Manual
Compiling
git clone https://github.com/pachterlab/kallisto
cd kallisto
mkdir build
cd build
cmake ..
make
make install
If you don’t want to install kallisto system-wide and only want the executable binary, don’t run make install – make will already have generated the binary and have put it at: kallisto/build/src/kallisto
kallisto
Typing kallisto produces a list of usage options, which are:
kallisto 0.50.0
Usage: kallisto <CMD> [arguments] ..
Where <CMD> can be one of:
index Builds a kallisto index
quant Runs the quantification algorithm
quant-tcc Runs quantification on transcript-compatibility counts
bus Generate BUS files for single-cell data
h5dump Converts HDF5-formatted results to plaintext
inspect Inspects and gives information about an index
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 0.50.0
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)
-d, --d-list=STRING Path to a FASTA-file containing sequences to mask from quantification
--make-unique Replace repeated target names with unique names
--aa Generate index from a FASTA-file containing amino acid sequences
--distinguish Generate index where sequences are distinguished by the sequence name
-t, --threads=INT Number of threads to use (default: 1)
-m, --min-size=INT Length of minimizers (default: automatically chosen)
-e, --ec-max-size=INT Maximum number of targets in an equivalence class (default: no maximum)
The Fasta file supplied can be either in plaintext or gzipped format. Note: Do not supply the genome Fasta file; the Fasta file must be a transcriptome Fasta.
quant
kallisto quant runs the quantification algorithm. The arguments for the quant command are:
kallisto 0.50.0
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:
-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
--single-overhang Include reads where unobserved rest of fragment is
predicted to lie outside a transcript
--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: -l, -s values are estimated from paired
end data, but are required when using --single)
-t, --threads=INT Number of threads to use (default: 1)
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). For reads that are produced by 3’-end sequencing, the --single-overhang option does not discard reads where the expected fragment size goes beyond the transcript start.
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:
- abundance.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
- abundance.tsv is a plaintext file of the abundance estimates. It does
not contains bootstrap estimates. Please use the
--plaintextmode to output plaintext abundance estimates. Alternatively,kallisto h5dumpcan 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
-
-t, --threadsspecifies 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-strandedruns 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-strandedsame as--fr-strandedbut the first read maps to the reverse strand of a transcript.
Legacy options:
These options are only supported in kallisto versions before 0.50.0. To use them install an older version of kallisto (recommended: 0.48.0).
kallisto quant:
-
--biaslearns parameters for a model of sequences specific bias and corrects the abundances accordlingly. -
--fusiondoes normal quantification, but additionally looks for reads that do not pseudoalign because they are potentially from fusion genes. All output is written to the filefusion.txtin the output folder.
--pseudobam outputs all pseudoalignments to a file pseudoalignments.bam in the output directory. This BAM file contains the pseudoalignments in BAM format, ordered by reads so that each pseudoalignment of a read is adjacent in the BAM file.
--genomebam constructs the pseudoalignments to the transcriptome, but projects the transcript alignments to genome coordinates, resulting in split-read alignments. When the --genomebam option is supplied at GTF file must be given with the --gtf option. The GTF file, which can be plain text or gzipped, translates transcripts into genomic coordinates. We recommend downloading a the cdna FASTA files and GTF files from the same data source. The --chromosomes option can provide a length of the genomic chromosomes, this option is not neccessary, but gives a more consistent BAM header, some programs may require this for downstream analysis. kallisto does not require the genome sequence to do pseudoalignment, but downstream tools such as genome browsers will probably need it.
A detailed description of the SAM output is here.
quant-tcc
kallisto quant runs the EM algorithm to produce estimated counts from a transcript-compatibility-counts matrix file (which is in a MatrixMarket format where each column is an equivalence class and each row is a sample). The necessary files can all be generated from bustools count in bustools.
