Basic data analysis in R Studio

So as I bring in trainees into the lab, I’ll want them to learn how to do some (at the very least) basic data analyses. I realize they may not know exactly where to start, so as I go about making my own basic analysis scripts for analyzing data relevant to me, I’ll post them here and make sure they’re reasonably well commented to explain what is going on.

OK, the specific backstory here. Back in UW, we had next-day IDT orders. It became very clear that was not going to be the case at CWRU, especially after talking to the IDT rep (who did not seem to really care about having a more thriving business here). So, I priced out my options, and Thermo ended up handedly winning the oligo price battle ($0.12 a nucleotide. which is a slight improvement over the $0.15 we seemed to be paying at UW *Shrug*). Thermo also does no shipping cost (another bonus), with the downside being that they only deliver on Tuesdays and Thursdays. I wanted to figure out how long it takes to receive primers are ordering them, so I’ve been keeping track of when I made each order, and how long it took to arrive. Now that I have a decent number of data-points, I decided to start analyzing it to figure out if there were any patterns emerging.

Here’s a link to the data. Here’s a link to the R Markdown file. You’ll want both the data and the R Markdown file in the same directory. I’m also copy-pasting the code below, for ease:

Primer_wait_analysis

This is the first chunk, where I’m setting up my workspace by 1) Starting the workspace fresh 2) Importing the packages I’ll need 3) Importing the data I’ll need

# I always like to start by clearing the memory of existing variables / dataframes / etc.

rm(list = ls())

#Next, let's import the packages we'll need.
#1) Readxl, so that I can import the excel spreadsheet with my data
#2) Tidyverse, for doing the data wrangling (dyplr) and then for plotting the data (ggplot)

library(readxl)
library(tidyverse)
## ── Attaching packages ─────────────────────────────────────────────────────────────────────────── tidyverse 1.3.0 ──
## ✓ ggplot2 3.2.1     ✓ purrr   0.3.3
## ✓ tibble  2.1.3     ✓ dplyr   0.8.3
## ✓ tidyr   1.0.0     ✓ stringr 1.4.0
## ✓ readr   1.3.1     ✓ forcats 0.4.0
## ── Conflicts ────────────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
## x dplyr::filter() masks stats::filter()
## x dplyr::lag()    masks stats::lag()
# Lastly, use readxl to import the data I want

primer_data <- read_excel("How_long_it_takes_to_receive_items_after_ordering.xlsx")

The idea is to make a bar-graph (or equivalent) showing how many days it takes for the primers to arrive depending on what day I order the primers. I first have to take the raw data and do some wrangling to get it in the format I need for plotting.

# I care about how long it took me to receive primers in a *normal* week, and not about what I encountered during the holidays. Thus, I'm going to filter out the holiday datapoints.

primer_data_normal <- primer_data %>% filter(holiday == "no")

# Next, I want to group data-points based on the day of the week

primer_data_grouped <- primer_data_normal %>% group_by(day_of_week) %>% summarize(average_duration = mean(days_after_ordering), standard_deviation = sd(days_after_ordering), n = n())

# Now let's set the "day_of_week" factor to actually follow the days of the week

primer_data_grouped$day_of_week <- factor(primer_data_grouped$day_of_week, levels = c("M","T","W","R","F","Sa","Su"))

# Since I'll want standard error rather than standard deviation, let's get the standard error
primer_data_grouped$standard_error <- primer_data_grouped$standard_deviation / sqrt(primer_data_grouped$n)

Now that the data is ready, time to plot it in ggplot2.

Primer_plot <- ggplot() + geom_point(data = primer_data_grouped, aes(x = day_of_week, y = average_duration)) +
  geom_errorbar(data = primer_data_grouped, 
                aes(x = day_of_week, ymin = average_duration - standard_error, 
                    ymax = average_duration + standard_error), width = 0.5) + 
  geom_text(data = primer_data_grouped, aes(x = day_of_week, y = 0.2, label = paste("n=",primer_data_grouped$n))) + geom_hline(yintercept = 0) + scale_y_continuous(limits = c(0,7), expand = c(0,0.01)) +
  theme_bw() + xlab("Day primer was ordered") + ylab("Days until primer arrived") + theme(panel.grid.major.x = element_blank())

ggsave(file = "Primer_plot.pdf", Primer_plot, height = 4, width = 6)
Primer_plot

Hmm, probably should have made that figure less tall. Oh well.

So the n values are still rather small, but it looks like I’ll get my primers soonest if I order on Monday or maybe Tuesday. In contrast, ordering on Thursday, Friday, or Saturday give me the longest wait (though well, some of that are the weekend days, which don’t matter as much). Thus, if I have any projects coming up where I have to design primer, it’s probably worth me taking the time to do that on a Sunday or Monday night instead of late in the workweek.

Writing a simple Python script (for Biologists)

Being able to write short scripts that help you do what you need to do is a very empowering ability for modern-day biologists like me.

