SU Course: Applied Programming for Life Sciences II

The Stockholm University follows a pattern of single full time courses, as opposed to Karolinska and KTH. However, this course for our masters programme is an exception.

The Applied Programming for Life Sciences is the continuation of python programming course from the Karolinska Institute. The course instructor Lars Arvestad conducts the classes distributed throughout the semester.

It is a very small course with 1.5 credits, with the focus on three main topics: Structuring and modularising python scripts, Classes and Objects and writing a command line tool.

The command line tool that has help options like the professional command line tool.

Each concept is covered in individual class at the Albanova campus of SU. Each class is 2 hours long and followed by an build up exercise to implement new concepts on the previous exercise.

 

Frozen lake next to our class building. We went walking on the lake during our breaks!

Another important aspect of this class was the instructors emphasis on writing codes in a standard format. The reason being, if we revert to the code after a year then we should be able to understand what the code does and how it does that. This practice has helped me a lot in the other courses where I was able to write codes with comments and doc strings, which not only helped me understand my code later but also my team mates who were doing the project with me.

SU Course: Methods in Molecular Life Sciences Part – 3

2. Lab Practicals

2. Data analysis – Dry Lab

The Data from Illumina sequencing was then transferred to the Uppmax server. Each of us made an Uppmax account and used to access this data on the server.

Uppmax account accessed through the terminal to access our sequencing data on the server

In order to get an idea of the data analysis pipeline, on the first day of the exercise, we were given sample data to practice the use of various tools and get used to the command line usage.

On the second day, we got our own sample data from the Illumina sequencing run. The Illumina converts the image files and provides them in the form of Fastq files. We then followed the general flow of the data analysis from the practice exercise and the tutorial provided.

This is an example of how the sequencing data looks like – Fastq file

We started by checking the quality of the data using FastQC tools available on uppmax. We then used BWA tool to map the reads to the reference genome. This step generates human readable ‘SAM’ format.

Alignment file – SAM format . An example of how the mapped file looks like. Each column represents different values

This was then converted to ‘BAM’ i.e. binary format using Picard tools. This reduce the size as well as increase the processing efficiency. Further, most of the tools used downstream are compatible with the binary format of input files.

An example of BAM file. Various tools can be used to extract information from this binary file. Samtools can be used to convert it into human readable format

BAM files were then used for variant calling using GATK tool. The GATK looks for the difference in the nucleotide present in the mapped reads as compared to the reference. These were stored in a file format call ‘vcf’ file or the variant calling file. GATK was instructed to look for SNP as well as Indels (Insertions and Deletions) using different commands.

This is a VCF file. It shows the position of the variant in the reference, the reference nucleotide and to what it is mutated to.

Once this was done, the reads were recalibrated and another round of variant calling was performed. The recalibration improves the alignment near the regions of Indels and improves the chances of detection of variants more efficiently and accurately.

Finally, this file was used to visualise the regions having Indels in a graphical manner. IGV tool was used for visualisation. This tool enabled us to look navigate to a specific gene (LYS2, LYS5 and LYS14 genes in our case) and look for the mutations i.e. variants.

IGV visualisation of mutations in the entire yeast genome

We then used the loci information from the reference to look for information i the SNP databases for yeast and other similar resources.

One such mutation observed in majority of the reads was T to C mutation in the LYS5 gene.

Finally, on comparing the information obtained from IGV analysis and complementation test we were able to confirm the mutation was in LYS5 gene (for our group named Cocolocos).

SU Course: Methods in Molecular Life Sciences Part – 2

Today I will talk about the wet lab aspect of the Practicals of the methods course.

2. Lab Practicals

The main aim of the lab exercise was to generate mutant strains of the Saccharomyces cerevisiae and look for the mutations in genes involved in Lysine production  pathway using Illumina sequencing.

1. Experimentation and Data generation – Wet lab

a. Yeast Culturing

We started by culturing yeast in plates from the culture flasks by using different OD to get a good growth that can be used for further experimentation. Saccharomyces cerevisiae or commonly known as Yeast takes about 2 days for good growth. Thus, after streaking on the YAPD (rich media) we let them grow for about 2 days.

b. Clonal Purification

The yeast cells were then clonally purified on nitrogen-poor media that contained high levels of α-Aminoadipate and lysine. This is an intermediate from the Lysine production pathway. However, downstream synthesis product derived from this intermediate is shown to cause the growth inhibition on nitrogen poor media. Thus, if cells do not have the 3 important lysine genes (LYS2, LYS5, LYS14) then the conversion of α-Aminoadipate to the toxic intermediate does not take place, allowing the cells to grow.

