File name Description Tcas5.2.fna.gz Tribolium castaneum reference genome (build 5.2) in fasta format. (X-chromosome and Autosomes only)

Tcas5.2.gff.gz Corresponding GFF file for the Tcas5.2 reference genome provided.

Control_R1.fastq.gz 3 Control replicates: R1, R2, R3 50bp single-end non-strand-specific reads

Control_R2.fastq.gz

Control_R3.fastq.gz

Primed_R1.fastq.gz 3 Primed replicates: R1, R2, R3 50bp single-end non-strand-specific reads

Primed_R2.fastq.gz
Primed_R3.fastq.gz

I have the following files and I need to return a. Provide a table listing all the genes that are significantly differentially expressed at adjusted p-value < 1e-10 and more than 4-fold up or down regulated (i.e. to be in the table it must meet both criteria)? The table should include columns for: 1) a unique gene identifier that can be looked up in NCBI databases (e.g. XM_001808014.3), 2) adjusted p-value, and 3) log2FC

b. Present a figure illustrating the differential expression between individuals and/or treatments. This could be as simple as the PCA plot from default DESeq2 output, but the possibilities are not limited to that. For example, you might learn how to do a volcano plot and include that.

c. For primed replicate #1 (Primed_R1.fastq.gz) how many SNVs show allele-specific expression bias? (define bias as SNV allele frequency > 0.7)

I know I have to use the RNA seq and The htseq .. Convert the GFF file to GTF file to do the htseq and then use the DESeq.

I want to know in what order should i do it and DO i need to use blast anywhere and what shoukd be the methods to solve this problem.