All the RNA-seq data used in this class were downloaded from NCBI Sequence Read Archive (SRA) database using wget. Note: the RNA-seq data and intermediate outputs are put in the folder of largedata.
cd glumipatula/
### Oryza glumipatula, leaf RNA-seq, PE, 101bp
wget get ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR117/SRR1174772/SRR1174772.sra
### Oryza glumipatula, panicle RNA-seq, PE, 101bp
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR117/SRR1174773/SRR1174773.sra
### Oryza glumipatula, root RNA-seq, PE, 101bp
wget ftp://ftp-trace.ncbi.nlm.nih.gov/sra/sra-instant/reads/ByRun/sra/SRR/SRR117/SRR1174777/SRR1174777.sra
### dump the SRA into fastq
fastq-dump --split-spot --split-3 -A SRR1174772.sra
fastq-dump --split-spot --split-3 -A SRR1174777.sraThe aim of this experiment is to compare the differentially expressed genes in two different tissue type: leaf vs. root. One million reads were extracted for each rep.
cd largedata/
### checking the total number of reads
wc -l glumipatula/SRR1174772.sra_1.fastq
#660468896 SRR1170742.sra_1.fastq
### split the fq files into three files as arbitrarily assigned replications
cat glumipatula/SRR1174772.sra_1.fastq | awk 'NR >= 4000001 && NR <= 8000000 { print }' > leaf/leaf.rep1_1.fastq
cat glumipatula/SRR1174772.sra_2.fastq | awk 'NR >= 4000001 && NR <= 8000000 { print }' > leaf/leaf.rep1_2.fastq
cat glumipatula/SRR1174772.sra_1.fastq | awk 'NR >= 14000001 && NR <= 18000000 { print }' > leaf/leaf.rep2_1.fastq
cat glumipatula/SRR1174772.sra_2.fastq | awk 'NR >= 14000001 && NR <= 18000000 { print }' > leaf/leaf.rep2_2.fastq
cat glumipatula/SRR1174772.sra_1.fastq | awk 'NR >= 24000001 && NR <= 28000000 { print }' > leaf/leaf.rep3_1.fastq
cat glumipatula/SRR1174772.sra_2.fastq | awk 'NR >= 24000001 && NR <= 28000000 { print }' > leaf/leaf.rep3_2.fastq
wc -l glumipatula/SRR1174777.sra_1.fastq
#665118764 SRR1174777.sra_1.fastq
cat glumipatula/SRR1174777.sra_1.fastq | awk 'NR >= 4000001 && NR <= 8000000 { print }' > root/root.rep1_1.fastq
cat glumipatula/SRR1174777.sra_2.fastq | awk 'NR >= 4000001 && NR <= 8000000 { print }' > root/root.rep1_2.fastq
cat glumipatula/SRR1174777.sra_1.fastq | awk 'NR >= 14000001 && NR <= 18000000 { print }' > root/root.rep2_1.fastq
cat glumipatula/SRR1174777.sra_2.fastq | awk 'NR >= 14000001 && NR <= 18000000 { print }' > root/root.rep2_2.fastq
cat glumipatula/SRR1174777.sra_1.fastq | awk 'NR >= 24000001 && NR <= 28000000 { print }' > root/root.rep3_1.fastq
cat glumipatula/SRR1174777.sra_2.fastq | awk 'NR >= 24000001 && NR <= 28000000 { print }' > root/root.rep3_2.fastqOpen vi, specify the relative location and names of the fastq files. And, the replications and treatments of your experimental design.
### copy the following text into your sample.txt file.
fq1 fq2 rep tissue
leaf/leaf.rep1_1.fastq leaf/leaf.rep1_2.fastq rep1 leaf
leaf/leaf.rep2_1.fastq leaf/leaf.rep2_2.fastq rep2 leaf
leaf/leaf.rep3_1.fastq leaf/leaf.rep3_2.fastq rep3 leaf
root/root.rep1_1.fastq root/root.rep1_2.fastq rep1 root
root/root.rep2_1.fastq root/root.rep2_2.fastq rep2 root
root/root.rep3_1.fastq root/root.rep3_2.fastq rep3 rootTwo reference genomes for rice are available. Oryza sativa indica is one of the them. The genome assembly and gene annotation can be downloaded from EnsemblPlants.
mkdir OS_indica
cd OS_indica
wget ftp://ftp.ensemblgenomes.org/pub/plants/release-25/fasta/oryza_indica/dna/Oryza_indica.ASM465v1.25.dna.chromosome.*.fa.gzGSNAP (Genomic Short-read Nucleotide Alignment Program) is a fast and splice-aware aligner. You can download and install it from here.
### load the module of gmap if you use farm
module load gmap/2014-05-15
### http://research-pub.gene.com/gmap/src/README
### Setting up to build a GMAP/GSNAP database (one chromosome per FASTA entry)
### Note: be careful about your dir specified by -D
gmap_build -D largedata/OS_indica/ -d ASM465v1.25_gsnap -g Oryza_indica.ASM465v1.25.dna.chromosome*fa.gz > gmapbuild.log