Document the exercise in your electronic lab notebook.
NOTE: text in quotes within commands must be changed based on your directory and naming of files.
1. Convert the wt and prg-1 alignment files generated by Bowtie in step 10 above to bam format (binary format) using
$ samtools view -bS -o 'output_file.bam' 'input_file.sam'
2. Sort and index Bam files:
$ samtools sort 'input_file.bam' -o 'output_file.sorted.bam'
$ samtools index 'input_file.sorted.bam'
3. Obtain small RNA read counts from each of several miRNA, siRNA, and piRNA loci using
$ samtools view -c 'input_file.sorted.bam' 'chr:start-end'
This is best accomplished by writing a bash script to get reads for each set of coordinates and could easily be expanded to capture every small RNA locus in the genome. A file containing the coordinates of several small RNAs,
smallRNA_Coordinates.xlsx, is in the
Copy the results into the Excel document and use
Excel for performing the normalization in step 4.
4. Normalize small RNA reads based on library size (use the value from bowtie, see step 10 from Exercise 10): divide the number of reads for each small RNA locus by the total number of mapped reads in millions in each library (reads per million total small RNA reads - RPM).
5. Plot the normalized data for each small RNA in
Submit your answer to the following questions on Canvas:
1. Which classes of small RNAs are depleted in prg-1 mutants?
2. Which small RNA pathway does prg-1 likely function in?