Case Study: You have been tasked by your PI to perform a secondary analysis on a Bacillus thuringiensis BMB$171,$ bacterial sample. In addition to the mapping metrics you returned to them in Homework $3($chr$1.$bam$),$ they would also like to know what variants are present in their sample. If you have not done so already, sort and index the BAM file from Homework $3.$ Please provide screenshots of your steps. Proceed with the following steps and respond to the questions as follows:

$1(0$ pts$)$ Install the following packages using "sudo apt $".$ Each package will require it's own command line. Packages: bcftools, default$-$jre, samtools $($if you have not done so already$).$

sudo apt install bcftools

sudo apt install default$-$jre

sudo apt install samtools

$2(4$ pts$)$ Using NCBI, download the FASTA and GFF sequence for Bacillus thuringiensis BMB$171($NC$\_014171)$ and upload it to your virtual lab space. Using samtools, index your FASTA file and attach it and it's corresponding index as part of your response deliverables.

$3(4$ pts$)$Create a variant call file using the following command. How many variants total are there? What was the command you used to determine that? Print the first $3$ variants.

bcftools mpileup $-$f REFERENCE.fasta BAM$\_$Name.bam $|$ bcftools call $-$mv $-$Ob $-$o STUDENT$\_$NAME.variants.bcf

bcftools view $-$Ov $-$o STUDENT$\_$NAME.variants.vcf STUDENT$\_$NAME.variants.bcf

$4(4$ pts$)$ Inspect the header of your newly created vcf file. In addition to the definition of header fields, what are the key fields and their subsequent definitions and data types? Why would this information be useful to have?

$5(8$ pts$)$ Using the newly generated VCF file and the gene annotation file you downloaded $($GFF$)$ do the following for the gene gyrB:

a$)$ Where is this gene in Bacillus thuringiensis?

b$)$ Count all variants that would map back to the gene gyrB$.$ Return this number and the command used to find it$.$

c$)$ Using a relational database, list two other species that contain this gene name. List the database you used and how you conducted your search.