Blog Series: WoG, Cesky Krumlov; Day 9: A Tale of RAD-taggin Sea Drag-ins

Dr. Bill Cresko
University of Oregon
Sensei of the Unix Ninja

Topic: Genomic analysis of non-model organisms, RAD-tags and STACKS

Greetings!

So today we launch into how to analyze critters that do not contain a reference genome or have relatively few annotations associated with their genome, the proverbial black box we bang our heads against.

What is a non-model versus model organism anyway?

Model organisms have 'an entire community trying to dissect one species usually in an effort to understand humans better.' The mouse is a model organism, E. coli K12 is a model organism.

Non-model organisms are studied by relatively few, don't contain good references usually and have no correlation to research going on in humans; meaning they don't necessarily further our knowledge of processes in humans.

However, despite not having the breadth of knowledge that model organisms have--the literal arsenal of annotations--we still have the same questions about non-model organisms as we do with model organisms!

1. How do major differences among lineages evolve
2. What is the relatedness between organisms?
3. What is adaptation like in these organisms?

There are fundamental processes in evolution:

1. Origin of genetic variation: via mutation and migration
2. Sorting of that variation: variation can be affected by genetic drift and natural selection for instance. For those drawing a blank on 'genetic drift'...it is the change in allele frequency over generations...for those of you drawing a blank on 'allele'...an allele is essentially a gene or better yet a specific 'version' of the gene. So one gene can have more than one 'flavor'. An allele is a particular flavor of that gene as determined by it's sequence data.  For those of you drawing a blank on gene...click over to the nyan cat you tube video--be entertained and don't come back to this blog until you know what a gene is.
3. Simultaneous genotyping of neutral and adaptive loci (for population genomics): neutral loci provide a genome wide background that gives you estimates of effective population size and can be used for phylogeography. Adaptive loci are outliers from the neutral background and can lend insight into selective sweeps or local adaptation.

So the naive solution when approaching a project with a non-model organisms is to just 'sequence everything'. Why? Because sequencing is still quite expensive and for many studies is pretty much a waste.

Genomes are generally organized as linkage blocks so essentially as long as you have well spaced markers that will work just as well for genotype for a fraction of the cost. Having genetic maps are very useful in genome studies and often times a great first step to guide you in whether full genome sequencing really needs to be done to answer the scientific question you have.

So the Cresko lab is heavily involved in an alternative approach that exploits these linkage blocks in the genome. It bypasses the need for a full genome and still provides tons of data about the genome in addition to building a genetic map and doing genotyping that can guide further studies/hypotheses.

The technique is called RAD-tags, the program/pipeline is called STACKS and the 'grand architect' our resident Unix Ninja--Julian Catchen.

Blog Series: WoG, Cesky Krumlov: "One *ome to rule them all...?" and other anecdotes from this evening

Apologies to the Lord of the Ring die hards who are probably outside my door with torches and pitch forks...but truly this evenings discussion really brought some things to light and well...tossed other things into the dark...

Ah Science...

Blog Series: WoG: Cesky Krumlov; Day 4: Assembly

Dr. Rayan Chikhi

Pennsylvania State University

Topic: De novo Assembly

A whole day of assembly!

There is no single program right now that is considered 'the assembler'. Different assemblers have advantages and disadvantages as well as things they are generally useful and not useful for. So one thing in todays assemblers is that they all take a lot of time and memory to run--especially when doing de novo assembly. One of the exceptions is the program Minia, developed by Dr. Chikhi which was designed to run efficiently using low memory requirement.

One of the important things that you need to know for assembly is what a k-mer is. A k-mer is any sequences with length k.

AGC is a k-mer with k=3

AGCT is a k-mer with k=4

AGCTT is a k-mer with k=5

You hopefully get the idea. 

There are two essential methods that assemblers use to assemble: de Bruijn graphs and overlap/string graphs. Now we sort of covered this in the Assembly prep blog...lets see if I can explain this better here now...

Blog Series: WoG, Cesky Krumlov; Day 2: So you want a NGS Sequencer eh?

Dr. Konrad Paszkiewicz
University of Exeter
Director of Wellcome Trust Biomedical Bioinformatics Hub

Topic: DNA Sequencing Technology: Past, present, future

Good morning bioinformatic campers! Well, afternoon for me, but morning for many of you back in the U.S.

So much of what was covered this morning is going to be redundant with the DNA sequencing preparation blog I wrote previously. So between this entry and that other one you will hopefully get a complete view of the 'state of the union' where sequencing is concerned and prospects for the future. The nice thing is that Konrad tossed in a lot of pro/con lists for different platforms, so those of you considering NGS in the future, this is a bare bones, get you started guide as to what's out there and whether 'it's worth it' for your own research to invest in a platform and which one to invest in. Again, I still highly recommend Dr. Elaine Mardis' talk that I linked in my DNA sequencing technology prep blog.

