Electron Microscopy

Just some brief updates of my latest muse - electron microscopy and immunogold labelling (of which I started last Thursday). Perhaps the antibody I probed was abundantly available, so it's pretty awesome to see that the results was as expected for the wild type. As much as I was disappointed that none of the mutants I'm currently looking at has different phenotype when compared to wild type, the data is definitely useful to form future questions for my work. Exciting times ahead!

IgL of my protein of interest - yay!

TEM image of my Chlamybaby - pweeety!!

Here's the paper which I mentioned about the techniques used for IgL. Jeremy is an excellent adviser to me when it comes to imaging techniques. I do my own fixing and cell embedding, so it's always great to have Jeremy around in CAIC whenever I go over. Thanks a lot, Jeremy! :)


Hope you guys find this post helpful :) Cheers!

Immunogold Labelling

Today I've got some peace by chilling at the imaging centre to run some immunogold labelling (IgL). My samples had been fixed in epoxy resin (I'll show you guys some pics in action when I'm running it next week). For now, I'm using older ones, mounted on nickel grids. I'm using a modified protocol from Skepper (2000), J. Microscopy 199, 1-36. (I'll link it later)

I'm having research fun and I think research should be fun. Science is mind blowing and fun. Unfortunately some people have very distorted and disillusioned vision of what "fun" and professionalism in science means.

Right now I'm battling a birdman who wants to kick me out because he's totally disapproving my idea of fun in science. Calling me unprofessional, unproductive and unknowledgeable is low if not bitchy. Telling untruths and causing me this much of troubles at this point only caused me to find him disgusting, and crazy. Stalking me under the disguise of taking my attendance without me knowing is mental harassment.

Maybe some would think that I'm wasting my time battling against a silent but dangerous birdman but if I don't fight until the last drop of my blood runs dry, I'd do myself the biggest harm. As much as I'd like to live in peace to do a PhD, the evil birdman thinks he can show me the door easily. Making my research life difficult is only making the birdman's life worse. I'm a resilient wild grass. Have always been that way.

The birdman said he'd not help with carrying of gas cylinders for me from now on. I'm perfectly happy that he refuses to help me, though I'm totally pissed with his unprofessional and childish behaviour. Thanks to this behaviour I have more conviction that I should fight till the end.

St. Paul mentioned about him running his race till the end. I need to put up a good fight too. If not, I'll be disappointing myself in the end.

Quantitative Western Blotting

I've been recently introduced to infrared fluorescence Western blotting and the video embedded is helpful to understand the differences between IR fluorescence Western blotting and the conventional chemiluminescence method. I'd have to work more on this method before I can be conclusive on its potential and usage but so far, it seems amazing and more cost-, labour- and time-effective. I discovered more from the video of its other functions which I would like to explore further, if given the opportunity to do so.

Two of my samples from the LICOR Odyssey system for the infrared fluorescence Western blot I did this afternoon. 

Does anyone know why there's a red band below the green band when I only use one IRdye?

Introduction to Chlamydomonas reinhardtii

I need to write a 5-page proposal today, so I'm getting myself acclimated by doing some free writing here.

An explanation - I'm switching department and supervisor for the rest of my PhD. A difficult decision to make after spending (almost with futile effort) almost 10 months in BioAnth. It's not that I hate human evolutionary genetics, but it's not my cup of tea. So, upon discussion with various people, especially with my current supervisor and college graduate tutor, I embarked on the search for a new project, new supervisor, new department... Hard to bid goodbye, but it's better for everyone, especially for myself. I've nothing to lose in the first place, except some loss of time, but it's for the better.

If the official approval is granted, then I will be joining Professor Howard Griffiths' lab to work on Chlamydomonas reinhardtii together with an awesome team to eventually contribute to the CAPP (Combining Algal and Plant Photosynthesis) project.

An brief introduction of this species which we work on - the Chlamy:

Source: http://upload.wikimedia.org/wikipedia/commons/1/19/Chlamydomonas_TEM_04.jpg

If you could see the area which is densest in the TEM photo, that's our group's interest. The pyrenoid story. There are various aspects which we are looking into, but for now, it shall be a suspense, as it's time for me to run back to the proposal.

