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Read Quantification

Learning objectives:

  • Learn mapping and differential gene expression analysis of rna-seq data

  • Interpret rna-seq analysis results

Make a new working directory and link the trimmed reads and assembly

We will be using the same trimmed fastq data as before (Tulin et al., 2013).

The following commands will create a new folder rnaseq and link the data in:

cd ..
mkdir -p rnaseq
cd rnaseq

ln -s ../quality/0Hour*.qc.fq.gz .
ln -s ../quality/6Hour*.qc.fq.gz .

ln -s /opt/rnaseq/assembly/nema_trinity/Trinity.fasta .

Note that this is a previously-assembly Trinity.fasta file that the whole class will use. Why is this important?

Index the assembly:

salmon index --index nema --type quasi --transcripts Trinity.fasta

Run salmon on all the samples:

  • What are the flags used in the salmon command?
  • Read up on libtype, here.
for R1 in *R1*.qc.fq.gz
do
  sample=$(basename $R1 extract.qc.fq.gz)
  echo sample is $sample, R1 is $R1
  R2=${R1/R1/R2}
  echo R2 is $R2
  salmon quant -i nema -p 2 -l IU -1 <(gunzip -c $R1) -2 <(gunzip -c $R2) -o ${sample}.quant
done

Take a look at quant output

head 0Hour_ATCACG_L002_R1_001.qc.fq.gz.quant/quant.sf

Look at all the mapping rates:

find . -name \salmon_quant.log -exec grep -H "Mapping rate" {} \;

More reading