First and foremost, if you are familiar with what molecular biology is and what sequencing is and don't know who Fred Sanger is...then you've probably been living in a hole...

Blog Series: Workshop on Genomics, Cesky Krumlov; Preparation--Genome Structure

Section 2: Genomic Structure

The prep section for genomic structure was small with two articles suggested, one of which you'll need a subscription to read; the other is freely available! Huzzah!

• Alkan, C; BP Coe and EE Eichler. 2011. Genome structural variation discovery and genotyping. Nature Reviews Genetics. 12:363-376. (subscription needed)
• Mills, RE et al., 2011. Mapping copy number variation by population-scale genome sequencing. Nature. 470:59-65. (FREE!)

So lets jump into Mills et al. and learn something...the one caveat to this article is they automatically assume you know what a 'structural variant' is and they are specifically talking about this with respect to the human genome. So lets back-track a little--skim if you're already a structural 'pro'...or better yet, add your two cents in the comments along with other links to clarify this topic.

Blog Series: Workshop on Genomics, Cesky Krumlov; Preparation--Modern Genome Sequencing Technology

Given the workshop will be moving quite quickly the organizer(s) suggested for those of us with less background to do some reading up in certain topics which they outline and include links to on the website in a section called Preparation.

This page, with respect to the workshop on genomics, is broken down into sections. Total there are a suggested 23 readings or websites, unfortunately many (11 of 23) of the links, you have to have a subscription to view the articles beyond the abstract; which unfortunately I do not have and my institution has limited subscriptions; besides most of my preparation is being done at home where I would't have access to the institutional subscriptions anyway. I'll be going section by section with the papers linked and offer some additional suggestions for those unable to access articles due to lack of subscription.

Time for a Tutorial...or how about 10

Greetings, Salutations and Happy New Years Eve!

So I have decided to run a 'live-blog' starting January 7, 2013 with the expressed intent of conveying whatever I learn to you--the blog reader.

For those unfamiliar with who I am, you can read my byline: aspiring infectious disease ecologist. I am currently a bioinformatician at Walter Reed Army Institute of Research (WRAIR) in Silver Spring, MD. My educational background is quite varied and if that interests you click over to my LinkedIn Profile which also has a list of my recent scientific publications.

Crib Sheet Bio: Grew up everywhere...ended up in Hawaii and graduated from Maui High School in 1997. I did my undergraduate degree at Pacific Lutheran University in Tacoma, WA--Major PreMed/Biology and Spanish Language and Literature, graduated in 2001. Worked for a plastic surgeon 2 years back in Hawaii then promptly opted out of medical school and in 2003 entered into a Ph.D. program in Ecology and Environmental Sciences at Montana State University in Bozeman, MT (microbiology focus; Advisor Dr. David M. Ward, Land Resources and Environmental Science Dept). I graduated in 2010 and immediately started a post-doctoral fellowship courtesy of the National Research Council in Bangkok, Thailand at the Armed Forces Research Institute of Medical Sciences (AFRIMS; Advisor: Dr/MAJ Richard Jarman, Virology Dept). In May 2012, I was offered a position at WRAIR helming the massive amounts of sequence data being generated through the introduction of their 454 Roche FLX and Ion Torrent sequencing platforms. We may also be obtaining a MiSeq at some point as well. So I am currently running the gauntlet of various sequencing technologies and implementing bioinformatic pipelines developed in-house as well as utilizing the best of what academia has been able to offer in terms of open source software. I am also coincidentally (or not so coincidentally perhaps) a 2013-2014 ASM/BWF Science Teaching Fellow, so as you might surmise--I enjoy research and teaching and aspire to an academic posting in the future.

My current research muse is dengue virus. Specifically, dengue virus quasispecies characterization and dynamics over time and space. I have also worked on sequence data from thermophilic Synechococcus spp. bacteria, influenza A (pH1N1, sH1N1, H3N2), influenza B (Yamagata and Victoria lineages) and Hepatitis E virus. 

WRAIR as well as other military institutions have certain restrictions on what software can be installed and there is a lengthy approval process to ensure information assurance policies are followed. For that reason, those of you following this blog may not be able to immediately use the software, pipelines or even access certain internet links. Fear not, I feel you pain. I will make every attempt to make sure links I provide will allow you to access the information no matter where you are at. This means I will provide links to the sites where you can find downloads for academic open source software, I will not be providing actual downloads through this blog (see disclaimer).