Yes, I'm a happier me after the 10 months of struggles, though I'd have tonnes of work to be completed in lesser time than others. Yet, I am still happy and glad. I am thankful to God for answering my prayers and to St. Jude for interceding for me. Miracles do happen, if we also work in tandem with Him.

Ubuntu on VirtualBox

I began to realise the need to eventually get a computer with Linux/Mac OS since many of the softwares developed for genomic analyses are Linux-based softwares. Though I might be an ardent supporter of Android phones and Windows 8 computers, for the sake of work, I probably am going to request for money to buy a computer with Linux or Mac environment.

Right now I'm in the midst of the troubling question if where I am currently is matching to my real interests. I felt ashamed, fearful and disappointed that I am where I am right now, and it didn't help when everything I proposed didn't work and I don't seem to be very enthusiastic about this. I know it is a sign I am not suitable to be in this area of research, as much as I wish to feel belonged here.

Back to what I've learned for the past nine months... More aware of computer language for sure. I might not speak any particular language yet. I am at beginner's level in Spanish and Japanese if I were to compare to "speaking" Python 3 and R.

Right now I'm installing Ubuntu on Virtual Box with the help of wikiHow's instruction. Very precise and convenient indeed. Now I can try to install some of the softwares which I desire to use. Let's see if it works.

My Windows desktop with Ubuntu running on VirtualBox

PLINK for Windows

As I'd have to pay for every Gigabyte of internet used, I was a little concerned about working in the comfort of my room. The move to a room with larger window overlooking the backyard where a couple of squirrels and a pair of pigeons reside at the pine tree really helps with a better working at home environment. I'd need to work in the department once in a while, but the alternative is as good as the office, so I feel comforted. And finally, a more sturdy desk chair is provided to me today! Yay!

This is the view from my desk: perfect, isn't it? Getting back to work :)

Back to the topic, since I'm using a laptop installed with Windows 8.1, I was thinking of running PLINK on the laptop, provided I have enough HDD space and RAM, of course. I think with 6GB of RAM, I should be able to do quite a bit before I crash it. A tiny search on PLINK website led me to the MS-DOS version of PLINK, which means I could do the genomic analysis offline without using my group's server! Another yay!

Here's the instruction as per PLINK website on how to download and install PLINK on Windows... I think the links on my page would lead you to the download site, further instructions and manuals for PLINK. Enjoy using the whole genome association analysis toolset!

