[MUSIC] Hello everyone, my name is Pimlapas Leekitcharoenphon. I'm a post doc from division for epidemiology and microbial genomics at DTU Food. Today, I'm going to be presenting you about the resistance gene detection, and the ResFinder tool it's description and it's applications. So if you have the isolate and the background isolate is a pathogen, it is important to find out how it can be treated. And traditionally, the antimicrobial susceptibility would be determined using phenotypical testing and additional characterization of their resistance determinants. For example using PCR for specific resistance genes. But the whole genome sequencing analysis tool can give the possibility of analyzing genotypic bases of researching gene directly from the genome, and infer the phenotype resistance as expected. And the price and the cost of whole genome sequencing has been decreasing dramatically, and this actually inspired us to develop a method for identification of acquired antiimicrobial resistance genes called ResFinder. The ResFinder if a tool, it's actually best curated database that constructed from resistant genes that we gathering from public database. Also as scientific papers, and the tool of the ResFinder is a web friendly interface and freely available. ResFinder will detect the presence of whole resistance genes, but not point mutations causing resistance in the whole genome sequence data. And the sequencing data can be either raw reads or assembled genomes. And based on the ResFinder results, a predicted phenotype may be inferred using phenotypes from original published studies of the genes that were found. And we actually tested tool with the phenotypic testing, and we found a high concurrence that 99.74% between phenotypic and predicted antimicrobial susceptibility. So the antimicrobial resistance testing based on whole genome sequencing is an alternative to conventional phenotypic methods. So how actually we can use the tool. So as the scientist or the medical doctor, you can actually, if you have sequencing data of your bacterial genome, you just submit your sequencing data to the ResFinder. And the ResFinder will identify If your genome contains any resistant genes and report the resistant genes back to you. At the moment, the database in the ResFinder contains genes for 14 major antimicrobial groups. As you can see here. And in total, there are more than 2,000 genes in the database. So the structure of the tool, it actually contains the main database that we're actually gathering all the available known resistance genes. And once the user uploaded their unknown genomes as a raw reads or assembled genomes to the ResFinder, the tool will use the genomes to align or compare to all the resistance genes in the database using alignment program called BLAST. And the program will the resistance genes that are present in your unknown genome. And here is a web link to the ResFinder website. And this is the web interface. So, first of all, when you start using the tool, your browse your genome by clicking browse here, and you upload the genome. Right now our service allows you to upload one by one. So you upload one of your genomes into the browser here. And the next option is the antimicrobial group that you can choose and search for. You can choose more than one group or if you want to search for all, you click all here. And the next is the, we have two cutoffs that you can choose. The first one is percent identity. The percent identity is the minimum percentage of nucleotides that are identical between the resistnce gene from the database and the gene from your genome. For example, if you set the percent identity at 98%, it means that any resistance genes having less than 98% to your genome will not be shown in the output pairs. And if you've got nothing in the output pairs, it might not mean that your genome doesn't contain any resistant genes. Your genome might contain the genes, but with some sequencing error. Or contain a gene with some mutation or where isn't in that. So, to be sure that your genome doesn't contain any resistance genes, or your genome is susceptible, you choose lower down the percent identity. We recommend to lower it down to 80% to make sure that your genome is actually susceptible. And the next cutoff that you can choose is the minimum length. The minimum length is the percentage of total resistance genes length that match to your sequence in a genome. And the last option that you can choose. If you had to choose to tie up your. If your is the assembled genome, then you choose assembled genome here, and the format of your assembled genome has to be in fasta format. The fasta format starts on the header. The header begins with the greater than sign and the ID, and the next line is the sequencing data. And if your genomes, if the data that directly comes from the sequencer, so we call it as the raw reads. And the raw read is normally in the Fastq format. The Fastq format is the fasta plus quality score. That's why it's called Fastq. And the Fastq is start by the header and the sequence. The first two line is the sequencing data and the ID, and the second last lines, they are the quality score. And you have to choose either single end or pair in reads. And you have to choose technology or the sequencing platform that you actually use for sequencing. OK, once you upload your genome, you choose other options here and you're ready to submit your genomes, and get your output by clicking the submit. And the web will lead you to another page. It tells you that you your job is being processed, and you have another option that you can fill in your email and click notify me with email. So when your job is done, the program will send you an email with the output link in the email. So if you want to star, submit your new genome. You can actually close this window and open another one. You don't need to wait until these stop down and start another one. Yes here is the output that you might get from the ResFinder. So It tells you, and a copy of group here, and the genes that match in your genome and the person's identity between the resistance genes and the sequence in your genome. The HSP and the query length means, the HSP is the length of the alignment between the best matching resistance genes and the corresponding sequence in your genome. The query length is the length of the best matching resistance genes in the database. So if the HSP and the query length they are equal, it means the alignment between the sequence from your genome and the resistance gene in the database, they cover entire length of the resistance genes in the database. Then it tells you which sequence in your genome that match to the resistance genes here. And the position from your genome that match to the genes and the predictive phenotype here. And if you want to know more detail about the resistance genes, you can click at this number, and it's going to direct you to the information of the gene in the NCBI. And we have some different color here. The dark green color, it indicates you a perfect match for a given gene. So green color means percent identity is 100. And a sequence in the genomes covers an entire length of the resistance gene in the database. It's mean 100% identity, and an SSP and a length, they are equal. Another color is red. If you get the red color, it means no resistance gene was detected for that class of anti micro genes. And there's a reason I told you earlier. If you got the output in red color, in all antimicrobial class. It might not be that your genome doesn't contain any genes. You have to go back and check again, how many percent identity that you actually set. If you set like 90% or 98%, that's too high. You lower it down, and read it again. You might see another color instead of the red one. We have another tool color, so if you get the grey color it means some warning of a non perfect match. The grey color, the HSP length is shorter than the resistance gene length. And the percent identity is less than 100. But if you get the light green color, it means a warning of percent identity is less than 100, but the HSP lands under resistance gene length, they are equal. And if you scroll down to the end of the output page, you will see this bar tool intended output. If you want to see an alignment between your sequence in the genome and the resistance gene, you can click here, intended output., You can see all the alignment between the sequence from your genome, and resistance genes. And if your genome that you submitted to the program is a or the sequence there directly from the sequencer, or the fast queue format. You also get assemble genome because the program will start assembling before it starts identifying duties in genes. So you would get assemble genome as well if your data is erroneous. And if you have any problem to use the tool, or any difficulties, you can just create technical problem and email to us. And thank you for watching. >> [MUSIC]