In the process of thinking about planning a new plasmid that encodes two proteins fused together by a flexible linker, I realized that I should just write a short script that, when run, gives me a random set of glycine and serine residues composing that linker (riffing off of the ol’ GGS-repeat linkers that seem to prevail in synthetic molecular biology). I’m also in the spirit of documenting the things I’ve learned so that future trainees could have them as reference for their own learning. So here goes my attempt at explaining how I’m approaching this:

First is thinking about the scope of the script you’re trying to put together. This should be an extremely simple one, where I’ll have lists of all of the glycine and serine codons, and a series of random numbers will determine which of those codons to use. Maybe I’ll also include in this script a user-feedback feature that will make it so you can tell the script how many codons it should be randomnly stringing together. Since it’s so simple, it shouldn’t require loading too many custom libraries / packages for performing more advanced procedures.

In the spirit of good practice, I’ll try to do this in iPython as well, though I’ll make a simple python script at the end for quick running from the command line. OK, here goes:

Note: I posted the final code at my Github page (link at the bottom). But for people wanting to just follow along here, here is the code in its final form at the outset (so you can see how I went about building it):

from random import randint

codons = ["GGT","GGC","GGA","GGG","TCT","TCC","TCA","TCG","AGT","AGC"]

length = input("What amino acid length flexible linker would you like a nucleotide sequence for?")

linker_sequence = ""
length = int(length)
for x in range(0,length):
    new_codon = codons[randint(0,9)]
    linker_sequence = linker_sequence + new_codon
    x = x + 1

print(linker_sequence)

1) Had to remind myself of this, but firs you type in “jupyter notebook” to open the interactive web-browser interface (assuming you’ve already installed it).

2) Go to the right directory, and then make a new iPython3 notebook.

3) Let’s start simple and make the random codon generator. First, let’s make a list of the codons we want to include.

codons = ["GGT","GGC","GGA","GGG","TCT","TCC","TCA","TCG","AGT","AGC"]

4) Next, let’s figure out how to choose a random index in the list, so that a random codon is chosen. I don’t remember how to do this off-hand, so I had to google it until I got to this page.

5) OK, so i guess I do have to load a package. I’m now writing that at the top of the script.

from random import randint   
randint(0,9)

It gave the correct output so I saw that it worked and commented out the randint call.

# randint(0,9) #This line won't run

6) Next is having the script randomly pull out a glycine or serine codon. This is simply done by now including:

codons[randint(0,9)]

7) Cool. All that has worked so far. Let’s now have the script take in a user-input for the number of residues so we can repeat this process and spit out a linker sequence of desired length. I don’t remember how to ask for user input in a python script, so I had to google this as well.

8) Allright, so I need to use the use the “raw imput” function and lead it to a variable. Well, tried that and it said “raw_input” wasn’t defined. Google that, and found this link saying that advice as deprecated, and instead it was just “input()”.

9) Thus, I typed in:

length = input("What amino acid length flexible linker would you like a nucleotide sequence for?")

And this asked me for a number like I had hoped. Great.

10) To make this process iterable, I went for my trusty “for” loop. Actually, I tried to make a “while” loop first, but realized I didn’t know how to make it work off the top of my head. So I just ended up making a “for” loop and putting a x = x + 1 statement at the end to effectively turn into a “while” loop. Personally, I think the ends justify the means, and going with what you know works well is a valid option for most basic scripts, when efficiency isn’t a huge priority.

length = 3  #Giving an arbitrary number for testing the script
for x in range(0,length):
    new_codon = codons[randint(0,9)]
    x = x + 1 

11) I then had to be able to keep track of the codons that were pulled during each iteration of the loop. I thus created an empty variable called “linker_sequence” and just added the string for the new codon at the back of “linker_sequence” during each iteration.

linker_sequence = ""  # A blank variable to keep track of things
length = int(length)  # To convert the input text into a number
for x in range(0,length):   
    new_codon = codons[randint(0,9)]  
    linker_sequence = linker_sequence + new_codon  
    x = x + 1

12) Lastly is putting a final print function so it returns the desired string of nucleotides to the user.

print(linker_sequence)

Nice, I think that does it for the script. Super easy and simple!

13) Finally, let’s test the script. I typed in 5 amino acids, and it gave me “TCTAGTTCAGGCTCT” as the output string. I google searched “Transeq” to get to the ebi for a simple codon translator, and translation of the nucleotide sequence above gave “SSSGS” as the protein sequence. So great, it worked! Sure, a little serine heavy, but that’s random chance for you. Should still be perfectly fine as a linker, regardless. Now to finish planning this plasmid…

Note: I’ve posted both the iPython notebook file (.ipynb) and a simple python script (.py) on my Github page. Feel free to use them!

Installing Enrich2

Installing Enrich2 / using a Conda environment

I have a couple of new Macs in the lab that need Enrich2 installed. The goal today is to go through the steps of making a Conda environment specifically for running Enrich2 (and installing Enrich2), developed by Alan Rubin.

