STAR

rna-seq

To resolve geneInfo.tab error, install pre-release version.

FASTA and GTF files

We use FASTA for alignment and GTF for quantification.

A FASTA file contains a reference genome or a reference transcriptome.

A GTF (Gene Transfer Format) is a tab-delimited text file that holds information about gene structure and annotations on a genome

• each row describes one specific feature
• key columns:
  • seqname, source, feature (gene, exon or CDS for coding sequence)
  • start and end of the feature on the chromosome
  • strand (forward + or reverse -)
  • attribute is a list of key-value pairs, giving extra information like gene_id and transcript_id

Command

STAR genome index

	STAR --runThreadN 8 \ 
		--runMode genomeGenerate \ 
		--genomeDir genome_dir \ 
		--genomeFastaFiles genome_dir/Saccharomyces_cerevisiae.fa \ 
		--sjdbGTFfile genome_dir/Saccharomyces_cerevisiae.gtf \ 
		--sjdbOverhang 99

Alignment

 
STAR --runThreadN 8 \ 
	--genomeDir genome_dir \ 
	--readFilesIn "$TRIMMED_READ1" "$TRIMMED_READ2" \ 
	--readFilesCommand gunzip -c \ 
	--outFileNamePrefix "$OUTPUT_SAM_PREFIX" \ 
	--outSAMtype BAM SortedByCoordinate \ 
	--outSAMunmapped Within \ 
	--outFilterType BySJout \ --outFilterMismatchNmax 999 \ --outFilterMismatchNoverLmax 0.04 \ --outFilterMultimapNmax 20 \ --alignIntronMin 20 \ --alignIntronMax 1000000 \ --alignMatesGapMax 1000000 \ --alignSJoverhangMin 8 \ --alignSJDBoverhangMin 1 \ --quantMode GeneCounts \ --sjdbScore 1 \ --limitBAMsortRAM 8000000000
 
 

_.final.out

NOTE

Bad example: reads that we are trying to align are twice longer than needed.

• Average input read length | 195
• Uniquely mapped reads number | 5
• Average mapped length | 96.40
• Number of reads unmapped: too short | 701512

head -n 4 SRR1553531_1.fastq: check whether the length of reads matches.

BAM files

echo "Indexing BAM file..." samtools index SRR1553531_Aligned.sortedByCoordinate.bam

head SRR1553531_ReadsPerGene.out.tab
wc -l SRR1553531_ReadsPerGene.out.tab # Count number of genes

Binary Alignment Map is a compressed, binary version of a SAM file (Sequence Alignment/Map).