Module: Mycobacterium tuberculosis NGS made easy: data analysis step-by-step

Webinars and hands-on tutorials on several aspects related to NGS in Tuberculosis

Introduction

There were unusually high rates of TB cases in your country this year. To characterize the underlying bacterial factors driving the epidemic, isolates have been sent for whole-genome sequencing. Doctors and public health authorities request information in order to take decisions. In this course it will be demonstrated how you would make use of NGS to answer several questions relevant for patient and public health system management such as:

• Are there cases of drug resistant bacteria?
• Is there transmission of drug resistance?
• Is there evidence of de novo emergence of resistance?
• Are there multiple infections per patient?
• Do we have on-going transmission?

We hope that at the end of the different training sessions you can answer this question on your own!

Part 1 - Overview of NGS technologies & TB specific NGS solutions

Part 2 - Mapping and Variant calling of short MTBC reads

The 20 strains isolated in your country have been sequenced with Illumina technology to obtain whole-genome sequences. In this part of the workshop you will learn how to analyse those sequences.

In a typical bioinformatic pipeline you would store your sequences in a computer server where all necessary software would be installed. This would be a server running the operating system LINUX, which is the most efficient way to run bioinformatics pipelines (more on this on Part 5). You will be running your analysis in a LINUX server from Galaxy, but instead of writing directly commands to execute operations in the server, you will be executing operations through a Galaxy graphical interface. This allows you to have access to a LINUX server and to run workflows without knowing LINUX. Importantly, for training purposes it also allows you to dedicate more attention in trying to understand what is being done in each of the steps without having to understand the programing behind. This being said, working directly on a LINUX cluster provides you always more flexibility, but if you don’t have access to one, Galaxy is a very good alternative for data analysis.

You will need to understand how to use Galaxy to run all the hands-on tutorials and therefore is highly recommended that you follow the next webinar and hands-on on Galaxy. The good thing about this is that once you know how it works, you can use it to run your own analysis with your own data.

Session 1: Learning Galaxy

Session 2: Mapping and Variant calling of short MTBC reads -hands-on

Once you have received your sequences, the first step is to assess the quality of sequencing. Once we are sure that the sequencing worked well, we typically compare our sequencing results to a reference genome (re-sequencing approach) by using a bioinformatics procedure usually called mapping. After, we will identify the genomic variants in our sequences with respect to the reference genome, a bioinformatics procedure called, variant calling. Once we are certain of the variants we have identified, usually we are interested in determining to what genes they belong, to what pathways or for instance if they are likely to disrupt protein function. This procedure is called annotation. Once we have gone through each of these steps we are ready to analyse drug resistant patterns, draw phylogenetic relationships or identify clusters of transmission M. tuberculosis.

You are now ready for performing bioinformatic analysis in Galaxy. Before we start we would like you to watch a short video on how Illumina sequencing works. Following that video we have prepared a webinar on mapping and variant calling of Illumina applied to MTBC. After watching it you will be hopefully able to know; how a reference genome is chosen, why we typically ignore some regions of the MTBC genomes or what is the difference between a fixed and a variable SNP and why do we care about it (among other things).

Part 3 - Evolutionary epidemiology: using phylogenetics to understand DR emergence and Mtb transmission

We are ready to analyse drug resistant patterns, draw phylogenetic relationships or identify recent transmission among the isolates we have sampled in our population. Before delving into the analysis of the genomes we would like to share with you some notions important to the inference of direct transmission and to the interpretation of drug resistant patterns.

Part 4 - Webtools dedicated to MTBC bioinformatics

The use of whole-genome sequencing (WGS) for antibiotic resistance prediction and routine typing of bacterial isolates has increased substantially in recent years. To date a multitude of solutions for analyzing WGS data of the Mycobacterium tuberculosis complex (MTBC) data have been developed. In the first 4th part, we introduce some freely available webtools and open source pipelines designed to analyze MTBC sequence data and we’ll provide some examples of how these tools work and how to interpret the results.

Part 5 - Be a bioinformatician in the jungle

On parts 2 and 3 of this training you have learned how you could use galaxy for analysing your own data. Establishing your own workflows in galaxy would allow you combining different tools and build your own pipeline without having to know how to program. If you are not so interested in having your own pipeline, webtools for WGS analysis can be very useful, as we have shown in part 4.

However, in this last part of the training we would like to convey to you what it would take if would want to run Linux via the command line. The Linux operating system will be introduced, how to perform basic tasks using the Unix shell and how to install and run pipelines on the command line. You will learn the power of the Unix shell in performing complex and powerful tasks, often with just a few keystrokes or lines of code. In fact, Unix shell helps users automate repetitive tasks and easily combine smaller tasks into larger, more powerful workflows (i.e. pipelines). Use of the shell is fundamental to a wide range of advanced computing tasks, including high-performance computing. These webinars will introduce you to this powerful tool. Which approach to choose, Galaxy workflows, Webtools or native Linux depends on your needs, your interests and what computer resources you have available.