Hi, everyone. My name is Pimlapas Leekitcharoenphon, I'm a Researcher at Research Group for Genomic Epidemiology at National Food Institute, Technical University of Denmark. Today I'm going to talk about Resistance gene detection: ResFinder tool description and applications. If the bacterial isolates that you're working with is a pathogen, it is important to find out how the patient can be treated. One way to find out is to identify if the bacterial isolate is actually resistant to any antibiotics or any antimicrobial resistance. Traditionally, the antimicrobial susceptibility would be determined using phenotypic testing. If you want to know more about the gene that's causing resistance, you have to do further thing, for example, to do PCR for some specific resistance genes, to understand more about the gene. But since we have whole genome sequencing data and whole genome sequencing analysis tool can actually give the possibility of analyzing of genotypic basis of resistance directly and infer the phenotypic resistance that you expected. Actually, there are plenty of the resistance tools and database for the resistant genes. For example, you can see from the list of the tools here, some of the tool, they are both tool and database. For example, the ResFinder that I will talk about later on, and some of the tools, it just a tool. There are also some of the databases available for antimicrobial resistance, as you can see from here. Let's talk about ResFinder. ResFinder is based on curated database, public databases as well as scientific papers that refer to the anti-microbial resistance or resistance genes. The ResFinder is a web-friendly interface and freely accessible, and is actually not only web interface tool, it is also stand-alone tool. The ResFinder will detect the presence of whole resistance genes, or what we call acquire resistance genes, and the chromosomal point mutations causing resistance in the whole genome data. This means the ResFinder can detect both resistance genes and the mutation that's causing resistance, and the tools can accept the data in the form of assembled genomes in FASTA format and in the form of raw reads in FASTQ format. Right now, the current version of ResFinder is 4.0. It actually provides in silico antibiograms as reliable, as those obtained by phenotypic antimicrobial susceptibility testing. We actually tested ResFinder 4.0 with many bacterial strengths and the testing result turn out that we found a very high concordance, more than 90 percent between phenotypic and the predicted antimicrobial susceptibility from the whole genome sequencing data. Of course, there are some discrepancies and those discrepancies is mainly linked to the criteria for interpretation of the phenotypic tests and also the suboptimal of the sequence quality, not much under ResFinder 4.0 performance. If you want to read more about how can we test the ResFinder 4.0 and how to build their databases, you can read it out from this paper of ResFinder 4.0. The concept of ResFinder 4.0 or ResFinder in general, as a scientist or a researcher, once you have whole-genome sequencing data, you submit your data to ResFinder and ResFinder will return you with the resistance phenotype. Let's go to more detail of the ResFinder 4.0, what is actually database behind the ResFinder 4.0? It contains four databases; first, the antimicrobial resistance genes, which you can get more detail of the whole database of genes from this link. The second database is chromosomal point mutation, and you can get more detail of what is available in the database from this link. The other two databases are: translation of genotype into the phenotype and the species-specific panels for the in silico antibiograms. The ResFinder 4.0 as I told you is contain the database of known resistance or known mutations. Then as a user, when you upload your unknown genomes, you can upload as a raw reads or assembled genomes. If you upload as an assembled genomes, the tool will use BLAST to do an alignment between your genome and the genes in the database. If you upload raw reads in FASTQ format, the tool will not assemble your data. But instead, use KMA or k-mer alignment, map your raw reads directly to the resistant gene database is here. What you get is the resistance gene and the phenotypic result. Let's have a look at the ResFinder 4.0. This is a link to the ResFinder 4.0 in the website version. You have the front page over here. There are some options that you can choose over here. Let's get through each of them. Firstly, you can choose, or if you don't want to choose, it's okay as well. First option is the chromosomal point mutation, and of course you have the percent Identity and the minimum length, if you want to change the parameters, over here. The second option is, if you want to search for the acquired resistant genes, you can go to this option. Then you can choose all the classes or you can choose any particular classes of AMR that you're interested in. Then again, you have the filter percent identity, which it mean percent similarity. For example, if you set is 90 percent similarity, or 90 percent identity over here, it means, any genes that's found less than 90 percent similarity it will not show in the output. It's like a cut off. Another cut off is minimum length. Minimum length is the percentage of total gene