Research Project RNA UTR Isoforms

Purpose of the project is to identify 3’ UTR isoforms from 3’ RNA sequencing data Pipeline created 2021 summer through Stanford Summer Research Program in colllaboration with the Das Lab. Picture shows a flowchart of the pipeline process. 

See the "APA Analysis Research Project Handbook" word doc in this repo for even more information.

Installation

brew install samtools
brew install bedtools
brew install wget

Other dependencies: FastQC, CutAdapt

Download reference files

Download Fasta and GTF files. We use Rnor6 for rat.

Comands to generate bed file for input to python program

# Make Folder Containing Data:
mkdir APA analysis

# Build bowtie index for genome
# Input rat genome, output bowtie index
bowtie2-build ref/Rattus_norvegicus.Rnor_6.0.dna.toplevel.fa Rnor_6

# Align FastQ to genome using bowtie
# input 
bowtie2 -x ref/Rnor_6 -U fastq/Proces_cleaned.fastq -b aligned/projections.bam
bowtie2 -x ref/Rnor_6 -U fastq/Proces_cleaned.fastq -S aligned/projections.sam

bowtie2 -x ref/Rnor_6 -U fastq/Soma_cleaned.fastq -b aligned/soma.bam
bowtie2 -x ref/Rnor_6 -U fastq/Soma_cleaned.fastq -S aligned/soma.sam

# Convert SAM to BAM File using Samtools:
samtools view -S -b aligned/projections.sam > aligned/projections.bam

samtools view -S -b aligned/soma.sam > aligned/soma.bam

# Convert BAM to BED File using Bedtools:
bedtools bamtobed -i aligned/projections.bam > aligned/projections.bed

bedtools bamtobed -i aligned/soma.bam > aligned/soma.bed

# Sort BED file using Bedtools:
command: sortBed -i aligned/projections.bed > aligned/sortedprojections.bed

command: sortBed -i aligned/projections.bed > aligned/sortedsoma.bed

# Filter GTF file for stop codon annotations:
command: grep "stop_codon" ref/Rattus_norvegicus.Rnor_6.0.104.gtf > ref/rnor6_stopcodons.gtf

# Use [Bedtools](https://bedtools.readthedocs.io/en/latest/content/tools/closest.html) closest command to find closest upstream stop codon:
command: bedtools closest -D ref -fu -a aligned/sortedprojections.bed -b ref/rnor6_stopcodons_sorted.gtf > closest/stopcodonsprojections.bed

command: bedtools closest -D ref -fu -a aligned/sortedsoma.bed -b ref/rnor6_stopcodons_sorted.gtf > closest/stopcodonssoma.bed

# Sort GTF file:
command: sortBed -i ref/Rattus_norvegicus.Rnor_6.0.104.gtf > ref/rnor6sorted.gtf (bedtools accept gtf)

# Grep filtering stop codons for sorted:
grep "stop_codon" ref/rnor6sorted.gtf > ref/rnor6_stopcodons_sorted.gtf

Detecting and QUanitifying 3' UTR

Implemented in jupyter notebook (link)