This page contains some important information regarding how to set up and use PLINK. Individuals familiar with using command line programs can probably skip most of this page. Download PLINK is now available for free download. Below are links to ZIP files containing binaries compilied on various platforms as well as the C/C++ source code. Linux/Unix users should download the source code and compile (see notes below). These downloads also contain a version of gPLINK, an (optional) GUI for PLINK. Please see these pages for instructions on use of gPLINK. Remember This release is considered a stable release, although please remember that we cannot guarantee that it, just like most computer programs, does not contain bugs... Platform File Version Linux (x86_64) plink-1.07-x86_64.zip v1.07 Linux (i686) plink-1.07-i686.zip v1.07 MS-DOS plink-1.07-dos.zip v1.07 (to be posted later today, 30-Oct) Apple Mac (PPC) plink-1.07-mac.zip v1.07 (to be posted next week) Apple Mac (Intel) plink-1.07-mac-intel.zip v1.07 C/C++ source (.zip) plink-1.07-src.zip v1.07 One more thing... If you download PLINK please either join the very low-volume e-mail list (link from Introduction page) or drop an e-mail to plink AT chgr dot mgh dot harvard dot edu letting me know you've downloaded a copy. For old versions of PLINK please visit the archive. Debian users PLINK is available as a Debian package, see these notes. Note, the executable is named snplink in the Debian plink package. Development version source code You can download the very latest development source code in this ZIP file. This is really, strongly not recommended for most users. The code posted here could change on a daily basis and is not versioned. Development source code versions have a p suffix, meaning pre-release. For example, if the current release is 1.04, the next stable release will be 1.05 and the development code will be 1.05p. Note that 1.05 may differ from 1.05p and as noted before, from day-to-day the 1.05 development code may change in any case. The principle reason for including the source code here is to allow access for specific users to specific, new features. These features are described here. General installation notes The PLINK executable file should be placed in either the current working directory or somewhere in the command path. This means that typing plink or ./plink at the command line prompt will run PLINK, no matter which current directory you happen to be in. PLINK is a command line program -- clicking on an icon with the mouse will get you nowhere. Below, on this page, is a general overview of how to use the command line to run PLINK. The next sections give details about how to install PLINK on different platforms. Windows/MS-DOS notes Unzipping the downloaded ZIP file should reveal a single executable program plink.exe. The Windows/MS-DOS version of PLINK is also a command line program, and is run by typing plink {options...} not by clicking on the icon with the mouse. Open a DOS windows by selecting "Command Prompt" from the start menu, or entering "command" or "cmd" in the "Run..." option of the start menu. The folders c:\windows\ or c:\winnt\ are typically in the path, so these are good places to copy the file plink.exe to. You can copy the plink.exe file using Windows, as you would copy-and-paste any file (e.g. using the right-button menu or the keyboard shortcuts control-C (paste) and control-V (paste). Alternatively, if you know that you will only ever run PLINK on files in a single folder, then you can paste plink.exe into that folder, e.g. C:\work\genetics\. The disadvantage of this approach is that PLINK will not be available from the command line if you are in a folder other than this one. Once you have copied plink.exe to the correct location, you can test whether or not PLINK is available (i.e. in your command path) by simply typing plink at the command line. You should see something like the following message: Microsoft Windows XP [Version 5.1.2600] (C) Copyright 1985-2001 Microsoft Corp. C:\>plink @----------------------------------------------------------@ | PLINK! | v0.99l | 27/Jul/2006 | |----------------------------------------------------------| | (C) 2006 Shaun Purcell, GNU General Public License, v2 | |----------------------------------------------------------| | http://pngu.mgh.harvard.edu/purcell/plink/ | @----------------------------------------------------------@ Web-based version check ( --noweb to skip ) Connecting to web... OK, v0.99l is current *** Pre-Release Testing Version *** Writing this text to log file [ plink.log ] Analysis started: Fri Jul 28 10:07:57 2006 Options in effect: ERROR: No file [ plink.ped ] exists. Do not worry about this error message -- normally you would specify your own PED/MAP file names to analyse (i.e. the default input filename is plink.ped). Please ask your system administrator for help if you do not understand this. HINT In MS-DOS, you can to increase the width of the window to avoid output lines wrapping around and being hard to read. To do this under Windows XP DOS: right click on the top title/menu bar of the window and select Properties / Layout / Window Size / Width -- increse the width value to a larger value (e.g. 120, or as large as possible without the window getting too big to fit on your screen!).

Anxiously Taking Off...

Source: http://upload.wikimedia.org/wikipedia/commons/6/64/Malaysia_Airlines_Boeing_777-200ER_9M-MRO_IST_2012-1-23.png

Finally, after 6.5 months, the first phase of my PhD is sort of taking off. I hope that this first phase will reach its final destination of a first year report which enables me to officially become a Cambridge PhD student, rather than the current probationary status. This post also serves as a tribute to all the passengers of MH370, the aircraft which also the last location where my cousin worked as the cabin crew. Despite not knowing him well when he was around because of our large age gap, I know David, my cousin, worked hard his whole life, and went through various struggles. He never gave up on life, on love. I wish I can learn this attribute of his.

I am nervous, and anxious too, especially after such a long hiatus on research due to my health, the travels, and the graduate conference (as much as I tried to deny the fact that I did put in much time and enjoyed doing it). Sometimes I dislike the fact that I could catch a cold too easily, and with the new addition to my medical history of acute sinusitis, my sinuses become infected twice this year alone. All these took time away from me trying to work hard because I sleep for days. Right now, I am worried I can't meet the par of passing my first year though I have another 3 months before the reality sets in. What if I have to go home because I can't complete year 1 and pass this probation? Everyone knows he/she has worked hard for a year, so it is natural passing first year isn't difficult. For me who spent most of my time sleeping and idling, it is as natural as them, only I'm at the other side of the fence. Working hard is not my forte. Yet, I know it takes hard work whether or not I'm good at it.

I saw this online when I was searching for the quote... "Don't be upset with the results you didn't get from the work you didn't do".

