I was pipetting it from the “B” tube to the “E1” tube
    • Added 400 µL High Salt Elution Buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E2” tube, and placed “E2” tube on ice
    • Added 400 µL High Salt Elution Buffer to “B” tube
  • For each tube:
    • Placed tube on rotating mixer for 3 minutes
    • Placed tube on magnetic rack for one minute
    • Pipetted supernatant from “B” tube into corresponding “E3” tube, and placed “E3” tube on ice
    • Discarded “B” tubes

I finished this stage at 11:05, and took a break until 12:05 to eat lunch. I covered the samples with extra ice to keep them cool.

Step 3: Ethanol precipitation

Kaitlyn helped me with this step, which was nice since I had 60 tubes!

  • Obtained glycogen from -20ºC freezer
  • Added 1 µL glycogen to each tube (all W0, WA, WB, E1, E2 and E3 tubes)
    • I had sets W0, WB, and E2. Kaitlyn had the others
  • Added 1/10th sample volume of pH 5.2 3 M sodium acetate (made by Sam in March 2007) to each sample
    • I measured the contents of the W0 tubes with a pipet, since those volumes were larger than the others (every other tube had a volume of 400 µL). The W0 samples were 700 µL
    • Added 70 µL of sodium acetate to W0 tubes, and 40 µL to all others (except for 31 WB, which I accidentally added 70 µL to instead of 40 µL)
  • Added 2 sample volumes of 100% ethanol (200 proof) to each sample
    • Added 800 µL to all sets except W0, which needed 1400 µL. The W0 tubes were extremely full, so I had to transfer each W0 tube to a corresponding 2.0 mL centrifuge tube. While I was doing this, Kaitlyn took care of sets WB and E2
  • Kaitlyn vortexed all sample centrifuge tubes while I put reagents away
  • Placed all tubes in a box, then placed box in the -20ºC freezer

Next Thursday, I’ll finish the precipitation process and quantify the reads. Almost done with labwork!

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Yaamini’s Notebook: Virginica MBDSeq Day 3

Dynabeads are dynamite

But first, a couple of things I forgot to mention I did yesterday:

  • Made 10 mL of the 1x Bind/Wash Buffer following the MethylMiner protocol
    • 2 mL 5x Bind/Wash Buffer mixed with 8 mL DNAse free water
  • Sample 106 had a very low concentration of DNA, so Sam set it in the speed vacuum to evaporate the liquid and further concentrate it before I used it

Today, I went through the protocol and stopped when incubating the DNA, dynabeads, and MBD-Biotin protein together. The reagent volumes I used in several steps, if not specified below, can be found here.

Step 1: Calculate new concentration for sample 106

  • Sam removed the sample from the speed vacuum yesterday and placed the sample back in the fridge.
  • Used a pipet to measure volume of sample
  • Calculated the new sample concentration using the original sample concentration, the original volume, and the new volume
  • Calculated the volume of DNAse-free water needed to dilute the sample to 25 ng/µL, which is the concentration specified by the protocol
  • Added DNAse-free water to sample

Step 2: Prepare beads

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “B” (ex. 31 B)
  • Obtained Dynabead stock from fridge
  • Resuspended beads by gently pipetting up and down
  • Added 10 µL of beads for each µg of DNA to each centrifuge tube
  • Brought volume in tube up to 100 µL with 1x Bind/Wash Buffer, and gently mixed tube contents by pipetting
  • For each tube:
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.
    • Repeated this procedure a total of two times.

Step 3: Bind MBD-Biotin protein

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “P” (ex. 31 P)
  • Obtained MBD-Biotin Protein from -80ºC freezer
  • For each µg of input DNA, I added 7 µL of MBD-Biotin Protein
  • Topped off volume to 200 µL with 1X Bind/Wash Buffer, gently pipetting to mix
  • Transfered MBD-Biotin protein tube contents to corresponding “B” tube
  • Placed all “B” tubes on a rotating mixer for one hour at room temperature

Step 4: Wash MBD and beads

  • For each tube:
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.
    • Placed tubes back in rotating mixer for 5 minutes at room temperature
    • Repeated this procedure a total of four times.
  • For each tube:
    • Placed tubes on a magnetic rack for 1 minute to concentrate beads
    • Kept tube in rack and removed supernatant. It’s important to not touch the beads in this step!
    • Immediately removed tube in the rack and added 100 µL of 1X Bind/Wash Buffer to resuspend beads. The beads cannot be left dry for more than a minute.