kallisto 0.50.0
Generates BUS files for single-cell sequencing
Usage: kallisto bus [arguments] FASTQ-files
Required arguments:
-o, --output-dir=STRING Directory to write output to
Optional arguments:
-i, --index=STRING Filename for the kallisto index to be used
(required if file with names of transcripts not supplied)
-T, --txnames=STRING File with names of transcripts
(required if index file not supplied)
-e, --ec-file=FILE File containing equivalence classes
(default: equivalence classes are taken from the index)
-f, --fragment-file=FILE File containing fragment length distribution
(default: effective length normalization is not performed)
--long Use version of EM for long reads
-p, --platform=STRING [PacBio or ONT] used for sequencing
-l, --fragment-length=DOUBLE Estimated average fragment length
-s, --sd=DOUBLE Estimated standard deviation of fragment length
(note: -l, -s values only should be supplied when
effective length normalization needs to be performed
but --fragment-file is not specified)
-t, --threads=INT Number of threads to use (default: 1)
-g, --genemap File for mapping transcripts to genes
(required for obtaining gene-level abundances)
-G, --gtf=FILE GTF file for transcriptome information
(can be used instead of --genemap for obtaining gene-level abundances)
-b, --bootstrap-samples=INT Number of bootstrap samples (default: 0)
--matrix-to-files Reorganize matrix output into abundance tsv files
--matrix-to-directories Reorganize matrix output into abundance tsv files across multiple directories
--seed=INT Seed for the bootstrap sampling (default: 42)
--plaintext Output plaintext only, not HDF5
bus
kallisto bus works with raw FASTQ files for single-cell RNA-Seq datasets. For each read the cell barcode and UMI information and the equivalence class resulting from pseudoalignment are stored in a BUS file output.bus stored in the output directory directory, along with matrix.ec and transcripts.txt which store information about the equivalence classes and transcript names for downstream processing.
kallisto 0.50.0
Generates BUS files for single-cell sequencing
Usage: kallisto bus [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
-x, --technology=STRING Single-cell technology used
Optional arguments:
-l, --list List all single-cell technologies supported
-B, --batch=FILE Process files listed in FILE
-t, --threads=INT Number of threads to use (default: 1)
-b, --bam Input file is a BAM file
-n, --num Output number of read in flag column (incompatible with --bam)
-N, --numReads Maximum number of reads to process from supplied input
-T, --tag=STRING 5′ tag sequence to identify UMI reads for certain technologies
--fr-stranded Strand specific reads for UMI-tagged reads, first read forward
--rf-stranded Strand specific reads for UMI-tagged reads, first read reverse
--unstranded Treat all read as non-strand-specific
--paired Treat reads as paired
--long Treat reads as long
--aa Align to index generated from a FASTA-file containing amino acid sequences
--inleaved Specifies that input is an interleaved FASTQ file
--batch-barcodes Records both batch and extracted barcode in BUS file
--verbose Print out progress information every 1M proccessed reads
To process the output.bus file further use bustools; examples of downstream processing can be seen in dataset specific notebooks available from the bustools repository.
Running kallisto bus -l gives a list of currently supported single cell technologies
List of supported single-cell technologies
short name description
---------- -----------
10xv1 10x version 1 chemistry
10xv2 10x version 2 chemistry
10xv3 10x version 3 chemistry
Bulk Bulk RNA-seq
SmartSeq2 Smart-seq2
BDWTA BD Rhapsody WTA
CELSeq CEL-Seq
CELSeq2 CEL-Seq version 2
DropSeq DropSeq
inDropsv1 inDrops version 1 chemistry
inDropsv2 inDrops version 2 chemistry
inDropsv3 inDrops version 3 chemistry
SCRBSeq SCRB-Seq
SmartSeq3 Smart-seq3
SPLiT-seq SPLiT-seq
SureCell SureCell for ddSEQ
Visium 10x Visium Spatial Transcriptomics
When specifying the input the short name can be used to indicate the technology.
Additionally kallisto bus will accept a string specifying a new technology in the format of bc:umi:seq where each of bc,umi and seq are a triplet of integers separated by a comma, denoting the file index, start and stop of the sequence used. For example to specify the 10xV2 technology we would use 0,0,16:0,16,26:1,0,0. The first part bc is 0,0,16 indicating it is in the 0-th file (also known as the first file in plain english), the barcode starts at the 0-th bp and ends at the 16-th bp in the sequence (i.e. 16bp barcode), the UMI is similarly in the same file, right after the barcode in position 16-26 (a 10bp UMI), finally the sequence is in a separate file, starts at 0 and ends at 0 (in this case stopping at 0 means there is no limit, we use the entire sequence).
Batch files
Using the –batch option in kallisto bus allows you to supply a list of fastq files in a separate text file (each line represents a unique sample or cell).
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
...
h5dump
kallisto h5dump converts
HDF5-formatted results to
plaintext. The arguments for the h5dump command are:
kallisto 0.46.0
Converts HDF5-formatted results to plaintext
Usage: kallisto h5dump [arguments] abundance.h5
Required argument:
-o, --output-dir=STRING Directory to write output to
inspect
kallisto inspect can output the Target de Bruijn Graph in the index in two ways, as a file in GFA format or it can map the contigs of the graph and and equivalence classes in a BED format that can be visualized using IGV
kallisto 0.50.0
Usage: kallisto inspect INDEX-file
Optional arguments:
-t, --threads=INT Number of threads
version
kallisto version displays the current version of the software.
cite
kallisto cite displays the citation for the paper.