Installing Kallisto on a Mac OS
If you’re running a Mac OS, then begin by downloading and installing Homebrew with this single line of code:
/usr/bin/ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)"
Now, installing Kallisto is simple:
brew tap homebrew/core brew install kallisto
Test whether Kallisto is properly installed by typing
kallisto, and you should see this output
kallisto 0.44.0 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 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>
Installing Kallisto on a Windows OS
- Obtain administrative access for your computer
- Go to https://pachterlab.github.io/kallisto/download
- Download the latest Windows release (v0.44 for Fall 2018)
- Extract to Program Files or Applications. Kallisto is now installed on your computer but it cannot be accessed from any location in the command prompt (Windows equivalent of Terminal) until you add it to your computer’s path system variable
- Add the kallisto directory to your PATH to allow for access from any directory
- Start the System Control Panel applet (Start -> Settings -> Control Panel -> System)
- Select the Advanced tab.
- Click the Environment Variables button.
- Under System Variables, select Path, then click Edit. Note: You’ll see a list of folders, as this example for my system shows: C:\Program Files\Windows Resource Kits\Tools\; etc
- Add the name of the path to your kallisto directory (e.g. including semicolon ;C:\Program Files\kallisto_windows-v0.43.1
- Relaunch command prompt and check for proper installation by typing kallisto
- although not required for running kallisto, you should install Cygwin to give your PC some linux functionality (like running shell scripts)
Build an index from reference transcriptome .fasta file
Get reference transcriptome files from here Choose your the cDNA file for your organism, then download the file that ends in “cDNA.all.fa.gz”
Build the index
kallisto index -i myNewIndex InputFasta.fasta
align single-end reads
Run the following command for pseudoalignment of single-end reads to index.
kallisto quant -i myHumanIndex -o Sample1.mapped -b 60 —-single -l 275 -s 20 read1.fastq.gz
Once read mapping is complete, you will see a short report printed to your screen that indicates the number of reads kallisto saw in the fastq file, and the number of these that mapped to the reference. Often times it is useful to automatically store this information in a log file so that it can be parsed by other programs, such as the incredibly useful multiQC. To do this, simply append the code below at the end of your alignment bit above. The outcome will be the same, but you will also produce a log file.
-targument followed by the number of threads you have on your machine. If you’re on a mac, you can find out the number of virtual cores (i.e. threads) using the
sysctl hw.ncpucommand directly in the terminal
align paired-end reads
kallisto quant -i myMouseIndex -o Sample1.mapped -b 100 read1.fastq.gz read2.fastq.gz
stranded alignments and bigwigs
In some cases, you may want to carry out a stranded alignment with the end goal of viewing read ‘pile-ups’ on a genome browser track. This can also be done using Kallisto, but requires a few other programs and steps to get from the Kallisto alignment to bigwig.
Stranded alignment using pseudobam. SAM creation is also possible at this step.
kallisto quant -i [yourindex] -o [outputname] --fr-stranded -b 60 --pseudobam [input1] [input2] | samtools view -Sb - > [kallisto.fr.bam] kallisto quant -i [yourindex] -o [outputname] --rf-stranded -b 60 --pseudobam [input1] [input2] | samtools view -Sb - > [kallisto.rf.bam]
Sort and index using samtools
samtools sort -@ 24 [kallisto.fr.bam] [kallisto.fr.sorted] samtools sort -@ 24 [kallisto.rf.bam] [kallisto.rf.sorted] samtools index [kallisto.fr.sorted.bam] samtools index [kallisto.fr.sorted.bam]
BAM to BigWig conversion using deeptools
bamCoverage –b [kallisto.fr.sorted.bam] –o [kallisto.fr.sorted.bw] -p max bamCoverage –b [kallisto.rf.sorted.bam] –o [kallisto.rf.sorted.bw] -p max
Open RStudio and install the rhdf5 package from the BioC website
install the devtools package (if you don’t already have it)
install the Sleuth package directly from Lior Pachter’s github page using:
- open Rstudio and load the Sleuth package
- read in a study design file with column called ‘path’ which lists the path to each Kallisto output folder
- set-up a study design using the model.matrix function in R, with an intercept:
myDesign <- model.matrix(~treatment)