The Enrich2 repository

The Enrich2 docs

Alan instructions for making a Conda environment specifically for Enrih2

Alan’s instrucitons for doing this is pretty good, if I remember correctly. But, Alan is a seasoned programmer / computational scientist, while people like me are novices and far less familiar with these steps. Furthermore, depending on the specifics of your computer / system, you may get different errors in the installation process. Thus, here’s my interpretation of this process for the benefit of others like me.

0) This supposes that you have already installed an updated version of Anaconda on your Mac. Go back and do this now, if you haven’t done so already.

1) Download the Anaconda Enrich2 environment file and put it somewhere you can access using Terminal.

2A) Update to the newest version of Anaconda, just in case. Hit “y” if prompted.

conda update conda

2B) OK, so now was installing the right version of Pandas it needs. To do this, I first went into Anaconda Navigator and made a new environent called python2, that uses Python 2. Then in terminal, I called:

conda activate python2

2C) This activated python2, so that now the prompt didn’t say “base”, but now it said “(python2)”. Cool. Envinroment activated. I then installed pandas 0.19.2 by typing in:

pip install pandas==0.19.2

2D) OK, this seemed to work too. I got an error when trying to re-run the “enrich2_env.yml file”, so I removed the “=0.19” part form the .yml file and ran “conda env create -f enrich2_env.yml”. This actually seemed to work giving me a list of packages being extracted. Now to actually test it out.

conda activate enrich2

3) Cool, that worked, and the terminal prompt now says “(enrich2)”. Now we’re in business. Next is actually installing Enrich2 now that we’re in the enviroment. Go to the Enrich2 repository, download the file, unzip it, and then move to its directory in terminal. Then run:

python setup.py install

That seemed to work since it didn’t throw any errors.

4) Next is actually trying to run the application. Type in the following:

enrich_gui

Oh Jesus christ. It literally crashes the Finder due to the following errors:

CGSTrackingRegionSetIsEnabled returned CG error 268435459
CGSTrackingRegionSetIsEnabled returned CG error 268435459
CGSTrackingRegionSetIsEnabled returned CG error 268435459
HIToolbox: received notification of WindowServer event port death.
port matched the WindowServer port created in BindCGSToRunLoop

Looks like this may have been a problem with the MacOS operating system. Updating my OS to Catalina and then trying again.

5) OK, MacOS has been updated to Catalina. Now let’s try running Enrich again. (You’ll likely want to run this command after you’ve navigated to the directory with your raw files to simplify the file locating process).

enrich_gui

Awesome. It worked!

PS. I followed these instructions for my second Mac and it worked like a charm. I even went straight for the Cataline update early on and didn’t run into the error in Step4.

Downloading JoVE videos

Part of my goal for this holiday break is to work on an exploratory research grant proposal for a high-throughput investigation studying how protein coding variants in inflammasome components lead to various autoinflammatory diseases. I heard there were supposed to be some cool-looking videos of ASC speck formation (like this video from Kuri et al, 2017, J Cell Biol), so I did a google search for such videos. This lead me to some videos at JoVE, the Journal of Visual Experiments. CWRU has institutional access to tons of journals including JoVE, but always having to log in to watch the video is kind of clunky, so I wanted to be able to download the relevant videos. Thus, I just looked under the hood at the html used to organize the webpage, and found where the video lived so I could download it. Here are some instructions for doing just that:

1) Using Google Chrome (though I’m sure other browsers like FireFox should do this as well), log into your institutional access service to get to the login-protected JoVE page with the full video.

2) Right click on the area with the video and hit “inspect”.

3) In the top inspector pane, go to the area that says something along the lines of…:

<video class=”fp-engine” playsinline=”” webkit-playsinline=”” preload=”none” autoplay=”” crossorigin=”anonymous” src=”blob:https://www.jove.com/d4697446-c6f4-4902-ab6b-37580284d671″ style=”display: block;”><source type=”video/mp4″ src=”https://cloudflare2.jove.com/CDNSource/protected/57463_Fink_051418_P_Web.mp4?verify=1576994641-Y2wt%2BLiPU3Iw9mTSO%2BNHPlX%2BjGEwXuULht5jH92%2FuzY%3D”><track kind=”subtitles” label=”English” srclang=”en” src=”/files/vtt/57463/57463.vtt” id=”en-English”></video>

… and click on the triangle to open up that section and display all of the sub-sections of it.

4) The first subsection should say something like …:

<source type=”video/mp4″ src=”https://cloudflare2.jove.com/CDNSource/protected/57463_Fink_051418_P_Web.mp4?verify=1576994641-Y2wt%2BLiPU3Iw9mTSO%2BNHPlX%2BjGEwXuULht5jH92%2FuzY%3D”>

… and right click on the “src” link and open in a new window.

