Category Archives: Tiny Tutorials

Cas9 – The Good, The Bad, and The Ugly of a Viral Hunter

The CRISPR/Cas9 system for viral defense in bacteria is a recently discovered system that is really fascinating.  Cas9 is useful for genome engineering and currently getting a lot of attention in this area.

Greene Lab Studios (a subsidiary of the Greene Lab) put together this video to the title music from the soundtrack of the film  The Good, The Bad, and The Ugly composed by Ennio Morricone.  The video briefly explains how the CRISPR/Cas9 system works in recognizing and cleaving foreign nucleotides in a cell.

My Philosophy On Attending Scientific Meetings

I enjoy going to academic meetings and very frequently post information about upcoming meetings and symposia.  Some people have recently asked me why I put so much emphasis on meeting information on the blog and others have asked how I decide which meetings to attend, including this recent comment.  I go to meetings for many reasons – some of which I will cover here – but, specifically for me, the main reason I go to meetings is to interact with other scientists in my discipline.

I believe going to scientific meetings is a vital part of mastering how to communicate your research.  The types of communication you have the opportunity to master can range from giving an oral presentation to hundreds (or perhaps thousands!) of other scientists to speaking with someone for 30 seconds in an elevator.  Academic meetings give you an opportunity to master your communication skills.

MSA 2011 presentations

Meetings are also great places to learn about the cutting edge of your research area.  Often scientists will present their most recent findings prior to publication.  You might be able to learn about new technologies or novel ways to analyze data before you read about them in publications.  Often during the question and answer portion of talks, theories and controversial topics are discussed, with a debate in real time.  I tend to return from meetings mentally energized with lots of new ideas to investigate and think about.

Meetings are great places to network and meet people in your research area.  It’s easier to strike up a conversation or collaboration via email if you’ve met someone in person at a meeting.  Also, if you are in academia, the people you speak with at meetings will likely be the ones who are reading your grant proposals and reviewing your manuscripts.  Future job contacts and impromptu pre-interviews happen at meetings.  Most importantly, there’s the added bonus of developing friendships with your fellow scientists.

Lastly, I enjoy traveling, so going to academic meetings is a way for me to mix business with pleasure, so to speak.  I’ve gotten to see some truly beautiful places under the auspices of attending scientific meetings.

rhodes_at_dawn

If you are a student or a post-doc – or have been asked to present a talk – there may often be registration discounts for meetings and symposia.  You may be able to get a discount if you volunteer to help out at the registration desk or other planned events.  You might also be asked to provide financial need.  You should check with the meeting organizers as soon after the meeting is announced for these types of discounts.

When it comes to choosing a meeting to attend, I personally prefer smaller more intimate meetings, with a range of participants in the hundreds, and not in the thousands like you tend to find at large meetings.  Large meetings can be valuable, but I personally find that it’s more difficult to attend all the talks you want to and to locate the people you want to speak with.  I will often speak to people who have attended specific meetings in the past and ask them about their experiences in order to gauge if a meeting will be a valuable one to attend.

Before going to a conference, I usually get an idea of which talks I would like to see and who I would like to speak with at the meeting.  This can happen before I leave for a meeting if the conference booklet is posted online, but it typically happens right after I arrive at the registration desk.  Some people prepare by bringing business cards, and although I haven’t used business cards, this could help people remember you after the meeting.  You also want to prepare your “talk” – whether it’s for a speaking presentation, a poster presentation, or just speaking in the hallways or at a dinner of the meeting – you should be able to communicate your research clearly in many different formats.

During the meeting, I have different strategies depending on the meeting size and the types of talks.  I try to pick the talks that are most relevant to my interests, but sometimes this leaves me running between sessions.  This can be a great time to “bump” into someone you want to speak with, but you may also miss important talks this way too.  Sometimes, particularly at the end of meetings when my mind is overwhelmed, I sit through entire sessions just to see if there is something interesting in a disparate research area that can be applied to my research.  I’ve gotten some great ideas listening to talks I thought would not be pertinent to my research.  I take notes and make sure to write down literature to look up and read when I get home from the meeting.

After the meeting is over it’s important to follow up with those you have started collaborations with and those whose research papers you want to read.  If you’ve taken notes, review them and think about posting them to a public forum, like a blog, so that others can share in on your meeting experience.