Selecting clones for plating and obtaining genetically purified (identical) colonies

Thus, we clonally select cells that have mutation in these Lysine genes or so called auxotrophic for Lysine.

c. Validation of auxotrophy

To verify that the cells thus selected actually have the mutations in the lysine genes, they are replica plated on Lysine free media to see for no growth as opposed to lysine rich media.

The third column shows a mutant strain, as it has no growth in Lysine deficient media

d. Complementation test

Plate containing the auxotrophic mutants was crossed with the tester strains (strains that are known to have mutations in LYS2, LYS5 and LYS14 genes) by replica plating.  This test is used to determine experimentally the specific gene in which each mutant has a mutation. This observation is used to obtain preliminary information if it is worth sequencing the sample and where to expect the mutation while analysing sequencing data. Good samples were selected and used to perform Illumina sequencing.

Replica plating for complementation test (crossing tester strains with the mutants from the velvet to the plate)

SU Course: Methods in Molecular Life Sciences Part-1

The next course in the Masters is of Methods in Molecular Life Sciences. This course is also a two part course like the Bioinformatics course.

The two parts are :

1. Theory lectures

2. Lab practicals

1. Experimentation and data generation – Wet lab

2. Data analysis – Dry lab

1. Theory lectures

There are 10 theory lectures each 2 and 1/2 hours long. The classes start at 10 in the morning until lunch time. Each class focuses on different techniques and methods in molecular biology – wet lab. All these methods are taught by experts in the field, who use these techniques in their labs on daily basis. The classes are conducted by faculties from the MBW department at Stockholm University and Scilife lab Fellows.

The main topics covered in the course are Protein-protein interaction. This class is complementary to the course on Lipids and bio-membranes. The next lecture is introduction to various Protein-nucleic acid interaction, where they show how various Transcription factors which are proteins recognize the nucleic acids for their function.

Following this, we had two lectures on Yeast as a model organism and molecular tools available for use in this organism. This also formed basis to the lab exercises that we performed in the afternoon, after the lectures. Continuing the discussion on the tools, we were introduced to the more tools that can be used on various model systems and in-vitro mutagenesis, ethical concerns and some interesting examples of the use of some of them in the on-going research in Dr. Claudia Kutter’s lab.

Dr. Claudia Kutter’s lecture on gene modification

While mentioning model systems, we were also about microbial growth systems, and a recent advancement in the technique of single cell imaging. Moving to higher Eukaryotic organisms we learned about Drosophila melanogaster, were we saw the first manual drawing of the polytene chromosome. Further, we learned the epigenetic basis of DNA modification and how it can be used to create desired mutations chromosome wide.

Manual drawing of the polytene chromosomes from Fruit fly

After the Easter break of about 2 weeks, we had the most interesting lecture of the course on the advancements in the next generation sequencing techniques by Dr. Vicent Pelechano. In this, we were fascinated to see how the same samples can be pre-processed in multiple ways to get different kind of information (TSS, TTS, splicing information, snap shot of the genes currently transcribed, and many more…) from it, by employing the same Illumina sequencing at the last step. Finally, we had a introductory lecture to the data analysis explaining its importance in the age of Big Data.

One such seuencing method – TIFseq, developed in Dr. Vicent Pelechano’s lab      Source: Nature journals

Appointment for Biometrics

Once you have submitted your application for the Residential permit, and paid the fees, the next step is to book an appointment for submitting your biometrics.

Swedish residential permit is in the form of a card. This card contains your biometrics information. This includes fingerprints, signature and photo. In order to submit this information with the migration agency, you need to go to the nearest Swedish embassy. You can find the information about the nearest embassy on the website here.

The option to select the embassy will appear after the payment section in your application. Select the embassy that is nearest to you from the above link and state that in the given column. Then you will have an option to select the slot convenient to you from the available slots. Note that the submission of biometrics does not affect the decision on your case. You can alternatively wait and book the appointment for biometrics after you receive the decision. However, I recommend to not wait that long as the slots might fill be full at a later time.

While at the embassy, you have an option to get you card delivered to your home address or to collect it from the embassy. To get the residential permit card delivered to your home address there is a form to be filled that is provided by the staff at the embassy. The residential permit generally takes about 2-3 weeks to arrive at your home address after the decision has been made and the biometrics have been submitted. You can find the information of the cards dispatched from the Sweden every week on the Sweden-abroad website in the waybills.

In case of queries you can write to the Swedish migration agency from their contact page. Alternatively you can call them on the given number. They generally answer in short time.