5) Right click again and download the video file to your hard drive. It will likely be a .mp4 file format. Now you can rewatch it without having to be logged into the JoVE website.

PS-1. Once you’re at the first part of step 3, you can just look in the “src” section and copy-paste the text starting at “https://…” up through “…mp4″ (and not ?verify…” and copy-paste that to a new window as well, and skip to step 5. Though I suppose this is actually the same amount of effort as actually doing step 4.

PS-2. Yes, I find it kind of funny that I just made a tutorial of saving a video from a visual tutorial.

PS-3. For the record, I’m not supporting / condoning bypassing the gatekeeping code this journal has for accessing the full-content. I’m mostly just trying to streamline science so people can get more / better work done without impediments. As far as I can tell, you still do need institutaional access to be able to access the full file (doing the above steps at the non-logged in site only links you to the “teaser” video).

Simulating sampling during routine lab procedures

TL;DR: Statistics is everywhere, and simulating bottlenecks that happen during routine lab procedures such as dilutions of cells can potentially help you increase reproducibility, and at the least, help you better conceptualize what is happening with each step of an experiment.

I’m still working on getting a cell counter for the lab. In the meantime, we’ve been using an old school hemacytometer to count cells before an experiment. Sarah had used a hemacytometer more recently than me, and knew to dilute the cells from a T75 flask 10-fold to get them into a countable range for the hemacytometer. She said she had performed the dilution by putting 10 ul cells in 90 ul media (and then putting 10 ul of the dilution into the hemacytometer). But as she said this, she asked whether it was OK to perform the dilution as described; a grad student in her previous lab had taught her to do it that way, but a postdoc there said it was a bad idea. My immediate response was that if the cells are sufficiently mixed, then it should be fine. And while that was my gut reaction, I realized that it was something I could simulate and answer myself using available data. Would the accuracy of the count be increased if we diluted 100 ul of cells into 900ul of media, or 1ml of cells into 9ml of media?

Here are the methods (skip if you don’t want to dive in and want to save yourself a paragraph of reading): To me, it would seem the answer to whether the dilution matters depends on how the cells are dispersed in the media / how variable the count is when the same volume is sampled numerous times. Sarah’s standard practice is to count four squares of the hemacytometer, so I had four replicate counts for each volume pipetted. She had repeated this process three times by the time I had performed the analysis, giving me a reasonable dataset I could roll with. I got the mean and standard deviations for each of the three instances, all corresponding to a volume of 0.1 ul. They were all quite similar, so I created a hypothetical normal distribution from the average mean and standard deviation. Next was seeing how different ways of performing the same dilution impacted the accuracy of individual readings. I recreated the 10 ul cells by sampling from this distribution 100 times, 100 ul cells by sampling 1,000 times, and 1 ml by sampling 10,000 times, and taking the mean. I repeated this process 5,000 times for each condition, and looked at how wide each distribution was.

I then turned the counts into concentration (cells / ml):

Instead of stopping there, I thought about the number of cells I was actually trying to plate, which was 250,000. The number the distributions were converging to was ~ 27.3 (black line), so I used that as the “truth”, and saw how many “true” cells would be plated if I had determined the volume needed to be plated based on each of the repeat concentrations calculated by each of the conditions of dilutions. The resulting plot looked like this:

So as you can tell based on the plot, there are slight differences in cells plated depending on imprecision propagated by the manner in which the same 10-fold dilution as performed: while all distributions are centered around 250k, the 10 ul dilution distribution was quite wide, while the 1 ml in 9 ml dilution resulted in cell counts very close to 250k each time. To phrase it another way, ~15% of the time, a 10 ul in 90 ul dilution would cause the “wrong” number of cells to be plated (less than 24k, or more than 26k). In contrast, due to the increased precision, a 100 ul in 900 ul dilution would never result in the “wrong” number of cells being plated. So speaking solely about the dilution, the way the dilution was being performed could have some light impacts on the accuracy of how many cells would be actually plated.

I was going to call this exercise complete, but I ran this analysis by Anna, and she mentioned that I wasn’t REALLY recreating the entire process; sure I had recreated the dilution step, but we would have also counted cells from the dilution in the hemacytometer to actually get the cell counts in real life. Thus, I modified the code such that each dilution step was followed by a random sampling of four counts (using the coefficient of variation determined from the initial hemacytometer readings), and taking the mean of those counts; this represented how we would have ACTUALLY followed up each dilution in real life. The results were VERY different:

In effect, the imprecision imparted by the hemacytometer counts seemed to almost completely drown out the imprecision caused by the suboptimal dilution step. This was pretty mind-blowing for me; especially considering that I would have totally missed this effect had I not run this post by Anna. Now fully modeling the counting process, a 10 ul in 90 ul dilution would cause the “wrong” number of cells (less than 24k, or more than 26k) to be plated ~ 42.5% of the time, and a 100 ul in 900 ul dilution would still cause a “wrong” cell number to be plated ~ 42.2 % of the time; almost identical! Thus, while a 100 ul in 900 ul dilution does impart some slightly increased accuracy, it’s quite minor / negligible over a 10 ul in 90 ul dilution. So while in a sense this wasn’t the initial question asked, it’s still effectively the real answer.

