1.Software Download

1).fastq-dump: convert sra file to fastq file.

 website:http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software 

2).bowtie:an ultrafast and memory efficient tool for aligning sequencing reads to long reference sequences.

 website:http://bowtie-bio.sourceforge.net/bowtie2/index.shtml 

3).cufflinks:assembles transcripts, estimates their abundances, and tests for differential expression and regulation in RNA-Seq samples.

 website:http://cufflinks.cbcb.umd.edu/ 

4).gffread: convert gff3 file to gtf file.

 website:http://cufflinks.cbcb.umd.edu/ (This program is included with cufflinks package)

2. Operation

1) Download genome.fa and genes.gff3 file from genome website; Download sra file from NCBI

2) Format conversion

 $ fastq-dump -I --split-files SRR123456789.sra # convert sra file to fastq file

 $ gffread -E genes.gff3 -o genes.gtf # convert gff3 file to gtf file

3) Index files

 $bowtie2-build genome.fa genome

4) Alignment

 $bowtie2 -x genome -1 SRR123456789_1.fastq -2 SRR123456789_2.fastq -S SRR123456789.sam

 $samtools view -bS SRR123456789.sam > SRR123456789.bam

 $samtools sort SRR123456789.bam SRR123456789

5) FPKM values

 $cufflinks SRR123456789.bam -G genes.gtf -o result

After these operations, we can extract FPKM values from genes.frkm_tracking file based on gene ID.