Week 5- One Step Backwards, Two Step Forwards [Re-running STAR Alignment and HTSeq]
Arnab M -
Hi guys, It’s Arnab and this is my fifth weekly update on my senior research project: Exploring the Genomic Effects of PNPLA7 Mutations on Cerebral Palsy through RNA Sequencing.
As expected from the start of this project there would be setbacks, trials, and tribulations; however, no obstacle should ever separate you from your goals especially when fighting for a cause as novel as children battling neurological movement disorders.
Previously, I anticipated running DESeq2 after our HTSeq-Counts outputs were finished processing, but when I opened the TSV file (a text-based file containing HTSeq-Counts outputs data) there were only columns of zeroes present. Baffled, I discussed with my advisor what might have been the root of our problems. We tracked back to our HTSeq-Counts code but found nothing. It wasn’t until we tracked back into STAR Alignment that we discovered the problem: I had mistakenly only added half our data files, coding a tediously long and inefficient line of code. With this newfound discovery, I quickly coded a for-loop in Shell Script so I wouldn’t have to individually list out every single directory pathway file like I previously did, leading to my error. The for-loop performed exceptionally as we ran it, and it let us place those output files for a test run in HTSeq-Counts, and when that was successful we let the HTSeq-Counts code run on every single data file (as shown in the attached image). To give you some insight into how long some of this code takes, our single HTSeq-Counts test run file was timed at 1 hour and 2 minutes, meaning processing the entire data we have will take around 31 hours.
As for some insight into my lab dynamics with the PhDs and Postdocs, previously in a lab meeting, we discussed the future of sequencing with PacBio’s HiFi Sequencing Machine and the future of DNA and RNA Sequencing. It was a very insightful and indelible experience that deepened my relationship with these incredible researchers. It furthermore reminds me how big and rapidly evolving this field of Bioinformatics is and why I wanted to join in the first place. See you next week!
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