At the end of the day, I think the more impactful aspect of this exercise is the idea that even routine aspects of wet-lab work are deeply rooted in stats (in this case, propagation of errors caused by poor sampling), and that the power of modern computational simulations can be used to optimize these procedures. There’s something truly empowering to having a new tool / capability that gives you new perspectives on procedures you’ve done a bunch of times, and allows you to fully rationalize it rather than relying on advice given to you by others.

Here’s the code if you want to try running it yourself.
Acknowledgements: Thanks to Sarah for bringing this question to my attention. Also, BIG THANKS to Anna for pointing out where I was being myopic in my analysis, which got me to a qualitatively different (and more real-life relevant) answer. It really is worth having smart people look over your work before you finalize it!

Software to download

I recently bought myself a new computer for the office, which meant that I had to download and install all of the key software I used for work. I decided to write down what these were so future me (or future employees) could have it as a reference. All of the following links / commands are for use with a mac.

Everyone should install these:

Google Chrome

Google Drive & Sync or Google Drive File Stream

Microsoft Office

Anaconda

R (requisite for R Studio)

R Studio 
packages worth installing: tidyverse, shiny, ggrepel

XQuartz (requisite for Inkscape on older macs)

Inkscape

Pymol

iTerm2

Sublime Text 3

FlowJo (v10)

Optional (but useful):

Box Sync Installer

Dropbox desktop app

MacDown

A plasmid Editor (ApE)

Snapgene Viewer

Flux

Sim Daltonism

ImageOptim

The Unarchiver

4 Peaks

Skim

GitHub Desktop

Zoom Desktop

Skype Desktop

OBS (Open Broadcast Software)

Useful installations from the command line

Samtools

First go to the Samtools directory

$ ./configure

$ make

$ make install

$ export PATH=bin:$PATH

Homebrew

$ xcode-select –install

$ ruby -e “$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)”

$ brew doctor

Gifski

$ brew install gifski

FFmpeg

$ brew install ffmpeg

PyPDF2

$ conda install -c conda-forge pypdf2

A note about editing your .bash_profile

Adding printers in WRB 5th floor

Note: You will not be able to set up the printer when on the “CaseGuest” wireless network. The printer can be setup while on the “CaseWireless” wireless network, though it may be hard to access it from the lab-space. In that case, a direct link to the ethernet is probably the best way to go. When connected to the land-line, you may first encounter an error saying the identity of any websites you go to cannot be confirmed. This may be because you are on a new computer, in which case you have to first register your computer with CWRU IT.

The most convenient printer is the black-and-white one near my office. To add this printer on a mac: 1) Go to Systems Preferences (such as through the apple icon op) 2) Go to Printers and Scanners 3) Press the “+” sign to add a printer 4) Type in the IP address “129.22.253.166”. 5) The protocol should be “Line Printer Daemon – LPD” 5) You can rename it to something like “Nearby Black and White” to make it easier to remember. 6) It’s fine to leave Use as “Generic PostScript Printer” 7) Click on Duplex Printing (Since it’s useful). Voila!

Make a movie of a pymol structure spinning for powerpoint

I’m sure there are many ways to do this, but this is the way I’ve been doing it most recently:

1) Set up your pymol session. That means importing your structure, turning the background to white, and enabling any other setting to make it the desired quality.

2) I use a custom python script to make a series of commands that make the structure turn slightly, ray-trace the structure (for a high quality image), and export the image to a PNG file. The script that I’ve linked to here makes pymol export 360 images, making the structure spin around completely.

Note: Depending on your settings, this process can take 10 minutes or 3 hours. An easy parameter to change would be the resolution (default setting in the script is 2000). Obviously the settings that are toggled during step 1 can drastically change how long it takes as well.

3) Use ffmpeg (at least, on a mac) to turn the 360 images into a video.

$ ffmpeg -framerate 20 -pattern_type glob -i '*.png' -c:v libx264 -preset slow  -profile:v high -level:v 4.0 -pix_fmt yuv420p -vf pad="width=ceil(iw/2)*2:height=ceil(ih/2)*2" -crf 22 -codec:a aac Output.mp4

I used homebrew to install ffmpeg last time I had to do it (brew install ffmpeg). I followed these instructions last time I had to install homebrew.

4) Drag into your powerpoint presentation, and voila!

VAMP-seq Tips

VAMP-seq is a valuable tool for identifying loss-of-function variants for a protein when you don’t have a specific assay for characterizing that protein’s activity. This is because almost all proteins can be “broken” by mutations that cause the protein to mis-fold, mis-traffick, or any other perturbations that may cause the protein to disappear from its normal cellular compartment. In the case of PTEN, we identified 1,138 variants that were lower than WT abundance. Notably, ~ 60 % of variants characterized as pathogenic in people were loss-of-abundance variants, confirming the importance of this property.

