Kallisto Tutorial

Overview of Kallisto

Kallisto is a bioinformatics tool designed for the fast and efficient quantification of transcript abundances from RNA-Seq data. It utilizes a novel algorithm for pseudo-alignment that allows it to process data much more rapidly than traditional alignment-based methods. The core idea behind Kallisto is to create an index of the transcriptome and then use this index to quickly determine the compatibility of reads with the transcripts, without the need for full sequence alignment.

The advantage of using Kallisto lies in its speed and accuracy. It can quantify 30 million human reads in less than 3 minutes on a standard desktop computer, which is significantly faster than many other RNA-Seq quantification tools. Moreover, Kallisto's pseudo-alignment approach reduces the computational resources required, making it possible to run analyses on less powerful machines, such as laptops.

Kallisto was developed by Nicolas L Bray, Harold Pimentel, P√°ll Melsted, and Lior Pachter and was first introduced in a publication in Nature Biotechnology in 2016. Since its release, Kallisto has become a popular choice for researchers needing to quickly and accurately quantify transcript abundances in RNA-Seq experiments.


To install Kallisto, you will need to have a compatible operating system such as Windows, Linux, or Mac OSX. The installation process is straightforward:

  1. Download the precompiled binaries for your operating system from the Kallisto GitHub repository.
  2. Extract the downloaded archive to a directory of your choice.
  3. Add the directory containing the Kallisto executable to your system's PATH environment variable to allow it to be run from any location.

Alternatively, if you prefer to compile Kallisto from source, you can clone the repository from GitHub and follow the compilation instructions provided in the README file. This approach may be necessary if you are using an operating system for which precompiled binaries are not available or if you wish to customize the build.

Quick Start

To get started with Kallisto, you need to perform two main steps: building an index of the transcriptome and quantifying the abundances of transcripts.

Building the Index

Before you can quantify transcript abundances, you must first build an index from a FASTA file containing the transcript sequences. This is done using the kallisto index command:

kallisto index -i transcriptome.idx transcripts.fasta.gz

This command will create an index file (transcriptome.idx) that Kallisto will use for pseudo-alignment.

Quantifying Transcript Abundances

Once the index is built, you can quantify transcript abundances from your RNA-Seq reads using the kallisto quant command:

kallisto quant -i transcriptome.idx -o output_dir -b 100 reads_1.fastq.gz reads_2.fastq.gz

This command will output the quantification results to the specified output directory (output_dir). The -b 100 option tells Kallisto to perform 100 bootstrap samples, which can be used for downstream analysis of quantification uncertainty.

Code Examples Of Popular Commands

Here are five popular commands that you can use with Kallisto:

1. Building an Index

kallisto index -i transcriptome.idx transcripts.fasta.gz

2. Quantifying Transcript Abundances with Single-End Reads

kallisto quant -i transcriptome.idx -o output_dir --single -l 200 -s 20 reads.fastq.gz

In this example, --single indicates that the reads are single-end, -l 200 specifies the average read length, and -s 20 specifies the standard deviation of read length.

3. Quantifying Transcript Abundances with Paired-End Reads

kallisto quant -i transcriptome.idx -o output_dir -b 100 reads_1.fastq.gz reads_2.fastq.gz

4. Estimating Transcript Abundances without Bootstrapping

kallisto quant -i transcriptome.idx -o output_dir --plaintext reads_1.fastq.gz reads_2.fastq.gz

The --plaintext option outputs the results in plaintext format, and bootstrapping is skipped by default if the -b option is not provided.

5. Using Kallisto with Single-Cell RNA-Seq Data

kallisto bus -i transcriptome.idx -o output_dir -x technology reads_1.fastq.gz reads_2.fastq.gz

The kallisto bus command is used for generating BUS files for single-cell RNA-Seq data, where -x technology specifies the single-cell technology used (e.g., 10xv2, 10xv3).

Kallisto is a powerful and user-friendly tool that has made RNA-Seq data analysis more accessible and efficient. Its speed and low memory requirements, combined with its accuracy, make it an excellent choice for transcript quantification in a wide range of research applications.