DISCLAIMER: Please do not attempt to install any software discussed on this blog without prior authorization and approvals if you work for an institution with enhanced security policies in place. 

This disclaimer will appear at the top of any blog post that contains links leading you to software download sites to remind you to be aware; annoying but necessary. If you work at WRAIR and need assistance with the approval process, please contact me directly. If you work elsewhere, please check the regulations of your institution for guidance on policies regarding software downloads, installation and implementation.

I am not a professional science blogger so apologies if the the prose is less than polished. As you also probably noticed, I opted for the fantastically bland and ever-so boring 'simple' display for this blog, but that was by design. I plan on conveying as much material (text, links, diagrams etc.) as possible and want that to be the highlight of this blog as opposed to fancy displays which can get distracting for me. I have always been the girl who gravitates to shiny objects--great for picking up pennies, not so great when I am attempting to focus.

The field of bioinformatics can be quite daunting for those with little or no background in computational biology. Bioinformatics is also a catch-all for all the specialized fields within the discipline. For instance, there are researchers who only focus on whole genome sequencing and comparision studies, others deal primarily in transcriptomics/proteomics, still others are metagenomic analysts. Additionally, those in the field of epidemiology have their own bioinformatic analysis packages; not to mention all the bioinformatic programmers who create the packages and pipelines used today. Personally, I have experience with whole genomes, single and multiple gene analyses, microbial/viral evolution analysis and some metagenomics. I have dealt with sequence data derived from sanger and next generation sequencing platforms. I don't program beyond shell and bash scripts, though I have some working knowledge of python and R (stats package) but I only program when I have to and my knowledge of transcriptomics is minimal; hence the attractiveness of the workshop I will be blogging about. The field itself moves at lightening pace and it is not unusual to feel as though you are in a constant state of 'catch up'. Just when you acquire useful knowledge, it suddenly becomes 'outdated'. This can prove to be an immense frustration.

The goal of this blog is to provide, hopefully, practical tutorials and resources on things I learn in real-time and to serve as an archive...it helps to have decipherable e-notes readily available. I also desire to make what I do more 'accessible' to other investigators who would like to manhandle their own data (or have a grad student or technician learn how to do it) instead of waiting several months for a potentially incredibly busy bioinformatic collaborator to get back to them. I am going to sheepishly raise my own hand on this as I have reports and data to get back to collaborators myself in addition to my own research and routine obligations at WRAIR. Granted I only started 7 mos ago, so I'll catch up--BUT it is easy to get bogged down, so let's dispense some knowledge and hopefully lighten everyone's load! The only way I personally learn things is to keep copious notes and re-write/view them in different ways--we'll see how this works out.

Blog Series #1: Workshop on Genomics, Cesky Krumlov, Czech Republic, 7-18 Jan 2013.
First Blog: 7 January 2013
Last Blog: 18 January 2013
Assumed Audience: Investigators, students, technicians who know what sequencing is, what a nucleotide is, what an amino acid is and if I showed them 'ATGC' and 'AUGC' they know what both are. Background in software packages, evolutionary or genomic analysis and programming would be helpful but will not be assumed. So for some this may be a little more of a hand-hold than you might like--feel free to skim. 

I have obtained approval to 'live blog' this workshop from Dr. Scott Handley, many thanks!

I will post a preparation blog prior to officially starting on 7 Jan 2013. Blog posts will arrive in the evening as I cannot blog what I have not learned!

Comments and questions are welcome, what I cannot answer I will ask someone at the workshop--please identify yourself in your comment if you don't use a profile, I'm not a fan of anonymous posting and will not bother to answer/reply to anonymous posts.

Following the workshop, I cannot guarantee how often posts will appear as I stated earlier; I'm not a science blogger really, but feel free to subscribe and then you'll get a notice when postings do happen. You will probably only see postings on this blog site when I feel I have something functionally useful to convey or I am attending a workshop where I have organizer permission to live-blog or a conference.

Ok! For those of you without 2 weeks to spare to flit off to the Czech Republic to learn the latest and greatest in genomic analysis, care to join the journey and have a somewhat 'virtual-workshop' experience?

Excellent, I wish us both luck then.

Kind Regards,
Mel
---
Melanie Melendrez, Ph.D.
Contractor-CNTS
Chief, Bioinformatics, Lead Scientist
2013-2014 ASM/BWF Science Teaching Fellow

Walter Reed Army Institute of Research

Viral Diseases Branch

Silver Spring, MD 20910

melanie.c.melendrez.ctr@us.army.mil

mmelendrez@gmail.com