Hi, everyone. My name is Pimlapas Leekitcharoenphon. I'm a researcher from the research group for Genomic Epidemiology at National Food Institute, Technical University of Denmark. Today I'm going to talk about, multipurpose detection of genetic markers, MyDbFinder tool description and application. As you know, whole genome sequencing is becoming a more practical, rapid, and less expensive form of typing. Tools intended for quick analysis of whole genome sequencing data are required for this. There are many purpose for identifying genes, and particular purpose for example, if I want to identify virulence genes in my species that I study with, then you have to have a set of Database that contain their virulence genes of your species. But not always that they're going to be available species or available tools that have exactly the Database of genes of your interests provided for you. To accommodate the researcher that may have interests in particular set of genes for which no Database is available, we developed a special versions of finder tool called MyDbFinder. MyDbFinder is a web friendly interface tool and freely accessible tool and user can generate their own Database of genes that contain genes of their interests and search for those genes in their input genome. My DB Database is a blank tool. It doesn't contain any Database, but the Database is from you as a user. The user provide Database to MyDbFinder and the user also provide unknown genome for the tool to search if any of the genes in your Database present in your unknown genome. Of course, your unknown genome can be assembled genomes, which is in FASTA format, then the tool we use, BLAST for the alignment. If the input genomes is raw reads of FASTQ format, the tool we use KMA for alignment. User Database have to be in FASTA format only, and the tool will give you the best matching gene from your own Database. Basically, this is like you have alignment, like BLAST with interface on it. If you are familiar with UNIX command line or Bioinformatics tool, you can actually use BLAST and install it by yourself, or you can actually use KMA and install it locally by yourself, you don't need this tool. For those that don't have Bioinformatics skill and really want to use our Finder gene for the web-based version of some easy to use tool, this might be a solution for you. The critical thing about MyDbFinder is the Database. First of all, you need to make your own Database. This is some suggestion for making your own database. I would suggest that the Database should be made in the pure Text Editor. You should not use Microsoft Word here because it contained a lot of hidden correctors that when you feed in to the tool, it wouldn't work or it would include some weird characters into alignment which you don't want. I suggest some pure Text Editor like Notepad or Jedit. The Database must contain of DNA sequences, and unfortunately it's only available for DNA sequences, not for the amino acid sequences or protein sequences. It has to be in FASTA format which like this. So you have the sequencing ID starting by the greater than sign like this and sequence ID. The second line is sequences. It can be a long sequences, long line, or it can be multiple lines like this. As long as it has greater than ID and a sequence, that is the FASTA format. Another thing that I would like to suggest is sometime you have lists of genes which have a very similar name and different by number like sequences one, sequences two and sometime you have a long explanation of what it is. Normally you can divide each what by space, that's what you normally do. But if you do that, when you have the result, it will cut the space. The output result is normally cut any space that it found. So, basically you hit the sequences number two, you will get only this. You will not get explanation of what a gene is. To avoid that, if you have any space in your FASTA Database, I would recommend you to add underscore space here. Then the tool will take everything with underscore here. Once you done with making your own Database, and made sure that is a correct form in FASTA format. Here is a link to their MyDbFinder https://cge.cbs.dtu.dk/services/MyDbFinder/. This is the front page. First of all, I would like to point out, so actually we used to include this in MyDbFinder, but now we separate them. We have a set of the Virulence genes that you can find it in the VirulenceFinder. We have a set of Virulence gene for E.coli and for Listeria. You can find it from here. We have the list of gene that you use for subtyping Vibrio Cholerae. You can go for here, so we separate them from MyDbFinder, the new version. Here, you first of all in MyDbFinder, you have to upload your database in FASTA format. You can upload over here, and then there's a number of options if you can choose this example, person identity, how many percent identity that you want as a cutoff of percent similarity. For example, if you say 90 percent that mean any alignment or any hit that less than 98 percent similarity, won't be shown here in the output page. The next one is the minimum length. It means, the percentage of the total gene length in the database that match to user sequence. If you set the minimum length at 60 percent, it means If this two gene and this is your sequence, if the alignment is less than 60 percent of the size of the genes, it also won't show here. This just to make sure that the alignment or the hit in the gene in your database is the real hit or is not just a fault hit. The next option is you have to specify the input is going to be contigs or assembled genome and it has to be in FASTA format like this or if you're impulse is Raw reads and it has to be in FASTQ format, and the Raw reads can be single end reads, which you have to upload one Fastq file or it can be paired end reads which you have to upload two Fastq files. If it assembled genome or Contigs is only one file and this is one submission per one genome and you can put it in here. Then you've chose your genome, you put it in here, you have your database over here already, then you're ready to upload everything into the server. Once everything upload to the server, you can have the option to put to email here once the tool is done, it will send output link directly to your email that are provided here. Here is example of the output from the MyDbFinder. It showed you the list of the genes in your own database that match with your input genome. Of course it tell you the gene ID, it tell you the sequence identity, it tell you how good the alignment; if you look at this, good example should be this one because it's not perfect match. The first letter; don't confuse with the header here, the first letter is the size of the gene in your database. The second number is the alignment length. Let's say the size of the gene T-O-X-R is 721, but your input genome is matched only 720. It just one base pair missing and that's the reason why if it turns gray, because it warns you that is not perfect match. Like this, a 100 percent identity and the size of the alignment and the size of the gene is equal. This is the perfect match, you can trust it completely. But it's an indication is not perfect match, but it's also very high similarity and it match almost everything is just one base pair missing, so you actually should consider that this gene also present in your data. You can have an option to actually extend this output to see alignment in detail. For example, if you can see here, is you can see the alignment in detail, you can actually see what is the missing base pair in here you scroll down to see the result. Another thing is you can get all this output as a text file here, like this one and you also get the hit in the genome sequences. It means you can get the hit, you can get the sequences from your input genome that's similar to the gene in your database example here. The sequence that you see here is the sequences from your input genome that's similar to this gene in your database. You can get the sequences of all the gene that hit with your genome which is the gene from your database anyway. Yes, that's all for the MyDbFinder, and thank you for watching.
