Manual
Typing kallisto
produces a list of usage options, which are:
kallisto 0.43.0
Usage: kallisto <CMD> [arguments] ..
Where <CMD> can be one of:
index Builds a kallisto index
quant Runs the quantification algorithm
pseudo Runs the pseudoalignment step
h5dump Converts HDF5-formatted results to plaintext
version Prints version information
cite Prints citation information
Running kallisto <CMD> without arguments prints usage information for <CMD>
The usage commands are:
index
kallisto index
builds an index from a FASTA formatted file of target sequences. The arguments for the index command are:
kallisto 0.43.0
Builds a kallisto index
Usage: kallisto index [arguments] FASTA-files
Required argument:
-i, --index=STRING Filename for the kallisto index to be constructed
Optional argument:
-k, --kmer-size=INT k-mer (odd) length (default: 31, max value: 31)
--make-unique Replace repeated target names with unique names
The Fasta file supplied can be either in plaintext or gzipped format.
quant
kallisto quant
runs the quantification algorithm. The arguments for the quant command are:
kallisto 0.43.0
Computes equivalence classes for reads and quantifies abundances
Usage: kallisto quant [arguments] FASTQ-files
Required arguments:
-i, --index=STRING Filename for the kallisto index to be used for
quantification
-o, --output-dir=STRING Directory to write output to
Optional arguments:
--bias Perform sequence based bias correction
-b, --bootstrap-samples=INT Number of bootstrap samples (default: 0)
--seed=INT Seed for the bootstrap sampling (default: 42)
--plaintext Output plaintext instead of HDF5
--single Quantify single-end reads
--fr-stranded Strand specific reads, first read forward
--rf-stranded Strand specific reads, first read reverse
-l, --fragment-length=DOUBLE Estimated average fragment length
-s, --sd=DOUBLE Estimated standard deviation of fragment length
(default: value is estimated from the input data)
-t, --threads=INT Number of threads to use (default: 1)
--pseudobam Output pseudoalignments in SAM format to stdout
kallisto can process either single-end or paired-end reads. The default running mode is paired-end and requires an even number of FASTQ files represented as pairs, e.g.
kallisto quant -i index -o output pairA_1.fastq pairA_2.fastq pairB_1.fastq pairB_2.fastq
For single-end mode you supply the --single
flag, as well as the -l
and -s
options, and list any number of FASTQ files, e.g
kallisto quant -i index -o output --single -l 200 -s 20 file1.fastq.gz file2.fastq.gz file3.fastq.gz
FASTQ files can be either plaintext or gzipped.
Important note: only supply one sample at a time to kallisto. The multiple FASTQ (pair) option is for users who have samples that span multiple FASTQ files.
In the case of single-end reads, the -l option must be used to specify the average fragment length. Typical Illumina libraries produce fragment lengths ranging from 180–200 bp but it’s best to determine this from a library quantification with an instrument such as an Agilent Bioanalyzer. For paired-end reads, the average fragment length can be directly estimated from the reads and the program will do so if -l is not used (this is the preferred run mode).
The number of bootstrap samples is specified using -b. Note that because of the large amount of data that may be produced when the number of bootstrap samples is high, kallisto outputs bootstrap results in HDF5 format. The h5dump
command can be used afterwards to convert this output to plaintext, however most convenient is to analyze bootstrap results with sleuth.