Step 5: Add DNA

  • Labelled ten 1.7 mL centrifuge tubes with sample number and “DNA”) (ex. 31 DNA)
  • Added 100 µL of 5X Bind/Wash Buffer to each tube
  • Added designated sample DNA volume from calcuations to each “DNA” tube
    • Here’s where I boofed a little. The first sample I added to its designated “DNA” tube was sample 106. I used all of the sample I had, since that’s what Sam and I discussed. I then decided to do the next three samples with the lowest concentrations: 108, 31, and 32. I added all of the sample volume from those tubes, since that’s what Sam and I discussed (normalizing our sample volumes to the ones with lowest concentrations). I went to pull 160 µL from the next sample, but there wasn’t enough sample volume! That’s when I realized that the rest of the samples had not been diluted to 25 ng/µL! I quickly used DNAse-free water to dilute the samples to the the appropriate concentration. For the DNA samples I had already into the “DNA” tubes, I diluted them in those tubes. I accidentally added the 60 µL of water I was going to use to dilute 108 DNA into 108 B. Seeing how the next steps involve me mixing everything anyways, I figured it should be okay. Just another case of me messing up in the best possible way.
  • Topped “DNA” tube volumes to 500 µL with DNAse-free water
  • Transfered “DNA” tube contents to corresponding “B” tubes
  • Mixed tubes on a rotating mixer overnight at 4ºC

One day of labwork done! Here’s what I need to do to first thing tomorrow:

  • Make 10 mL more of the 1X Bind/Wash Buffer
  • Label six sets of 1.7 mL centrifuge tubes for non-captured DNA, washes, and elutions

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Yaamini’s Notebook: Virgnica MBDSeq Day 2

A fulfilling day of labwork!

JK.

This morning I was prepared to start my labwork, but when I talked to Sam about my time restrictions for the day, we both decided it would be better to start working tomorrow instead. I went through the MethylMiner protocol and calculated the amount of DNA I would need from each sample, and the amount of a 1X Wash/Buffer needed for tomorrow. You can find my calculations here.

Tomorrow’s work entails preparing the beads that bind to the DNA. I need to wash the beads, then bind the MBD-Biotin protein to them. Finally, I’ll incubate the beads and protein to my sample DNA overnight. I’m ready to get started!

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Laura’s Notebook: Oly Genetics 104, NF GenePop Analysis

Tried to do the html to .md trick for this notebook, but it did not function. No biggie, since there are no pretty plots in this notebook. Original notebooks: R markdown version, NF-GenePop-Analysis.Rmd; HTML version, NF-GenePop-Analysis.html

In this notebook I will use the GenePop R program to analyze the 2016/2017 Fidalgo Bay (NF) Ostrea lurida microsatellite data; the results from each analysis are housed in a series of .txt files.

Prior to importing the data, prepared the 2016/2017 NF data in Excel and exported into GenePop format; resulting file is available on 2018-01-22-Preparing-for-Genepop.md

First, install GenePop program;

  install.packages("genepop") library(genepop)  

Pull basic information on allele and genotype frequencies per locus and per sample

  basic_info(inputFile="Data/Oly2016NFH+2017NFW_Merged.txt", outputFile = "Analyses/NF-Basic-Info.txt", verbose=T)  

Resulting file: “NF-Basic-Info.txt” Hetero- and homozygosity info pasted here:

NF Wild

Loci Olur10 Olur11 Olur12 Olur13 Olur15 Olur19
Expected Homozygotes 4.99 24.59 11.83 6.68 6.66 6.64
Observed Homozygotes 2 28 12 7 6 7
Expected Heterozygotes 92.01 70.41 86.17 91.32 91.34 90.36
Observed Heterozygotes 95 67 86 91 92 90

NF Hatchery

Loci Olur10 Olur11 Olur12 Olur13 Olur15 Olur19
Expected Homozygotes 5.02 23.03 13.44 7.14 7.22 7.10
Observed Homozygotes 7 27 5 6 6 7
Expected Heterozygotes 92.98 70.97 86.56 88.86 90.78 88.90
Observed Heterozygotes 91 67 95 90 92 89

Assess whether loci are in Hardy-Weinberg Equilibrium

  test_HW(inputFile = "Data/Oly2016NFH+2017NFW_Merged.txt", which="Proba", outputFile = "Analyses/NF-HWE.txt", enumeration = FALSE, dememorization = 10000, batches = 500, iterations = 2000, verbose = interactive())  

Resulting file: “NF-HWE.txt” All P-values across loci in each population are »0.05, do not reject the null hypothesis that all loci are in HWE.

 Pop : NFW-2017

Laura’s Notebook: Oly Genetics 103, preparing microsat data for analysis in GenePop

New day, new genetics analysis work flow. This time I’m going to use GenePop, a standard program that (apparently) does everything I need it to do!

Checking 2016/2017 Fidalgo Bay raw data for correct binning

Crystal rounded the raw microsat data for the Fidalgo Bay 2016-hatchery and 2017-wild data. She provided both raw and rounded data. Before moving forward with the rounded data, I’ll check out the binning method she used.

In the Excel file Olympic Oyster NFH_NFW (1).xlsx she includes data from both wild and hatchery NF samples. She houses raw data for each locus in separate tabs, creates a list of “bins” at 0.2 increments, calculates frequencies for each bin and visualizes with histograms.

image

Then, using the frequency distributions she assigned alleles, for example:

image image

One question I have is regarding the assignment of all even-numbered alleles for Oly10, Oly11 & Oly12, while alleles are odd for Oly13, Oly15 & Oly19.

