Day 1 - Breakout 02

Aligning All Samples Exercise (Breakout):

15 Minutes

We just learned about how to use RSEM & STAR, but now we need to align all of the rest of our samples to the reference genome. We’ll use the concepts we’ve learned earlier in this breakout exercise.

Instructions:

• One group member should share their screen in the breakout room. If nobody volunteers, a helper may randomly select someone.
• The group members should discuss the exercise and work together to find a solution.
• After a solution is found, allow time for all members to complete the exercise.

• Review what we learned about running RSEM + STAR, to construct an appropriate command for aligning one of our samples.
• Use a bash variable in our alignment command to quickly and easily align samples 2-4.
• View the output, and verify that we have the files we need.

Solution - Aligning All Samples Exercise

One solution is to define a bash variable for the sample, use that variable in the alignment command, and then redefine the variable before repeating the command for each change .

# Define a variable $SAMPLE
SAMPLE=SRR7777896
rsem-calculate-expression --star --num-threads 1 --star-gzipped-read-file \
--star-output-genome-bam --keep-intermediate-files \
out_trimmed/${SAMPLE}_R1.trimmed.fastq.gz \
../refs/GRCm38.102.chr19reduced \
out_rsem/${SAMPLE}
SAMPLE=SRR7777897
# Use the up arrow key to repeat the same command as above with the variable reassigned to sample_03
SAMPLE=SRR7777898
# Use the up arrow key to repeat the same command as above with the variable reassigned to sample_04
SAMPLE=SRR7777899
# Use the up arrow key to repeat the same command as above with the variable reassigned to sample_04
SAMPLE=SRR7777900
# Use the up arrow key to repeat the same command as above with the variable reassigned to sample_04

Another solution is to create a for-loop with our bash variable and alignment command. E.g.

for SAMPLE in SRR7777896 SRR7777897 SRR7777898 SRR7777899 SRR7777900
do
    rsem-calculate-expression --star --num-threads 1 --star-gzipped-read-file \
    --star-output-genome-bam --keep-intermediate-files \
    out_trimmed/${SAMPLE}_R1.trimmed.fastq.gz \
    ../refs/GRCm38.102.chr19reduced \
    out_rsem/${SAMPLE}
done

Helper Hint: If suggesting a for-loop approach, it can be helpful to build up a “dry-run” command as a test case, to get learners to be more cognizant of what their code will do. Echoing filenames first might be a good suggestion.