kallisto quant
produces three output files by default:
- abundances.h5 is a HDF5 binary file containing run info, abundance esimates, bootstrap estimates, and transcript length information length. This file can be read in by sleuth
- abundances.tsv is a plaintext file of the abundance estimates. It does
not contains bootstrap estimates. Please use the
--plaintext
mode to output plaintext abundance estimates. Alternatively,kallisto h5dump
can be used to output an HDF5 file to plaintext. The first line contains a header for each column, including estimated counts, TPM, effective length. - run_info.json is a json file containing information about the run
Optional arguments
-
--bias
learns parameters for a model of sequences specific bias and corrects the abundances accordlingly. -
-t, --threads
specifies the number of threads to be used both for pseudoalignment and running bootstrap. The default value is 1 thread, specifying more than the number of bootstraps or the number of cores on your machine has no additional effect. -
--fr-stranded
runs kallisto in strand specific mode, only fragments where the first read in the pair pseudoaligns to the forward strand of a transcript are processed. If a fragment pseudoaligns to multiple transcripts, only the transcripts that are consistent with the first read are kept. -
--rf-stranded
same as--fr-stranded
but the first read maps to the reverse strand of a transcript.
Pseudobam
--pseudobam
outputs all pseudoalignments in SAM format to the standard output. The stream can either be redirected into a file, or converted to bam using samtools
.
For example
kallisto quant -i index -o out --pseudobam r1.fastq r2.fastq > out.sam
or by piping directly into samtools
kallisto quant -i index -o out --pseudobam r1.fastq r2.fastq | samtools view -Sb - > out.bam
A detailed description of the SAM output is here.
pseudo
kallisto pseudo
runs only the pseudoalignment step and is meant for usage in single cell RNA-seq. The arguments for the pseudo command are:
kallisto 0.43.0
Computes equivalence classes for reads and quantifies abundances
Usage: kallisto pseudo [arguments] FASTQ-files
Required arguments:
-i, --index=STRING Filename for the kallisto index to be used for
pseudoalignment
-o, --output-dir=STRING Directory to write output to
Optional arguments:
-u --umi First file in pair is a UMI file
-b --batch=FILE Process files listed in FILE
--single Quantify single-end reads
-l, --fragment-length=DOUBLE Estimated average fragment length
-s, --sd=DOUBLE Estimated standard deviation of fragment length
(default: value is estimated from the input data)
-t, --threads=INT Number of threads to use (default: 1)
--pseudobam Output pseudoalignments in SAM format to stdout
The form of the command and the meaning of the parameters are identical to the quant command. However, pseudo does not run the EM-algorithm to quantify abundances. In addition the pseudo command has an option to specify many cells in a batch file, e.g.
kallisto pseudo -i index -o output -b batch.txt
which will read information about each cell in the batch.txt
file and process all cells simultaneously.
The format of the batch file is
#id file1 file 2
cell1 cell1_1.fastq.gz cell1_1.fastq.gz
cell2 cell2_1.fastq.gz cell2_1.fastq.gz
cell3 cell3_1.fastq.gz cell3_1.fastq.gz
...
where the first column is the id of the cell and the next two fields are the corresponding files containing the paired end reads. Any lines starting with #
are ignored. In the case of single end reads, specified with --single
, only one file should be specified per cell.
When the --umi
option is specified the batch file is of the form
#id umi-file file-1
cell1 cell_1.umi cell_1.fastq.gz
cell2 cell_2.umi cell_2.fastq.gz
cell3 cell_3.umi cell_3.fastq.gz
...
where the umi-file is a text file of the form
TTACACTGAC
CCACTCTATG
CAGGAAATCG
...
listing the Unique Molecular Identifier (UMI) for each read. The order of UMIs and reads in the fastq file must match. Even though the UMI data is single end we do not require or make use of the fragment length.
When run in UMI mode kallisto will use the sequenced reads to pseudoalign and find an equivalence class, but rather than count the number of reads for each equivalence class, kallisto counts the number of distinct UMIs that pseudoalign to each equivalence class.
h5dump
kallisto h5dump
converts
HDF5-formatted results to
plaintext. The arguments for the h5dump command are:
kallisto 0.43.0
Converts HDF5-formatted results to plaintext
Usage: kallisto h5dump [arguments] abundance.h5
Required argument:
-o, --output-dir=STRING Directory to write output to
version
kallisto version
displays the current version of the software.
cite
kallisto cite
displays the citation for the paper.