Mapping & quantification

Analyzing RNA-seq data with Salmon/STAR/RSEM

Strategies

Methods	Tools	Output Quantification	Total Time (hr)
Alignment-free	Salmon	Transcript-level (gene-level aggregation using tximport)	3.4
Alignment-free	Salmon +bootstraps	Transcript-level (gene-level aggregation using tximport)	16.0
Alignment-free	Kallisto +bootstraps	Transcript-level (gene-level aggregation using tximport)	6.1
Alignment-based	STAR	Gene-level (with --quantMode GeneCounts option)	14.8
Alignment-based	STAR+Salmon	Transcript-level (gene-level aggregation using tximport)	16.3 STAR: 14.8 Salmon: 1.5
Alignment-based	STAR+RSEM	Gene-level and Transcript-level	57.2 STAR: 14.8 RSEM: 42.3

CPU usage is limited to 6 threads and memory to 32 GB where applicable

LibType and strandedness

Tools	dUTP, NSR, NNSR	Ligation, Standard SOLiD	Standard Illumina
TopHat/Cufflinks	fr-firststrand	fr-secondstrand	fr-unstranded
HISAT2	--rna-strandness RF (Paired) --rna-strandness R (Single)	--rna-strandness FR (Paired) --rna-strandness F (Single)
Salmon	-l ISR (Paired) -l SR (Single)	-l ISF (Paired) -l SF (Single)	-l IU (Paired) -l U (Single)
Kallisto	--rf-stranded	--fr-stranded
RSEM	--strandedness reverse	--strandedness forward	--strandedness none
FeatureCounts	-s 2	-s 1	-s 0
HTSeq	--stranded reverse	--stranded yes	--stranded no