Command line BLAST

One of the pseudo-projects in the lab requires looking for a particular peptide motif in genomic data. While small scale searches can be done using the web interface, the idea is to do this in a pretty comprehensive / high throughput manner, so shifting to the command line makes sense for this work. I last did this back in 2018 for some preliminary studies, so I’m going to have to re-install the software on my new computer and re-run some of those analyses. I figure I’ll write down my notes as I re-do this, so that I (and others) can use this post as a reference.

Installing BLAST+

The instructions on how to download the program can be found here. I’m on a mac, so I downloaded “ncbi-blast-2.13.0+.dmg” and double clicked and ran the package installer.

Assuming it’s been correctly installed, writing the command …

blastp -task blastp-short -query <(echo -e ">Name\nAAWLIEKGVASAEE") -db nr -remote -outfmt 1

… into the terminal should actually reveal some BLAST-specific output, rather than throw an error.

Running protein motif-specific blast searches

Type in the following into your terminal:

psiblast -phi_pattern PHI-Blast_2A_pattern.txt -db nr -remote -query <(echo -e ">Name\nGATNFSLLKQAGDVEENPGP") -max_hsps 1 -max_target_seqs 10000 -out phi_blast_output.csv -outfmt 10

Note: The above command will require having a text file specifying the pattern constraint (“PHI-Blast_2A_pattern.txt” above), which can be found here. This should yield a 25 KB file csv output, like so.

Extracting just the accession numbers

I don’t remember if there are other BLAST+ outputs that give you the full hit sequence. If so, the method I ended up taking back in 2018 would seem to be unnecessarily roundabout. But, until I figure that out, I’ll follow the old method. As you can see in the aforementioned output format, it doesn’t output the hit protein sequence, and instead just gives the accession number. Thus, the next step is using the accession number to actually figure out the protein sequence. To do this, we’ll use Entrez Direct. To install Entrez Direct, follow the instructions here. Briefly, type in the following into the terminal:

sh -c "$(curl -fsSL ftp://ftp.ncbi.nlm.nih.gov/entrez/entrezdirect/install-edirect.sh

In order to complete the configuration process, execute the following:

echo "source ~/.bash_profile" >> $HOME/.bashrc
echo "export PATH=\${PATH}:/Users/kmatreyek/edirect" >> $HOME/.bash_profile

OK, now that it’s installed, here’s how I’ve used it:

First, the output file above has more info than the accession number. To have it pare down to only the accession number, I used this script, which can be run by entering the following into the terminal, assuming you have the previous output csv file somewhere in the directory with the script (can even be in other folders within that directory):

python3 3_Blast_to_accession.py

This will create a file called “3A_prot_accession_list_complete.txt” (example output file here) which will be the unique-ified list of accession numbers to give to Entrez Direct. (Uniquifying is important if you have multiple .csv outputs you wanted to compile into a single master list).

This can be fed into Entrez Direct using this shell script, which you can run by typing in:

sh 4_Accession_to_fasta.sh

You should now have an output file called “4A_prot_fasta.txt” with the resulting protein sequences in fasta format, like so.

Now you can search for your desired sequence (in its full protein context) within the resulting file.

To be continued…

Are there other steps in this process related to this project? Sure. Like what do you do with all of these full sequences containing the hits? Well, that’s beyond the scope of this post.

ODs on the spec and nanodrop

So there are two ways to measure bacterial culture ODs in the lab. The first is to use the nearby ~ $10,000 Thermofisher Nanodrop One (no cuvette option). The second option is to use a relatively cheaply made cuvette-based spectrophotometer I bought off of Amazon for ~ $100. To make it clear, this comparison is not a statement about the value of a Nanodrop (though I will say that having an instrument like a Nanodrop is essentially a must in a mol biol lab). This is more about if the Nanodrop is already being used by someone and waiting would get in the way of some bacterial speccing timepoints, can I purchase a $100 piece of equipment to relieve such a conflict? Especially for bacterial cultures, where volume isn’t really an issue and the measurement is simply the reading at 600 nm, not even requiring some algebra to make a conversion to more practical units (like ng/uL for DNA).