VAMP-seq uses a genetic fusion between your protein of interest and a fluorescent moeity (eg. EGFP) to assay its steady-state abundance. Fusions with unstable variants of your protein results in cells expressing unstable fusion proteins that don’t really fluoresce. In contrast, fusions with stable variants (such the WT protein) may fluoresce brightly. Unlike western blots which are ensemble measurements, this assay can be performed at a single-cell level. Correspondingly, this single-cell fluorescence readout makes it possible to test a large library of variants in parallel, in a multiplexed format.

Two VAMP-seq orientation discussed here

Still, when thinking about using VAMP-seq, you must first consider its limitations. While strong loss-of-abundance variants will be non-functional, variants of intermediate abundance may still be sufficiently functional in many contexts. Furthermore, while low abundance correlates with inactivity (and pathogenicity, in many clinical genetics contexts), WT-like abundance in no way indicates activity (or benignity when observed in people). For example, active site mutants often destroy protein function while having little to no effect on protein folding and abundance.

There are also proteins that are inherently incompatible with VAMP-seq. Secreted proteins won’t work because you lose the single-cell, genotype-phenotype link needed for the single-cell assay to work. Marginally stable or intrinsically disordered proteins likely won’t work due to a lack of destabilizing effect. Obligate heterodimers won’t work, though you may be able to get around it by overexpressing the protein partner, such as what I did with MLH1 for assessing PMS2 (See Fig 6). Proteins that cannot be tagged are problematic; this likely includes proteins that normally exist in crowded complexes, or that have key trafficking motifs on their termini. Proteins that are toxic to cells when overexpressed also poses problems, though one of my new landing pad platforms may help with that.

If your protein of interest passes those criteria, the rest is empirically confirming that there is enough signal over background to run the assay. A good literature search is a great place to start. You should look for 1) evidence that an N- or C-terminal tag works well (both good expression and normal protein activity), and 2) known destabilized variants that could serve as controls when performing the preliminary experiments. Ideally, there will be a clear difference in fluorescence distributions that are separable by thresholds used in FACS sorting, and that these differences are physiologically meaningful (as far as we know). Comparing /correlating the results of western blotting with the fluorescence distributions of EGFP-tagged protein is helpful (see below for PTEN). If the initial EGFP distributions between WT and the destabilized variants don’t seem super crisp, see what it looks like when you take the EGFP:mCherry ratio. As you can tell in the below figure, the EGFP:mCherry ratio is quite handy for increasing the precision of each distribution, as it divides out much of the heterogeneity in transcription / translation between cells.

Western and flow results

Regardless, I recommend to most people that they clone both N- and C- terminal fusions, and minimally look at the MFI values of the cells expressing each fusion, as low MFI will likely mean low dynamic range of the assay (from too little signal over background). Ideally, both WT and controls will be tested in both contexts. If the signal is relatively high but there’s concern that the large GFP fusion is causing problems, you could try 1-10/11 split EGFP. This only requires fusion with the ~ 15aa beta-strand 11 of EGFP (and separately co-expressing the larger fragment in the cells), though it’s not completely free of steric hindrance as it requires the spontaneous complex formation of the two subunits for fluorescence. This format also worked with PTEN (See SFig 1d), though I noticed a ~ 10-fold hit in overall fluorescence using the splitGFP format.

Fluorescence levels from different formats

Once it passes all those tests, then follow the steps in the Nature Genetics paper. Good luck!

Landing Pad Cell Line Generation

My favorite method of landing pad cell line generation is with lentiviral transduction. The original method was with site-specific knock-in using genomic disruption (Using Cas9 or TALENs), followed by homology-directed repair from a transfected circular plasmid template. It worked, but it was slow, with a fair number of false positives, including cells that had likely still expressed BFP transiently during single cell sorting, or even more annoyingly, cells where numerous landing pads had integrated per cell. I’ve since gotten some potentially helpful tips (eg. linear template supposedly degrades faster than circular plasmid), but I still don’t think this method is practical for routine transgenic knock-in. Moreover, I’m not convinced the AAVS1 locus is even that great for cell engineering.

This brings me to Lenti landing pad transduction, which I’ve developed a bunch of supported tools for. It’s easy (few steps), fast (cells can be single-cell sorted 2 – 7 days following transduction), broadly applicable (many different cell lines are easily transduced), with low false positives (low MOI transduction, due to Poisson behavior, ONLY gives single integrants). Pretty good arguments, no?