Source: http://www.savingadvice.com/articles/wp-content/uploads/2013/08/insecurity.jpg

I guess me browsing through the Wellcome Trust Sanger Institute is a cause of my anxiety. There is a group who is doing an awesome job with malaria. I saw their papers and found those interesting. Who am I to compare to them? All highly-skilled bioinformaticians and biologists; big brains gathering big data as a team of researchers. I need to overcome this insecurity I feel from within, really.

This is my behind-the-scene, nothing too bright or shiny, as how anyone would see a Cambridge graduate student should be. We all are still graduate students, just like anyone else in the world. We struggle too, and maybe, more than the rest of the world.

The Big Q

Finally, I've arrived to the stage of forming the Big Q for my PhD research! *tears of joy*

MALARIA shall be my journey for the next three years (and whatever that might pop up as this continues).

I have a report deadline in 1.5 hours with the MA to earn the upgrade from "cause for concern" to "fair" on his supervisor report. Scary!!!!

Source: http://www.euvaccine.eu/sites/default/files/uploads/images/diseases/EVI_Pf_cycle_logo_110225.png

One's Beauty Might be Another's Thrash

Source: http://files2.coloribus.com/files/adsarchive/part_1095/10957605/file/solitaire-anti-reflective-coating-rubbish-dump-large-86735.jpg

My recent input of research questions is officially thrash which is deemed unfit to be done at this point. Too backwards, pointless and over-simplified for this group I'm in. I wouldn't say what I want to do is something stupid, it is just more industrious rather than academic, more focused on forensics than evolutionary. So it's unsuitable, and uninteresting from another point of view. So, bye-bye full-on forensics genetics for now. I guess this can't be help, when I'd rather try to find what would cost less for a third world, developing country to apply, where I am placed right now has excellent methods, human resources, and hardware to execute sophisticated analyses. I guess I need to discard the third-world mentality and embrace sophistication for now.

A negative progress is still a progress. Indeed. It is.

First Year Report Training

The day was gloomy when I woke up after the multiple alarms went off. It was a dread to try to wake up these days but I knew how important it is for me to connect with people studying Life Sciences and to know about first year report. The people in........***censored***........... It has been a lonely journey.

After a series of missing bus, getting lost and finally getting there to listen to Dr. Geraint talking and the PhD probationary students speaking of first years' worries, though more questions popped up but I'm assured that I'm not alone in my struggle to bear future responsibility as a Cambridge grad. Perhaps it doesn't take a super genius to get a PhD done here.

There's still some time left. Let's see how it goes.

PLINK: Association Analysis / Accounting for Clusters

It wasn't one of the best days. Had a word with the man above when I bumped into him in the pantry and he didn't seem pleased that it took me two weeks to look at the paper on placing individuals in their geographical location. I'm taking very long because the methods are fundamental, but it wasn't a knowledge that I am born with, unfortunately. I've long suspected that everyone is assumed to be a genius of some sort where I am based and I did feel stupid as I am taking longer time than "most people" to pick up the basics. I know he thinks I'm too slow to learn the skills needed. It's ok, it is not the first time I felt like a complete idiot in this group of geniuses. I have been feeling stupid for a while now. This stupidity is suffocating me from within. I guess this is a price to pay to eventually able to carry the responsibility of having the Cantab post-nominal and the permanent effect of a head damage for ramming into the world of graduate life. Why in the world did I decide to do this? I no longer am in the mood of discern it right now.

Source: http://thegradstudentway.com/blog/wp-content/uploads/2012/07/PhdComics2.jpg

Anyway, back to my learning progress. Any form of progress is better than nothing at all. After the long break from learning, finally I'm back at PLINK tutorial. Today I plotted a multi dimensional scaling (MDS) plot using the HapMap example of two population.

Population stratification is the presence of systematic difference in allele frequencies between subpopulations in a population due to different ancestries, also known as population structure. Stratification analysis use whole genome SNP data to cluster individuals into homogeneous groups. In the tutorial, simple stratification was performed, but the details of it could be found in another chapter in the PLINK website. I think it is worth me spending the whole afternoon going through the main documentation on population stratification as I probably would need to perform this as a routine data treatment procedure and if I don't get it now, I won't get it later. I don't want to form bad habits in programming and jeopardize the quality of my research in future.

