kallisto | bustools

kallisto, bustools, and kb-python are free, open-source tools used together to perform fast, lightweight RNA-seq quantification and preprocessing. These tools support the analysis of both bulk and single-cell RNA-seq data.

kallisto performs pseudoalignment, a method that assigns reads to transcripts without fully aligning them. This enables extremely fast and accurate transcript quantification.

bustools processes barcode and UMI information and provides utilities for manipulating BUS—a compact representation of barcodes, UMIs, and transcript equivalence classes.

kb-python offers high-level workflows that automate common tasks using kallisto and bustools, including reference generation and the processing of both bulk and single-cell RNA-seq experiments. It automatically handles downloading and formatting reference transcriptomes, manages file organization, and ensures that kallisto and bustools are invoked with consistent parameters.

You may run kallisto and bustools directly, or use kb-python to streamline complete workflows. Many users benefit from the convenience and reproducibility provided by kb-python, especially for multi-step analyses.

Note

This is unofficial documentation that is under active development.

Overview:

• Introduction
• Background
• References
• Dependents

Quick Start:

• Installing kb-python
• The kb-python workflow

Index generation:

• Generate a reference index
  • Downloading a premade index
  • Making an index
  • Advanced

Bulk RNA seq:

• Pseudoalignment of bulk RNA seq data
  • Quantification
  • Differential transcript expression
  • Differential gene expression
• Tutorials
  • Bulk RNA-seq (with differential transcript expression)

Single-cell RNA seq:

• Pseudoalignment of single-cell RNA seq data
  • Single-cell RNA-seq quantification
  • Single-nucleus RNA-seq
  • Nascent and mature RNA quantification
  • Counting Multimapped Reads
  • Output Files
  • Batch file processing
• Technologies and the -x string
  • -x string
  • strandedness
  • parity
  • on-list
  • replacement list
• Tutorials
  • Introduction to single-cell RNA-seq: Seurat (R)
  • Introduction to single-cell RNA-seq: Scanpy (python)
  • Introduction to single-nucleus RNA-seq: Scanpy (python)
  • Nascent and Mature RNA quantification
  • SPLIT-Seq preprocessing

Long read RNA seq:

• Long read pseudoaligment
  • Long read pseudoalignment of bulk RNA-seq data
  • Long read pseudoalignment of single-cell RNA-seq data
• Tutorials
  • lr-kallisto single-nuclei data analysis tutorial

Translated alignment:

• Pseudoalignment of RNA seq data against a protein reference
• Tutorials
  • Viral proteins in bulk RNA seq data
  • Viral proteins in single-cell RNA seq data

seqspec:

• seqspec

Troubleshooting:

• Interpreting quality and run information

Manuals:

• kallisto
• bustools
• kb-python

Advanced installation:

• Installing from source
• Releases

Advanced bustools:

• Barcodes on-list format

FAQ:

• Frequently asked questions