Next, we will be looking at the tool for variant calling, namely bcftools. bcftools usually operates on the output from the tool and up program and uses that information to make a call as to whether assembly position contains a little variation or not. So let's look at bcftools package and again I'm going to use bcftools one, because I have multiple ones on the system and these the most recent one. So bcftools common argument that's the usage line. And just like SAMtools, this is a suite of packages for manipulating VCF files including for variant coding. And this one include operations for indexing, for manipulating vcf and bcf files Reheader. An important one that we're going to look at more closely is view. And then we also have the very important part, the VCF/BCF analysis, and particularly the call function. So we'll be looking to start with, in the view command and the call command. To obtain more information about view comment, we simply type just like before bcftools one view. And this gives us the option for this particular package. There are a number of, so if you're looking at the usage its BCF tools view a number of options, a VCF which can be compressed or not and then a number of regions from which to do analysis only on a restricted region. There are a number of options that are in common to all of the BCF tools and most of them are listed here in the beginning. They refer to the input and output. For instance the output file the type of the output and therefore options here, Bis for compressed BCF, U is for uncompressed BCF, Z is for compressed VCF, and V for uncompressed VCF. So that's fairly important then dash r can specify the regions from the command and the dash capital R can allow the program to read the regions from a file. And so on. So let's try one more example, the view, the bcftools is used to convert from one format to another primarily. So in this scale you might recall that we have the sample BCF file. And we would like to see what's inside, because as you might remember, it is all in binary. So let's just bcftools1. View sample.bcf. And that transforms it into a form of CFR. You will see the quantity information so that's for every genomic sequence, then followed by an odd information, a presentation for the allele or alternate allele. A set of info lines. A format line and then one line for every position in the genome for which we have reads. Recall this was produced by empire so that's how we can use view in a very simple way. The second package from the VCF 2 suite that we are interested in is the actual genotype quantum pack. Package so that bcftools1 call. And let's look at the common like options for this package. Call.log So we're using just bcftools call, a number of options, followed by the bcf file in compressed form or not. And again, we have a number of options that refer to the input and output and which I mentioned are common to all the bcftool packages. Some additional ones here, and then some options we learned in the variant column algorithm. So one can specify one of two algorithms, a -c, the consensus caller. However, this method is now obsolete. And the other one is -m for multi [INAUDIBLE] and rare variant column. And this one is currently being used in most practical applications. So let's try now, using the [INAUDIBLE] output. With information on every location in the genome within lien base, and looking at that information and the summary to make variant calls using bcftools1. We're going to save bcftools1, with the option core. And then we have a number of comments like parameters, dash v would say then we should only write the license corresponding to fairness, dash m which says let's use the mortgage in any corner, the the mortgage and version. Dash o which says use these formula for the output, so l am going to use Z for bcf compressed, dash o to lower case to specify that the output should be put into sample.vcf.gzz and lastly sample.bcf. So that is the output that we produce from the package. So now we can look at a sample, the vcf.gz, and we can do so without having to unzip the file, so we can do it with z cap sample vcf.gz. And use information, so we have the preamble with information about the reference genome. We have the ALT template specifying the alternate alleles. The informants, which gives information about the algorithms that were used for variant coding. The format lines again, scores reproduced in variant coding. And then lastly we have one line for every variant that has been called. And if you're counting here you are going to see that there are eight variants so. Let me just do these. Just saying wrap dash v. So we're going to move all the lines that start with a double pound sign. And we're going to see, actually just a pound sign to remove the header as well and that's how many variants. And that's eight. Then we have represented here. Most of these are substitutions of snips, T to C, C to G, and so on. But then, we also have a deletion here and the more complex substitution. Then, you can see the qualities, no information about the filter, and then information about the depth 36 bs for the first variant, 22, 3, 1, 101 and so on. So this concludes our demonstration on how to use bcftools to produce variant cause. And next we are going to show how to put this together so that way we have a pipeline for a very simple pipeline for variant calling from a