If two or more individuals have identical nucleotide sequences in a DNA segment, it is known as identical by state (IBS). If this segment is inherited without recombination by a common ancestor, and is found in two or more individuals, then it is identical by descent (IBD). I find that both IBS and IBD are of the few jargons often mentioned during lab meeting, so it is important that I have these two definition registered in the brain and here. The clustering which I did today was based on pairwise identity-by-state (IBS) distance clustering. No constraint was applied to the process. Usually phenotype criterion and cluster size restriction, plus external matching criteria are specified.

In order to create the MDS plot for the HapMap example of two populations, I first created matrix pairwise IBS distances using this command line:

plink --bfile mydata --cluster --matrix --out myplot

A few files were generated: myplot.mibs, myplot.cluster0, myplot.cluster1, myplot.cluster2, myplot.cluster3, myplot.log. Information are stored in different formation within the four output files resulted from performing the --cluster option.

Using RStudio (it means I used R statistical tool), I created the MDS plot with the code given:

m <- as.matrix (read.table ("myplot.mibs"))
mds <- cmdscale (as.dist (1-m))
k <- c( rep ("purple", 45), rep ("orange", 44) )
plot (mds, pch=20, col=k)

# RStudio was used on Win8 laptop while PLINK was used on UNIX server.

Here's how my plot looked like:

Very interesting combo to use both PLINK and R to generate the plot. According to the tutorial, I could also generate the MDS plot using the --mds-plot option. I have not tried it, so I'm unsure how does it work. I guess it is best that I keep to one which I will be good at, rather than to learn 101 alternatives. I am certain I can beautify my MDS plot. That I can wait.

I better sign off now. I am attending training on "How to Write First Year Report" in the Clinical School later.

Sketches

Source: http://fc04.deviantart.net/fs71/i/2012/312/6/c/how_to_paint_digitally_over_a_pencil_sketch__gimp__by_crystal_89-d5kbt1t.jpg

Every artist knows that the first step to amazing paintings is sketching the art framework. The counterpart of artists would usually be the scientists (very loosely I defined it here). I'm guilty as charged when it comes to strictly sketching my experiments, since we somewhat know what is the concepts. I'm not saying that all scientists are like me, I know of many colleagues who have meticulous planning methods.

Upon attending programming classes for absolute beginners, I realised one repetitive warning from all lecturers - PLAN the logic of programming even before starting. For the past two days, I worked on the basis that I need to know this and that, without realising how dodgy it has become. This morning, I woke up thinking about it after some hiccups last night trying to figure out the meaning of the error I got trying to run ADMIXTURE. Apparently, I need to apply filters to the dataset. Yes, I have NO idea how to apply filters. Froze me on track.

Then, I began to liken myself as an artist, of a different field. Not the colourful flowers blooming in the middle of spring, but a starter of another type of bloom - the programming one. Running back to the previous checkpoint, I'm giving myself some time this morning to sketch the structure of the whole process before I get into the minute details of each step. Yes, ADMIXTURE is just part of the whole process and I was obsessed with it the whole of yesterday.

Source: http://isaiahbowling.com/wp/wp-content/uploads/2011/02/Example_looping_program-full.jpg

This is an example of planning a looping program. Not sure what language they are using. So any computer scientist who might know, please drop a comment! Thanks!

Upgrade Exam

I'm changing the format of this blog. My FB chronicles which I've posted here until October last year will still be available (wade through the entries please) but I will focus more on my PhD progress in this blog. After all, this is the blog of a wimpy scientist (aka me).

I saw myself as an infant of my field the day I stepped into the office and met my supervisor. After the 4 months of swimming in the sea of overwhelmed emotions (both at work and in my personal life), I've finally moved on from infancy to maybe kindergarten stage when I'm sort of done attending the important Absolute Beginners classes for most of the programming tools I need.

Today, I passed a little benchmark. I finally able to run some basic analyses using the example dataset on ADMIXTURE. Strictly following the examples given, and my brain would crash when it doesn't work. I guess I'm officially in Primary 1 right now. Hopefully they would make more sense as I explore this tomorrow. It is already Friday!!

I wonder how long I will stay in Primary 1? A week? Hopefully shorter than that, but I need to fully understand what I am doing with ADMIXTURE before I proceed to analyse the data provided for the papers I'm supposed to "emulate".

This is my very first barplot of Q estimates using R after running ADMIXTURE. Very raw and inadequate. Yet, a benchmark!