bustools is a program for manipulating BUS files for single cell RNA-Seq datasets. It can be used to error correct barcodes, collapse UMIs, produce gene count or transcript compatibility count matrices, and is useful for many other tasks. See the kallisto | bustools website for examples and instructions on how to use bustools as part of a single-cell RNA-seq workflow.
If you use bustools please cite
Melsted, Páll, Booeshaghi, A. Sina et al. Modular and efficient pre-processing of single-cell RNA-seq. BioRxiv (2019): 673285, doi.org/10.1101/673285.
For some background on the design and motivation for the BUS format and bustools see
Melsted, Páll, Ntranos, Vasilis and Pachter, Lior The Barcode, UMI, Set format and BUStools, Bioinformatics, btz279, 2019.
bustools works with BUS files which can be generated efficiently from raw sequencing data, e.g. using kallisto.
Binaries for Mac, Linux, Windows, and Rock64 can be downloaded from the bustools website. Binary installation time is less than two minutes.
To compile bustools download the source code with
git clone https://github.com/BUStools/bustools.git
Navigate to the bustools directory
cd bustools
Make a build directory and move there:
mkdir build
cd build
Run cmake:
cmake ..
Build the code:
make
The bustools executable will be located in build/src. To install bustools into the cmake install prefix path type:
make install
To see a list of available commands, type bustools in the terminal
> bustools
Usage: bustools <CMD> [arguments] ..
Where <CMD> can be one of:
capture Capture records from a BUS file
correct Error correct a BUS file
count Generate count matrices from a BUS file
inspect Produce a report summarizing a BUS file
linker Remove section of barcodes in BUS files
project Project a BUS file to gene sets
sort Sort a BUS file by barcodes and UMIs
text Convert a binary BUS file to a tab-delimited text file
whitelist Generate a whitelist from a BUS file
Running bustools <CMD> without arguments prints usage information for <CMD>
bustools capture can separate BUS files into multiple files according to the capture criteria.
Usage: bustools capture [options] bus-files
Options:
-o, --output Directory for output
-c, --capture List of transcripts to capture
-e, --ecmap File for mapping equivalence classes to transcripts
-t, --txnames File with names of transcripts
BUS files can be barcode error corrected with respect to a technology-specific whitelist of barcodes using bustools correct.
> bustools correct
Usage: bustools correct [options] bus-files
Options:
-o, --output File for corrected bus output
-w, --whitelist File of whitelisted barcodes to correct to
-p, --pipe Write to standard output
BUS files can be converted into a barcode-feature matrix, where the feature can be TCCs (Transcript Compatibility Counts) or genes using bustools count.
> bustools count
Usage: bustools count [options] bus-files
Options:
-o, --output File for corrected bus output
-g, --genemap File for mapping transcripts to genes
-e, --ecmap File for mapping equivalence classes to transcripts
-t, --txnames File with names of transcripts
--genecounts Aggregate counts to genes only
A report summarizing the contents of a sorted BUS file can be output either to standard out or to a JSON file for further analysis using bustools inspect.
> bustools inspect
Usage: bustools inspect [options] sorted-bus-file
Options:
-o, --output File for JSON output (optional)
-e, --ecmap File for mapping equivalence classes to transcripts
-w, --whitelist File of whitelisted barcodes to correct to
-p, --pipe Write to standard output
--ecmap and --whitelist are optional parameters; bustools inspect is much faster without them, especially without the former.
Sample output (to stdout):
Read in 3148815 BUS records
Total number of reads: 3431849
Number of distinct barcodes: 162360
Median number of reads per barcode: 1.000000
Mean number of reads per barcode: 21.137281
Number of distinct UMIs: 966593
Number of distinct barcode-UMI pairs: 3062719
Median number of UMIs per barcode: 1.000000
Mean number of UMIs per barcode: 18.863753
Estimated number of new records at 2x sequencing depth: 2719327
Number of distinct targets detected: 70492
Median number of targets per set: 2.000000
Mean number of targets per set: 3.091267
Number of reads with singleton target: 1233940
Estimated number of new targets at 2x seuqencing depth: 6168
Number of barcodes in agreement with whitelist: 92889 (57.211752%)
Number of reads with barcode in agreement with whitelist: 3281671 (95.623992%)
bustools linker removes specified section of barcode in BUS files.
Usage: bustools linker [options] bus-files
Options:
-s, --start Start coordinate for section of barcode to remove (0-indexed, inclusive)
-e, --end End coordinate for section of barcode to remove (0-indexed, exclusive)
-p, --pipe Write to standard output
If --start is -1, the removed section begins at beginning of barcode. Likewise, if --end is -1, the removed section ends at the end of the barcode. BUS files should contain barcodes of the same length.
The kallisto bus command maps reads to a set of transcripts. bustools project takes as input kallisto's (sorted) output and a transcript to gene map (tr2g file), and outputs a BUS file, a matrix.ec file, and a list of genes, which collectively map each read to a set of genes.
Usage: bustools project [options] sorted-bus-file
Options:
-o, --output File for project bug output and list of genes (no extension)
-g, --genemap File for mapping transcripts to genes
-e, --ecmap File for mapping equivalence classes to transcripts
-t, --txnames File with names of transcripts
-p, --pipe Write to standard output
Raw BUS output from pseudoalignment programs may be unsorted. To simply and accelerate downstream processing BUS files can be sorted using bustools sort
> bustools sort
Usage: bustools sort [options] bus-files
Options:
-t, --threads Number of threads to use
-m, --memory Maximum memory used
-T, --temp Location and prefix for temporary files
required if using -p, otherwise defaults to output
-o, --output File for sorted output
-p, --pipe Write to standard output
This will create a new BUS file where the BUS records are sorted by barcode first, UMI second, and equivalence class third.
BUS files can be converted to a tab-separated format for easy inspection and processing using shell scripts or high level languages with bustools text.
> bustools text
Usage: bustools text [options] bus-files
Options:
-o, --output File for text output
bustools whitelist generates a whitelist based on the barcodes in a sorted BUS file.
Usage: bustools whitelist [options] sorted-bus-file
Options:
-o, --output File for the whitelist
-f, --threshold Minimum number of times a barcode must appear to be included in whitelist
--threshold is a (highly) optional parameter. If not provided, bustools whitelist will determine a threshold based on the first 200 to 100,200 records.