The general steps are as follows:

  1. Transfect HEK 293T cells with a mixture of lentiviral vector plasmids to produce lentivector particles. Do this in no dox media.
  2. Change media the next day, and then replace and collect supernatant over a ~ 72 hour period. Do this with no dox media. It’s fine to keep collecting into the same collection tube.
  3. After all of your collections are done, spin out any cells that may have been accidentally collected, and pass the supe through a 0.45 micron filter.
  4. Plate out your target cells, and mix with a series of volumes of the filtered supernatant. This can be in dox media.
  5. At least 2 days after switching into dox media, read out how many cells are fluorescent protein (often BFP) positive. Go with the well closest to but lower than ~5%. If the positive cells are incredibly few, treat cells with antibiotic (often blasticidin) to enrich for transductants.
  6. Using FACS, sort individual positive cells into wells of a 96 well plate. Use conditioned media if your cells may require it.
  7. Allow clonal lines to grow out. Transfect with a mixture of Bxb1 expression plasmid, attB-EGFP, and attB-mCherry to confirm no stable double positives exist. Cells that pass this test should be good to go for library work

Key viral non-coding elements are white rectangles. Coding regions are colored rounded rectangles. 2A sequences are red inverted triangles. Promoters are stemmed arrows. Terminators are thick black bars.

Now for the flavors of lenti-landing pad that I’ve developed. It’s very choose your own adventure, based on your needs. The original (LLP) is the most conservative choice to start with in non-HEK cell lines. LLP-Int-Blast can make your life easier with easier / higher recombination rates, but requires a bit more care in its use. If you want to study a potentially toxic gene, use LLP-rEF1alpha. For everyone else, use LLP-iCasp9-Blast. The selection with AP1903 is so quick and easy that it allows you to brute force recombinations quite easily. I used to chuckle whenever anyone used the phrase “game changer”, but I totally find it appropriate for this landing pad.

Gene knockouts by exon deletion with Cas9 transfection

Cas9 is a dream biotechnology, since you can use it to quite easily and specifically disrupt almost any DNA sequence you’re interested in modifying. Its been particularly helpful in work with cultured mammalian cells. In this document, I will describe how I use Cas9 to make gene knockouts in HEK 293T cells.

So although I’ve heard good things about transfecting guide-RNA loaded Cas9 ribonucleotide protein complexes into cells, I haven’t done this yet. Instead, I’ll describe a likely less optimal, but super cheap and easy method of co-transfecting Cas9 and guide-RNA expressing plasmids into cells to create knockouts. Specifically, I’ll describe my method of knocking out a gene by removing a big chunk of coding sequence, either ablating its start codon, or getting rid of an important exon to only create truncated or frameshifted non-functional proteins.

Equipment & Materials:

1) Tissue culture hood & incubator
2) Transfection reagent
3) Fluorescence Activated Cell sorter (FACS)
4) PCR machine, polymerase, Gibson master mix.
5) Agarose gel electrophoresis apparatus and some form of GelDoc.
6) Sanger sequencer (or sequencing service)

Step1: Make custom guide-RNAs for your gene of interest

As mentioned above (and shown in the figure), to create genetic knockouts, I like to delete out entire exons. The main reason is that it’s really easy to genotype with PCR. It’s also nice in that if the change is made, even if the repair is an in-frame indel, the deletion will be so large that the protein should still be loss-of-function. Lastly, exon deletion can often be achieved by targeting adjacent introns; this means that even if you stably express the Cas9 / gRNA combo, you can re-express a cDNA version and have it not be susceptible to disruption.

I first find guide RNAs that will target the DNA I want to disrupt. Addgene seems to have a pretty handy list of guide-RNA sequence design applications here: (https://www.addgene.org/crispr/reference/). By default, I still tend to go to this classic website: (http://crispr.mit.edu/). Have it come up with a pair of seemingly specific guides, per the strategy above. Note: When using a U6 Pol3 promoter, I’ve read that if your guide RNA doesn’t start with a G, you should plan to include an extra G in front of the guide to increase transcription.

Addgene lists a bunch of protocols from the original labs here: (https://www.addgene.org/crispr/reference/#protocols). Depending on how you like to do your molecular cloning, you may want to follow one of those. I’ll describe making a custom guide-RNA using Gibson Assembly, which is absolutely amazing.

Using a really basic U6-promoter guide-RNA expressing plasmid (like so: XXXX), design a pair of long primers that overlap in their 5′ ends with the guide-RNA sequence, but hybridize to sequences adjacent to the guide-RNA sequence but point in the opposite directions. The ~20nt guide-RNA sequence makes for a great Gibson overhang.