Adding Dropbox To Remote Machines At The Command Line

When I was recently at Titus Brown’s Next-Generation Sequencing and Data Analysis Workshop we were using remote computers at the command line for our workshop exercises.  In the workshop we used Dropbox to transfer files from one computer to another.  I had no idea that I could easily install and utilize Dropbox at the command line on remote machines.  I’ve reproduced the tutorial from the workshop here with some minor changes of my own.

Knowing how connect Dropbox to remote machines has saved me some time transferring files and it’s been extremely helpful on many different levels.  I can quickly pipe or send output text or images right to numerous shared devices.  I can check on the progression of a pipeline running on a remote server with my mobile phone by looking at output images or files (or even quickly checking file sizes).  Visualization at the command line is non-existant, so if I want to see an output figure, I can look at data output graphs quickly from Dropbox, and, if I choose to do so, can put images in a shared folder for a colleague to inspect in a matter of seconds.  Before you use Dropbox at the command line, you’ll have to set up a Dropbox account.

To link Dropbox at the command line on your home computer or, perhaps more importantly, on a remote machine, you should start at the location where you want to put your Dropbox folder, such as your home directory on your machine.

$ cd

Next, you’ll want to download Dropbox (here, for Linux-based machines):

$ wget -O dropbox.tar.gz "http://www.dropbox.com/download/?plat=lnx.x86_64"

…and extract the zipped file:

$ tar -xvzf dropbox.tar.gz

Next, you should run the dropboxd program:

$ ~/.dropbox-dist/dropboxd &

After running the program, you should see a message like this:

$ This client is not linked to any account... Please visit https://www.dropbox.com/cli_link?host_id=XXXXX to link this machine.

You will next want to copy and paste that URL into your Web browser.  While in your browser log into dropbox.  BAM!  The folder ~/Dropbox is now linked to your home directory!

By accessing Dropbox in your browser you can modify which computers will be linked to your Dropbox account by accessing the “My Computers” tab in Account Settings option.

I prefer to use Dropbox and most of my colleagues do too, but I’m sure you can do this with other file sharing platforms such as Google Drive or SugarSync.

What Are Fosmids?

I’m starting a new series of short tutorials.  In a selfish way, these posts are for me – a vehicle for me to clarify my own comprehension of a given topic.  I might also tell you about a solution to a problem I have been troubleshooting.

The first post in this series is about fosmids.  There seems to be some public confusion as to what they are or what they can be used for – basically, I have been confused.  Apparently I had forgotten my microbiology coursework along the way.  Many genome sequencing projects – such as the human genome project – have utilized fosmids to create libraries prior to sequencing, but it wasn’t until hearing about JGI’s fungal and metagenomic sequencing initiatives did I hear the term fosmid mentioned frequently.

Fosmids are used when preparing genomic libraries for genome sequencing.  Fosmids are circular DNA of bacterial origin – technically plasmids – but where typical plasmids exist in high copy number (up to 100 copies per cell) and possess small (3 to 6 kb) inserts, fosmids are present as a single copy in a cell and may possess inserts upwards of 40 kb.  Fosmids are advantageous because they produce stable libraries for genome sequencing.  They have a tendency to provide fairly uniform coverage, so they are optimal for closing gaps in whole genome alignments.  In addition to genome sequencing, they have also been used for metagenomics and expression studies.

Fosmids are derived from the fertility plasmid (or F-plasmid) and are responsible for the formation of the sex pilus during bacterial conjugation.  This plasmid contains both origin and partitioning genes derived from the F’-episome and as a result, the plasmid is kept as a single copy clone, which comes in handy during genomic DNA library construction.  Fosmid vectors are derived from random shearing – which yields more uniform coverage when comparing against other library cloning methods.

Cosmids may also be useful for genome sequencing projects, but unlike fosmids, they are multi-copy vectors that are generally present at anywhere from 20-70 copies per cell and this high copy number leads to instability and lost segments of genomic DNA.  This can be an issue for closing gaps in genome alignment, but if you’ve got high sequencing depth and a small genome to sequence, it may not be much of an issue.  Most importantly, with high copy number plasmids, such as cosmids, the chance of recombination increases which can disrupt and rearrange genomic DNA inserts prior to sequencing.

fosmid figure

Lastly, fosmids can be useful for chromosome specific sequencing and as cytological markers for chromosome identification.  The image above — which comes from this paper — shows the identification of chloroplast genome isolation and sequencing from fosmids; a similar technique can be used to isolate and sequence specific chromosomes.  Also, fosmids may be used as cytological markers with in situ hybridization on metaphase karyotypes and sorted using flow cytometric methods.