– Don’t count…quantify!
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•free, quick, little memory
•takes into account experimental attributes and biases typical of RNA-seq data, including
positional biases in coverage, sequence-specific biases at the 5′ and 3′ end of sequenced
fragments, fragment-level GC bias, strand-specific protocols, and the fragment length distribution
•input: set of target transcripts
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MAPPING-BASED MODE
•Mapping instead of alignment
•Salmon index
•Decoy-aware transcriptome
•Outputs .fs file
•
> ./bin/salmon index –t transcripts.fa –i transcripts_index –decoys decoys.txt –k 31
> ./bin/salmon quant -i transcripts_index -l <LIBTYPE> -1 reads1.fq -2 reads2.fq --validateMappings
-o transcripts_quant
> ./bin/salmon quant -i transcripts_index -l <LIBTYPE> -r reads.fq --validateMappings -o
transcripts_quant
Building index:
Paired-end reads:
Single-end reads:

MAPPING-BASED MODE
Multiple samples:
Zipped files:

•Pre-aligned data
•.bam files
•Outputs .fs file
ALIGMENT-BASED MODE
> ./bin/salmon quant -t transcripts.fa -l <LIBTYPE> -a aln.bam -o salmon_quant

LIBTYPE
•automatic library type detection in alignment-based mode
•argument -l A
•
1.the relative orientation of the reads (only if the library is paired-end)
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LIBTYPE
2.the strandedness of the library
3.
3.
3.
3. the directionality of the reads (only if the library is stranded)

LIBTYPE



EXAMPLES
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•alignment-based mode: 8-12 (4 threads for BAM decompression, rest for quantification) for maximum
speed
•mapping-based mode: more threads = faster quantification
•-p argument (default = maximum number of available threads)
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THREADS

OUTPUT
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•transcripts per kilobase million
•how to calculate:
1.divide the read counts by the length of each gene in kilobases. This gives you reads per kilobase
(RPK)
2.then count how many RPK values you have in a sample and divide this number by million to get your
“per million” scaling factor
3.and then divide each RPK value by the scaling factor and the result is TPM
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TPM

•https://combine-lab.github.io/salmon/
•https://salmon.readthedocs.io/en/latest/salmon.html
•https://nf-co.re/modules/salmon_quant#input
•https://sailfish.readthedocs.io/en/develop/salmon.html
•https://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/
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SOURCES