MLST Typing: MLST tool description and applications

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En provenance du cours de Technical University of Denmark (DTU)

Whole genome sequencing of bacterial genomes - tools and applications

166 notes

Technical University of Denmark (DTU)

Whole genome sequencing of bacterial genomes - tools and applications

166 notes

This course will cover the topic of Whole genome sequencing (WGS) of bacterial genomes which is becoming more and more relevant for the medical sector. WGS technology and applications are high on international political agenda, as the classical methods are being replaced by WGS technology and therefore bioinformatic tools are extremely important for allowing the people working in this sector to be able to analyze the data and obtain results that can be interpreted and used for different purposes. The course will give the learners a basis to understand and be acquainted with WGS applications in surveillance of bacteria including species identification, typing and characterization of antimicrobial resistance and virulence traits as well as plasmid characterization. It will also give the opportunity to learners to learn about online tools and what they can be used for through demonstrations on how to use some of these tools and exercises to be solved by learners with use of freely available WGS analysis tools . By the end of this course you should be able to: 1. Describe the general Principles in typing of Bacteria 2. Give examples of the applications of Whole Genome Sequencing to Surveillance of bacterial pathogens and antimicrobial resistance 3. Apply genomic tools for sub-typing and surveillance 4. Define the concept of Next-Generation Sequencing and describe the sequencing data from NGS 5. Describe how to do de novo assembly from raw reads to contigs 6. Enumerate the methods behind the tools for species identification, MLST typing and resistance gene detection 7. Apply the tools for species identification, MLST typing and resistance gene detection in real cases of other bacterial and pathogen genomes. 8. Describe the methods behind the tools for Salmonella and E.coli typing, plasmid replicon detection and plasmid typing 9. Utilize the tools for Salmonella and E.coli typing, plasmid replicon detection and plasmid typing in real cases of other bacterial and pathogen genomes. 10. Explain the concept and be able to use the integrated bacterial analysis pipeline for batch analysis and typing of genomic data 11. Demonstrate how to construct phylogenetic tree based on SNPs 12. Apply the phylogenetic tool to construct phylogenetic trees and explain the relatedness of bacterial or pathogen strains 13. Describe how to create your own sequence database 14. Utilize the MyDbFinder tool to detect genetic markers of interest from whole genome sequencing

À partir de la leçon

Module 3

Whole genome sequencing tools- demonstration of analysis tools for species identification, MLST typing and finding resistance genes

Species identification: KmerFinder tool description and applications8:53

MLST Typing: MLST tool description and applications8:45

Resistance gene detection: Resfinder tool description and applications12:22

Rencontrer les enseignants

Lina Cavaco
Ext Researcher
Copenhagen University- ICMM /Statens Serum Institut - Department for Bacteria, Parasites and Fungi
Pimlapas Leekitcharoenphon
PostDoc
Research Group for Genomic Epidemiology, DTU Food

[MUSIC]

Hello, everyone, my name is Pimlapas Leekitcharoenphon.

I'm a Postdoc from Research Group for Genomic Epidemiology at DTU Food.

Today I'm going to be presenting you MLST Typing,

MLST tool description and applications.

For many species, multilocus sequence typing or

MLST is considered for a gold standard for typing.

And traditionally it is quite a very expensive and

time consuming to actually identify sequence type in the traditional way.

And because the cost of whole genome sequencing right now,

it continue to decline and it's become increasingly available to scientists and

routine diagnostic laboratories.

And it actually, right now, the cost of whole genome

sequencing is actually below the cost for traditional MLST typing.

That's why we have developed a web-based tool for identify an MLST and

do sequence typing directly from whole genome sequencing data.

And the database of the MLST tool is actually updated every month.

And the best-matching MLST alleles of specified

MLST scheme are actually found using BLAST-based ranking method.

And the sequence type is determined by the combination of the alleles number.

The method, the tool, has been tested on different set of data.

We used the preassembled genome from 336 isolates covering 56 MLST schemes.

And we also tested short sequence reads from 387 isolates covering 10 schemes.

And a small test set of short sequence reads from 29 isolates, which we actually

know the sequence type, that been identified by the traditional methods.

And it turned out that the MLST tool actually can determine

the sequence type of these isolates, basis on the polynomial sequencing data.

And the idea of the MLST tool.

As a scientist, or the medical doctor, or

any researcher that has their own genome of bacteria that their interest.

They can submit their own genomes into the MLST tools and

the MLST it that's contained in the MLST alleles.

If you give it back, we can type after it's submitted back to your genomes.

The MLST tool actually consists of the, of course,

MLST database that actually this set of allele

sequences that we got from the public MLST database.

So once you submit your unknown sequences and

your unknown sequence can be raw reads or assembled genomes.

If your sequences are raw reads the tool will automatically.

Do the novel assembly of your raw reads into contigs or assembled genomes.

And the tool will take your contigs or assembled genomes and

do BLAST to all the alleles in the database.

And with the combination of the alleles that file the combination of the alleles

numbers that fall in your unknown sample will be determined the Sequence Type.

So here's the link to the MLST tool.

And here's the webpage for the MLST so first of all you

have to choose the MLST configuration that also,

according to your species of your sample.

So you have to choose over here and then you choose the type

of your list of sample if your sample is Assembled Genome of Contigs.

Then you have to put the genome in Fasta format.

We start by enzyme, ID, and the sequencing data.

So two lines for one sequence in the Fasta format.

But if your genomes that you submit to the database is raw reads or

the data that coming from the sequencer that is going to be in Fastq format.

Fastq format it's a faster plus quality score, that's why it's called Fastq.

And in the Fastq you have the first two lines is the sequence ID and

the sequencing data, and the last two lines is the quality score.

That's why it's called Fastq.

So if your genome then you had to upload to Fastq and

they have to choose if the Fastq are single end.

Or pair end and then you have to choose the corresponding

technology of sequence and platform that you would use for sequencing.

So, and then you Upload your sample here.

And then you can click Upload.

After the program already transferred your genome to the server successfully.

If you have an option for

you to actually put your email over here and click Notify me via email.

So, once the program finished running the analysis on your sample.

It is send you an email with the output link in the email.

So you can actually open another window and start another job,

or even close this one down because you will get the output anyway by the email.

And here is the output that we get from the MLST.

Of course you get the most important thing over here as the Sequence Type

of your sample and you'll get the detail of the output.

Of course you will get a new number and this combination of this

number is used for determination of the ST type of your genome.

And you also have some of the alignment quality,

for example person identity and HSP length.

So the HSP length is a line length between the best matching

allele in the database and the sequence in your genome.

So if you have, The alignment length 501 over here and

the length of the alleles is 501,

meaning that every position in the alleles in

the database actually align with the sequence in your sample.

And if the alignment is 100% identity, meaning that every position of

the alleles in a database match perfectly with the sequence from your sample.

In this case, it's a perfect match.

And if you want to see the detail of the alignment between the alleles and

the sequence in your sample, you can click over here, extended output.

You can see the alignment between the allele in the database and

the sequence in your genome.

And at the end of the web page, do you have the technical problem over here?

So if you have any problem using the tools,

you can click technical problems and email us any problems that you have.

And thank you very much for watching.

[MUSIC]

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