The steps here are:
1) Amplify plasmid, appending the guide-RNA sequence to the termini of the amplicon. I use Kapa Hifi, with 40ng template plasmid (30ul total), and ~7/8 cycles.
2) DPNI digest. I pipet 1ul of DPNI directly into the 30ul reaction tube, and incubate at 37*C for 2 hours (I suspect you don’t need all two hours).
3) “Purify” your intended plasmid. At first, I used to run my amplifications out on an agarose gel and gel extract (using a Qiagen gel extract kit). Nowdays, I just use a Zymo Clean and Concentrate kit (first setting aside some sample to run on a gel afterwards, for diagnostic purposes only). I elute in a small volume (6 ul for the Zymo kit).
4) I then make equal volume mixtures (eg. 1ul “insert”, 1ul “vector”), and mix in the corresponding amount of 2x Gibson Master Mix (So 2ul for the above example). I incubate this at 50*C for 0.5 to 1 hr. Standard protocol here.
5) I mix the sample (usually 4ul) into 50ul of chemically competent E.coli, and transform (eg. “Heatshock” transformation).
6) In the case of the Church lab plasmids, they’re Kan resistant, so don’t forget to recover before plating onto Kan plates.

Normally, I end up seeing tens to hundreds of colonies the next day. I then usually pick 2 colonies to grow up and miniprep. When I run Sanger, usually at least one colony contains plasmid DNA with the intended guide-RNA encoded. If neither colony has the intended plasmid, I normally pick two more and screen those. I don’t remember the last time this protocol failed (possibly hasn’t in my dozen attempts), but I think if it does it’s likely going to be from there being too much template, so I would then re-do the process really making sure DPNI is doing its job (and maybe gel extracting if I have to).

Step2: Transfecting the guide-RNA

I’ve only ever done this in HEK 293T cells, since that’s where I do most of my tissue culture work (since they’re so easy to handle). Next step is to transfect cells with the guide-RNA and Cas9 encoding plasmids. I tend to do this in 6-wells, using Fugene 6.

For this transfection, I either mix the guide-RNA plasmids (half plasmid for guide 1, the other half plasmid for guide 2) with PX458 (which encodes Cas9 2A-linked with EGFP), or a plasmid expressing untagged Cas9 along with a plasmid encoding a fluorescent protein. Since transfection usually results in many many copies (hundreds? thousands?) of plasmid getting in per cell, cells expressing the fluorescent protein (generally) should have been transfected with guide RNAs and Cas9 also (and thus are the cells most likely to have been edited).

Then, approximately two days after transfection, I sort for transfected cells using the fluorescent marker. I usually do this in a 96-well plate, sorting single cells into individual wells (to create clonal lines), while taking one of the corner wells and sorting 100+ cells into this one. This gives me an improved bulk population as backup in case the single clone growouts fail, and gives me a well that makes focusing onto the right focal plane a lot easier when using the light microscope (since hard to correctly focus on wells where it’s not clear if there are even any cells there).

If in a rush, I’ll check on the wells in approximately a week. By then, you can definitely see colonies of cells growing by brightfield microscope. I would then trypsinize and replate to keep them growing happily. If I’m not in a rush, I’ll sometimes let the cells grow for a couple weeks before checking; at this point, the media color starts becoming yellowish in wells containing growing cells, and clonal lines that grew out are large enough that if I transfer them to a new well, I’ll be able to do an expt with those cells in a few days.

Step3: PCR Genotyping

Once I have enough cells (say, at least half a 24-well plate well’s worth), I’ll extract genomic DNA using a Qiagen DNA easy kit. I’ll then use the primer combinations shown above in the figure. Notably, primer combo BC should only amplify if there is a wild-type copy present (DNA from unmanipulated cells should serve as a positive control). This should be a binary “amplifies or doesn’t” readout. The other primer combo also gives a slightly orthogonal perspective on what might be present at the intended locus within the genome. If unmodified, combo AC should give a large band (or no band, if these are really large introns). If modified, combo AC should give a much smaller product (I aim for a few hundred bases for easy visualization by agarose gel electrophoresis). To verify that this small band is what I think it is, I gel extract the band and sequence using Sanger. Conveniently, you can even use either primer A or primer B as the Sanger sequencing primer if you didn’t order a dedicated internal primer. In the handful of times I’ve done this, I think the predominant modified product that I’ve seen is literally a blunt-ended Non-Homologous End Joining of the two DNA fragments without any resection / removal of terminal nucleotides. Thus, if this is indeed the major modified product, I’m able to design my guides such that NHEJ ligation results in a frameshift.

Using the above PCR, I go through and screen the colonies to find one that looks to be a complete knockout (no amplification with primers BC, only small band with primers AC). Of course, actual protein knockout should be confirmed by western blot.

Voila! Knockout cells to now use in your experiments!

Some references:

I think this project / paper from super grad student Molly Gasperini in Jay Shendure’s lab is what originally made me think about using paired gRNA deletions to make knockouts.

I’ve tried to link to specific protocols in the above descriptions. For any details that are ambiguous, the methods described in my 2018 NAR paper should largely describe the basic steps / reagents used.

PS: Ed Anderson, a Postdoc at UNC Chapel Hill, made the excellent point that the molecular cloning in this post (ie. Gibson assembly steps) could be performed with In-Vitro Assembly (IVA). We certainly had some success with this method in my postdoc lab (albeit we used it for parallelized NNK library generation), though could certainly be used here for simpler and most cost-effective routine molecular biology.