RNA-Seq pipeline

Converting Illumina run to FASTQ

• bcl2fastq

bcl2fastq 
	--runfolder-dir <path-to-nextseq-run>
	--output-dir <path-to-save-fastq-samples>
	--no-lane-splitting
	--loading-threads 8
	--demultiplexing-threads 8
	--writing-threads 8
	--sample-sheet /path/to/SampleSheet.csv

Aligning reads to genome

• STAR

START
	--runMode alignReads
	--runThreadN 8
	--genoeDir <path-to-genome-star-index>
	--readFilesIn <path-to-fastq-file>
	--readFilesCommand zcat
	--outStd Log
	--outSAMtype BAM SortedByCoordinate
	--outSAMstrandField intronMotif
	--outFilterIntronMotifs RemoveNoncanonical
	--alignSoftClipAtReferenceEnds No
	--outFilterScoreMinOverLread 0.25
	--outFilterMatchNminOverLread 0.25

Note: sometimes depend on your seq. library you will need to update the parameters to optimise the alignment. More parameters can be found in the manual.

Annotate alignments

• FeatureCounts

featureCounts
	-a <path-to-GTF-genome-annotation>
	-o <path-to-save-counts>/counts.txt
	-t exon
	-Q 255
	-T 8
	$(ls <path-to-BAM-file>/*.bam)

Differential expressions with R

• DESeq2
• EdgeR

Other useful tools

• samtools
• bedtools
• tabix