So to do this comparison, over a number of independent instances, I took the same bacterial culture and put 1mL into a cuvette and ran it on the old spec, and took 2 uL and put it on the Nanodrop pedestal and measured there. I made a table of the results, and graphed it in the plot below.

So the readings on the two instruments certainly correlate (that’s good), although it’s not an exact 1:1 relationship. In fact, the nanodrop gave numbers roughly 1.5 times higher than the spec. But if the two instruments give two different readings, then the question becomes “which is right?”

And to that, I essentially say there is no right answer. Each is a proxy for bacterial cell density (ie. Billions of bacteria / mL), but there’s no “absolute” information encoded in the OD number that tells us that specifically for our bacteria, and we’d still have to come up with a conversion factor either way (ie. my doing limiting dilutions of specc’d cultures and counting colonies), and once we have that, both will be right with that context. Sure, it would be nice if we had a method that was the most in-line with whatever ODs that were being described by various papers in the literature, but who knows what they used (recent papers may be using ODs from the nanodrop [with some perhaps using the cuvette option but many others not], while the older publications certainly didn’t have and instead likely used some old-school form of spec). But even that’s going to be heterogeneous, and will only give limited information anyway.

Well, good record-keeping to the rescue. We’ve transformed the positive control plasmid enough times to sample a range of various ODs just by chance, to see if certain bacterial ODs correlate with transformation efficiency. And boy, there’s been a whole lot of nothing there so far (which is actually quite notable; see below).

(FYI: I don’t remember which instrument I used to measure the OD A600 readings. Probably mostly the old spec, tho).

So yea, I’ve generally used cultures with ODs at the time of collection between 0.1 and 0.45, and they’ve collectively given me transformation rates of ~ 20,000 using our standard “positive control” plasmid. So there seems to be a pretty wide window of workable ODs. But generally speaking, I see no issue with having a culture of 0.1 to 0.4 OD as measured with either machine for use with chemical transformation.

Setting up a hybrid lab meeting

Both due to child-care and pandemic reasons, our originally 100% in-person lab meeting for a time was 100% remote and for the last few months have been 100% hybrid. For overall accessibility reasons, I’ll likely have hybrid remain the default option, and only not bother to set up the Zoom when it’s clear absolutely everybody is going to be in attendance in-person. Over time, I think I’ve better learned how I should be setting up the hybrid lab meeting, and I figure I’d write down the steps here so I can remember (and anybody else can do so if they’re setting things up).

Standard flexible format (ie. Nobody needs presenter mode)
Here, the laptop plugged into the projector is providing the sights and sounds of the conference room, but is simply serving as a “viewer” of the slides in Zoom. Here, I’ll assume this is being done with the common lab laptop (Kenny’s old laptop from 2019), although anyone’s laptop should work.

  1. Plug in the 360 degree camera into the common lab laptop. If you also want to use a different external microphone (ie. if you don’t want to use the microphone associated with the 360 degree camera), then plug that in now too.
  2. Log into the lab meeting on Zoom. Confirm that the right camera and microphone are selected. Make sure the sound is up to the maximum, and that this computer remains unmuted.
  3. Using the USB-C adapter, hook up the laptop to either the projector or the wheeled TV. The adapter will allow for connecting to the projector with the existing VGA cord, or the TV with an HDMI connection.
  4. Make sure the Zoom screen is showing on the projector / TV screen.
  5. To actually use this setup, the idea will be that 1) anybody with their own computer can log into the lab meeting Zoom and share their desktop or window with the presentation (powerpoint file or google slides, for example), or 2) if it’s someone without their own computer, they can use Kenny’s or Anna’s computer to screen share (assuming the presentation file is somewhere easily accessible, like the “Lab_meetings” directory of the lab Google Drive).
    Note: While these computers are being used to share the slides, all sound (input and output) should be happening from the common lab computer connected to the projection device.

