Lab 07: Pseudocounts

Pseudocounting with Salmon

Salmon documentation

Required R packages

These packages aren't required for running Salmon, but it would be good to try and install them before we get to the downstream steps.

install.packages("tidyverse")
install.packages("pheatmap")
BiocManager::install("tximport")
BiocManager::install("GenomicFeatures")


library(tidyverse)
library(tximport)
library(GenomicFeatures)
library(pheatmap)

Prepare a directory for pseudocounting:

mkdir 07_pseudocount
cd 07_pseudocount
ln -s /pickett_shared/teaching/RNASeq_workshop/final_outputs/02_trimming/cut_SRR* .
ln -s /pickett_shared/teaching/RNASeq_workshop/raw_data/reference/Athaliana_447_Araport11.cds.fa .

First we must create a Salmon index of our transcripts:

Note: For the salmon commands, we're going to be calling the salmon binary directly, which means we don't have to load the package using module or any other package manager. The file we'll be using is located at /pickett_shared/software/salmon-latest_linux_x86_64/bin/.

(~1 minute to run)

/pickett_shared/software/salmon-latest_linux_x86_64/bin/salmon index -t Athaliana_447_Araport11.cds.fa -i salmon_index

Now we can run the actual counting step on a single sample:

(~2 minutes to run)

mkdir salmon_results
/pickett_shared/software/salmon-latest_linux_x86_64/bin/salmon quant \
  -i salmon_index \
  -l A \
  -1 cut_SRR17062759_1.fastq.gz \
  -2 cut_SRR17062759_2.fastq.gz \
  -p 2 \
  --validateMappings \
  -o salmon_results/SRR17062759_quant

Run a loop to count all samples:

ln -s /pickett_shared/teaching/RNASeq_workshop/final_outputs/02_trimming/cut_SRR170627* .
for i in cut_SRR170627*_1.fastq.gz ; do
  base=${i%%_1.fastq.gz}
  base=${base#cut_}
  /pickett_shared/software/salmon-latest_linux_x86_64/bin/salmon quant \
  -i salmon_index \
  -l A \
  -1 cut_${base}_1.fastq.gz \
  -2 cut_${base}_2.fastq.gz \
  -p 2 \
  --validateMappings \
  -o salmon_results/${base}_quant
done