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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. Download the small RNA data from the montgomery lab server using
FileZilla. The path to the folder containing the data is
2. Change into the
3. There are two small RNA libraries: 1)
wt_fastq.txt.gz and 2)
prg-1_fastq.txt.gz. Examine the contents of the two files using
4. Decompress the files using
5. Examine the fastq files in
FastQC. How many reads are there in each library and what is the read length? Record this information in your lab notebook.
Trimmomatic to remove adapter sequences and reads shorter than 16 nt:
$ trimmomatic SE -phred33 'input_file' 'output_file' ILLUMINACLIP:/Users/bz360/Documents/Trimmomatic_Files/TruSeq-smallRNA.fa:2:30:10 MINLEN:16
What proportion of reads were dropped?
7. Examine the contents of the two original files and the two new files using
more. What is different between the trimmed and original files?
8. Examine the trimmed fastq files in FastQC. Now how many reads are there in each library and what is the read length? Record this information in your lab notebook.
9. Index the C. elegans genome for Bowtie2 (notice that a fasta formatted file containing the genome sequence is in the prg1 folder):
$ bowtie2-build 'genome_input_file.fa' 'genome_name'
10. Create a new folder within the prg1 directory called bowtie_cel using
mkdir. Move all the files related to the bowtie index and the fasta formatted genome sequence into the bowtie_cel folder using
$ mv c* bowtie_cel
11. Map reads from each adapter-trimmed library (wt and prg-1 from step 6) to the C. elegans genome using
Bowtie2. While waiting on bowtie, if you haven't done so already, examine the original and trimmed fastq files in
$ bowtie2 -x 'path_to_bowtie_index/prefix' -U 'fastq_file_name' -S 'strain.sam'
NOTE: record the total number of reads and total mapped reads for each library in your lab notebook.
> TOTAL READS TOTAL MAPPED READS
wt: prg-1: wt: prg-1:
Submit your answer to the following question on Canvas:
What proportion of reads in each library was mappable?