I also noticed that Oly18 data was initially processed, then not completed nor included in the “rounded” tab. I emailed Crystal to see what’s up (I presume it was an oversight).

Next step is to export the data into a GenePop format. GenePop is one of the most commonly used programs used to analyze microsatellite data. There are several ways to use GenePop: on the web, at the command line, and in R. I like to work in R. I could not find an R-based function to convert .csv format to GenePop format, however thre is an Excel plug-in caled GenAlEx that one can use. I download version 6.503 (Dec 5, 2016). Then, I merged the wild and hatchery data into one spreadsheet. I also found online that the commonly used “genind” format has a few key formatting requirements, which I point out in the following screenshot:

snip20180122_24

With this merged file open, I also opened the GenAlEx program. Then, I used the GenAlEx plug-in to export the file as a GenPop formatted .txt file:

image

A window pops up, which should automatically ID the #loci & #samples if you formatted the spreadsheet like I did; I edited the “Title” to include 2016/2017 info.

image

Saved the file as a .txt file under Oly2016NFH+2017NFW_Merged.txt; here’s what the resulting PopGen formatted file looks like:

image

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Yaamini’s Notebook: Comments and Tags

I pimped out my notebook!

I finally sat down and enabled Disqus commenting for my lab notebook posts and figured out how to tag my lab notebook entries. A quick how-to on both:

Comments:

  • Register on Disqus
  • At the top of each entry, add the text “comments: true”
  • Copy and paste the following code at the end of the lab notebook entry between an and
 
/** * RECOMMENDED CONFIGURATION VARIABLES: EDIT AND UNCOMMENT THE SECTION BELOW TO INSERT DYNAMIC VALUES FROM YOUR PLATFORM OR CMS. * LEARN WHY DEFINING THESE VARIABLES IS IMPORTANT: https://disqus.com/admin/universalcode/#configuration-variables*/ /* var disqus_config = function () { this.page.url = PAGE_URL; // Replace PAGE_URL with your page's canonical URL variable this.page.identifier = PAGE_IDENTIFIER; // Replace PAGE_IDENTIFIER with your page's unique identifier variable }; */ (function() { // DON'T EDIT BELOW THIS LINE var d = document, s = d.createElement('script'); s.src = 'https://the-responsible-grad-student.disqus.com/embed.js'; s.setAttribute('data-timestamp', +new Date()); (d.head || d.body).appendChild(s); })(); <noscript>Please enable JavaScript to view the <a href="https://disqus.com/?ref_noscript">comments powered by Disqus.</a></noscript>
  • Use the following code to count comments
 //the-responsible-grad-student.disqus.com/count.js  

Tags:

  • Add “tags: “ at the top of each lab notebook entry
  • List some tags after the colon! Helps if they are lowercase. Use a space between words to differentiate between tags (ex. “DNR labwork” sets two tags: “DNR” and “labwork”). Use a hyphen for multiword tags (ex. “mass-spec,” not “mass spec”).

Yet another thing I can cross off of my to-do list.

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Laura’s Notebook: Oly Genetics 102, preparing microsat data for analysis in GenePop

New day, new genetics analysis work flow. This time I’m going to use GenePop, a standard program that (apparently) does everything I need it to do!

Checking 2016/2017 Fidalgo Bay raw data for correct binning

Crystal rounded the raw microsat data for the Fidalgo Bay 2016-hatchery and 2017-wild data. She provided both raw and rounded data. Before moving forward with the rounded data, I’ll check out the binning method she used.

In the Excel file Olympic Oyster NFH_NFW (1).xlsx she includes data from both wild and hatchery NF samples. She houses raw data for each locus in separate tabs, creates a list of “bins” at 0.2 increments, calculates frequencies for each bin and visualizes with histograms.

image

Then, using the frequency distributions she assigned alleles, for example:

image image

One question I have is regarding the assignment of all even-numbered alleles for Oly10, Oly11 & Oly12, while alleles are odd for Oly13, Oly15 & Oly19.

I also noticed that Oly18 data was initially processed, then not completed nor included in the “rounded” tab. I emailed Crystal to see what’s up (I presume it was an oversight).

Next step is to export the data into a GenePop format. GenePop is one of the most commonly used programs used to analyze microsatellite data. There are several ways to use GenePop: on the web, at the command line, and in R. I like to work in R. I could not find an R-based function to convert .csv format to GenePop format, however thre is an Excel plug-in caled GenAlEx that one can use. I download version 6.503 (Dec 5, 2016). Then, I merged the wild and hatchery data into one spreadsheet. I also found online that the commonly used “genind” format has a few key formatting requirements, which I point out in the following screenshot:

snip20180122_24

With this merged file open, I also opened the GenAlEx program. Then, I used the GenAlEx plug-in to export the file as a GenPop formatted .txt file:

image

A window pops up, which should automatically ID the #loci & #samples if you formatted the spreadsheet like I did; I edited the “Title” to include 2016/2017 info.

image

Saved the file as a .txt file under Oly2016NFH+2017NFW_Merged.txt; here’s what the resulting PopGen formatted file looks like:

image

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