length in database that match to the users sequences. What that means, if you set 60 percent as a cut off, if the resistant genes that match with your genome have less than 60 percent alignment length. Let's say that the size of the gene is 1,000. If it has less than 600 alignments in the length of the gene, it will not show here. It's another cut off as well, to make sure that the gene that you found is the correct one. If you scroll down, you can see two more options. One, is the select species, this only matter if you choose the chromosomal point mutation in the option before. You can see here, there are not many species available here. The limitation here is because the availability of the mutation, or the known mutations that we currently know, there are not many. It's limit by our knowledge about the available of the known mutation that we know about in the bacteria. This option will not matter if you don't look for the chromosomal point mutations. If you want to look for only the resistant genes, you can put any species here. It only matter when you do the chromosomal point mutation. The last option is, you have to select what is the type of your input data. If your input is contigs or assembled genomes, it needs to be in FASTA format like this. If you choose any of the reads, either paired end or single end reads, your input needs to be in FASTQ format. If you choose paired end reads, you need to upload two files, two FASTQ files. If you choose single end reads, you only upload one FASTQ file, and then you upload your data. Click here and it shows here. This is the assembled genome. It's only one file, per one submission. - Then when you click upload the data, if your data upload successfully, you will see this page. You can have an option to put your email over here and click notify by email. When the tool is done, it will send out an output link directly to your email that you provided here. Have a look at the example of the output page for the ResFider 4.0 You have the summary of the output in the table over here. You have the antimicrobial, you have the antibiotic classes, you have whole-genome sequencing predicted phenotype, either resistance, or no resistance. If there's resistance, you will see the genetic background. That Mean you'll see what gene they're responsible for this resistance for example or sometimes is not a gene but is the point mutation. You will see what gene is responsible for this point mutation and what position. You see all this in this table as well. But if you want to see more detail of the result, you scroll down, you're going to see more detail of each of the resistant gene that found in your genome. For example, you have our gene here, you have percent identity. You have alignment length compared to the gene length. You see if green perfect match because the size of the alignment is equal to the size of the gene. What that mean, it means if this is the resistance gene, it means, your sequences are actually aligned from the first position to the last position of the gene that you can see here. When you have the equal between the alignment length and the gene length, and every alignment is all the match, you will have 100 percent identity. If those thing combine you can have perfect match. That mean you can trust this result completely. There are also another color that you can see over here is in gray. Is lower here, is the result of sul1 in the gene. It's gray because that person Identity is 100 percent. Every Match is Match, there's no mismatches. But the alignment length here is less than the size of the genes. You see the gene length is 840, the alignment length is 761. That means there are some part of the gene that missing from this. When you have something like this, that the alignment length is less than the size of the gene, but percent identity is 100 percent, or nearly 100 percent, it will you turn gray. Another color is in the light green color is mean. You have the alignment length is the same as resistance gene length. That means you align from the first position to the last position. But the some of the alignment, there are some mismatches. That's why the percent identity is not 100 percent. You will see it in the light green. When you see these two colors, it's just a warning that is not perfect Match, it's imperfect Match. You can consider if you want to include this result or not. But it doesn't mean that there is no genes at all in this color. It just warn you that is not a perfect match. In your scroll down below, you'll see more things that you can choose. You can get all the results that you see previously in the text and also in the tab spreadsheet, and in the result for the AMR in the text, the point mutation either in the table, in the text as well. You'll see hit in a genome sequences. You can get the output, or the sequences of your own sequence that match who the gene in the database. If you click here, this is the sequence from your genome that match to the gene in the database and of course, you can also get the sequences of the resistant gene that we found in your genome here. Anyway you have the option to click more extended output to see the alignment. You see if you have some mismatches. You can see it over here as well. You want to see the alignment detail. That's all for the ResFinder 4.0, and thank you for watching