One speaker / Longer-form talk where someone needs presenter mode
The main difference here is that the laptop presenting the slides has to be plugged into the projector / monitor, and is thus not simply a “viewer” in the Zoom call. Here, I’ll assume this is being done with the common lab laptop, a

  1. Plug in the 360 degree camera into the common lab laptop. If you also want to use a different external microphone (ie. if you don’t want to use the microphone associated with the 360 degree camera), then plug that in now too.
  2. Log into the lab meeting on Zoom. Confirm that the right camera and microphone are selected. Make sure the sound is up to the maximum, and that this computer remains unmuted.
  3. Open the presentation file. Windows may get much harder to navigate once connected to the second screen, so you may as well get everything set up beforehand.
  4. Using the USB-C adapter, hook up the laptop to either the projector or the wheeled TV. The adapter will allow for connecting to the projector with the existing VGA cord, or the TV with an HDMI connection.
  5. Now, go to Zoom and hit “Share screen”. Probably makes sense to choose the screen with the presentation on it, though it doesn’t really matter at this point since you can adjust it later.
  6. Once the screen is sharing, go to the slide / presentation software you’re using. Assuming it’s Powerpoint, then hit “presenter view”.
  7. If the wrong screen is showing on the projector / TV monitor, then hit swap displays in the Zoom panel until it does.
  8. Now, if the wrong screen is being shared on Zoom (ie. people in the Zoom call are saying they’re seeing your presenter view), then hit the “Share screen” button again and choose the correct screen to cast.

That should do it!

COVID cases at CWRU

I’ve been keeping track of what the COVID situation has been like at Case since they first started posting the data every week, back in the fall of 2020 (https://case.edu/covid19/health-safety/testing/covid-19-testing-vaccination-and-case-data). Whenever the cases seem to be higher than usual, I’ve been messaging the below graph out to my group, so they can be informed and make the best risk assessments about their activities on campus.

Anyway, figured other people may be interested in this information too, and I’m getting kind of tired of sending the same exact message out like the last four weeks, so I figured I’d just post the plot here so people can see the current stats.

As of writing this (first week of May), cases have been the highest they’ve ever been, although at least almost everyone should be vaccinated and perhaps even boosted. Still, would certainly be nice to see that number come down some…

Where lab funds go

As you can tell from the above graph, the people in the lab (including me) are by far its most costly resource, accounting for the majority of all lab expenditures. Thus, while there are other important reasons, there’s always this very “bottom line” reason for me wanting to minimize how much personnel time and effort is wasted by confusion and mismanaging!

Some Expected Yields

Here is some real-world data describing expected yields we may expect from some of these routine lab procedures or services.

Obviously the above plot is about how much total plasmid DNA we get from the miniprep kit we use in the lab.
The plot above show the expected total yields of DNA based on the extraction type / method
And this is the pretty wide range of reads we’ve gotten from submitting plasmids to plasmidsaurus
The above graph shows how many (raw) reads we’ve gotten from Azenta / Genewiz Amplicon-EZ.

Oh, and this is a good one:

How well my determination of flask “confluency” actually correlated with cell counts. I mean, sure, there must be some error being imparted by the actual measurement of the cells when counting, but I think we all know it’s mostly that my estimate really isn’t precisely informative.

Designing Amplicon-EZ primers

Amplicon-EZ is a pretty convenient service from Genewiz. In short, they’ll perform Illumina sequencing on a 150-500 nt DNA fragment you send them (they’l perform 2 x 250 cycles of sequencing, so fragments smaller than 500nt will have paired read regions). For $50 per sample, they’ll return ~50,000 reads (although in our experience, they tend to return more than this). Turnaround times can be kind of slow (while one can minimize the delay if you time things perfectly, it’s taken between 14 and 19 days to get data back following submission). That said, we’re still only running full kits a couple of times a year, so obviously a lot faster turnaround than that. Thus, definitely good for getting an initial look into something you may want to sequence more deeply later. My general policy for that lab is that if you make any library, it’s worth submitting the library to Illumina sequencing via Amp-EZ pretty early on so you can be confident that the library is good and worthy of further experiments.

Designing primers

Primers are pretty simple to design. Essentially, you’ll want to make a pair of PCR primers with Amp-EZ adapters on the 5′ ends (and of course, DNA hybridizing sequences on the 3′ ends). As shown in the above link, the adapter sequences are:

For the forward sequencing read: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’

For the reverse sequencing read: 5’-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’. <- Reverse strand on plasmid map

Here’s an example of a map and corresponding annotated primers used to sequence the ACE2 Kozak library plasmid, and a similar map for sequencing the library after its been integrated into landing pad cells.

