Skip the mapping step with Kallisto  

*Thanks to Anna Battenhouse to the text and figures!

Kallisto is a tool from the Pachter lab that performs quanitfication of transcripts without requiring alignment. 

• No explicit alignment to reference genome or transciptome 
• Instead, uses “pseudoalignment” to transcriptome 
  • For each read, determine not where in each transcript it aligns, but rather which transcripts it is compatible with 
  • Simultaneously addresses 2 aspects of “multi-mapping” reads in traditional RNAseq pipelines 
    • Multiple possible genomic loci (addressed during alignment) 
    • Multiple possible transcripts of origin (addressed during quantification) 
• Pseudoalignments are sufficient to quantify transcript abundances 
  • Expectation Maximization (EM) algorithm is applied to a “simple” RNAseq Likelihood function 
  • Report estimated abundances as Transcripts per Million (TPM) + counts 

No P-value reported or differential expression (DE) support, but… 

• kallisto re-runs EM on multiple bootstrap re-samples to estimate variance 
• then kallisto bootstraps are used by add-on sleuth DE package

Why Kallisto?

Speed and performance are greatly improved with Kallisto.