Amplifying your fragment

For the actual protocols to do this, it’s probably worth asking Sarah or Nidhi how they do it. The basic steps are going to be PCR, gel extraction of the band, and Qubit quantitation of the extracted DNA. Some things to keep in mind are that they’ll want a fair amount of DNA (500 ng), so you’ll either want to make sure you do a lot of cycles and extract a pretty hefty band, or you’ll need to do a second amplification from the initially extracted DNA.

Cell surface localization assay

About the same time I got inspired to try making the vesicular protease assay, I figured I’d also build an assay to try to look at cell-surface localization of proteins.

Going back to that post about dL5 and malachite green (MG), I believe that MG doesn’t cross the the plasma membrane particularly well (Although now that I look at it, there are both ways to increase and decrease it’s cell permeability). Thus, I figured I’d test the dL5 fluorogen activating peptide in two forms; one where it’s sent out to the cell surface using a signal peptide (but anchored to the plasma membrane with a transmembrane domain), and another where its expressed as an intracellular, cytoplasmic protein.

Well, we made the constructs, and Sarah recombined the cells and tested them, and here are the results.

Well, ignore the blue distribution for now (since this is a construct testing a different hypothesis), and only look at the red and orange distributions for now. As you can tell, in the absence of MG, the signal is pretty low (I should probably through in some unrecombined landing pad cells on that plot to show what the background level is). On the other hand, MG addition causes cells encoding the extracellular dL5 to exhibit ~ 3e4 near infrared MFI, while the intracellular dL5 cells had about 1e4 MFI. So while that’s only a 3-fold difference, the standard deviations of those distributions were pretty tight, such that there was rather small overlap between those two distributions. So while this is a single, one-off experiment, it looks like this assay format may work.

Probably some additional knobs that can be turned to try to improve signal over background. First, is maybe this effect is somewhat MG concentration dependent, and reducing the amount of MG that is added may add some more dynamic range. Also, there are those less cell permeant MG derivations, which will likely improve the range (albeit, these are likely far harder to get than OG MG)

Vesicular protease cleavage assay

Obviously we make a lot of recombinant DNA constructs and create a bunch of different assays to try to understand biology. Well, at some point I became curious if I could come up with a protease cleavage assay for measuring how various peptide sequences could be substrates for vesicular proteases inside cells. I figured we’d start to test this by looking at furin cleavage, since 1) furin is quite ubiquitous, 2) furin cleavage is relevant for multiple disease-relevant membrane proteins, like many viral entry glycoproteins, and 3) including SARS-CoV-2 spike, where the furin cleavage site in that protein is thought to contribute to its dynamics of spread and pathogenicity during infection.

Well, the first construct that may have worked is a pretty simple one, where I have EGFP targeted to extracellular space using an N-terminal signal peptide, but retained on the cell surface by having it C-terminally fused to a transmembrane domain. Anh has actually been using this construct as part of his undergraduate research project. Well, to turn this construct into one that can study Furin cleavage, I modified it to encode a R-R-A-R peptide between EGFP and the transmembrane domain. Thus, if Furin cleaves this construct, the EGFP protein is now no longer tethered to the cell, and presumably escapes into the media once it reaches the cell surface.

So this is an N=1 experiment so far, so it’s not the most conclusive. That said, there does seem to be decent separation between the construct with and without the furin cleavage site, where cells with the construct with the EGFP that potentially gets released had ~ 10-fold less fluorescence than the construct that could get released. I suppose if this reproduces, it could be worth trying to turn this into a library-based experiment for studying protease cleavage within intracellular compartments.

… Though now that I’m thinking about it further, I’ll definitely need to ask how these cells were prepped. If they were prepped with trypsin (which is likely), this was likely more of a trypsin cutting assay rather than a furin one.

Edit 10/27/22:
Well, so to avoid the whole trypsin complicated, recombined the above constructs in suspension HEK cells that are floating and don’t ever need to be detached off the plate for flow. Here’s what that plot looks like.

So ya, we’re still seeing the ~ 10-fold effect there, so it looks like that’s the real dynamic range of the assay for measuring furin protease cleavage.