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bustools

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, efficient and constant-memory single-cell RNA-seq preprocessing. Nature Biotechnology, 2021.

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, 2019.

BUS format

bustools works with BUS files which can be generated efficiently from raw sequencing data, e.g. using kallisto.

Installation

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

Usage

To see a list of available commands, type bustools in the terminal

> bustools 
Usage: bustools <CMD> [arguments] ..

Where <CMD> can be one of: 

sort            Sort a BUS file by barcodes and UMIs
correct         Error correct a BUS file
count           Generate count matrices from a sorted BUS file
inspect         Produce a report summarizing a sorted BUS file
allowlist       Generate an on-list from a sorted BUS file
capture         Capture records from a BUS file
text            Convert a binary BUS file to a tab-delimited text file
fromtext        Convert tab-delimited text file to a binary BUS file
extract         Extracts reads from input FASTQ files based on BUS file
umicorrect      Use a UMI correction algorithm
compress        Compress a sorted BUS file
decompress      Decompress a compressed BUS file

Running bustools <CMD> without arguments prints usage information for <CMD>

sort

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.

correct

BUS files can be barcode error corrected with respect to a technology-specific "on list" of barcodes using bustools correct.

> bustools correct
Usage: bustools correct [options] bus-files

Options: 
-o, --output          File for corrected bus output
-w, --onlist          File of on-listed barcodes to correct to
-p, --pipe            Write to standard output

count

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
--cm                  Count multiplicites instead of UMIs

inspect

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, --onlist          File of on-listed barcodes to correct to
-p, --pipe            Write to standard output

--ecmap and --onlist 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 on-list: 92889 (57.211752%)
Number of reads with barcode in agreement with on-list: 3281671 (95.623992%)

allowlist

bustools allowlist generates an on-list based on the barcodes in a sorted BUS file.

Usage: bustools allowlist [options] sorted-bus-file

Options: 
-o, --output        File for the on-list
-f, --threshold     Minimum number of times a barcode must appear to be included in on-list

--threshold is a (highly) optional parameter. If not provided, bustools allowlist will determine a threshold based on the first 200 to 100,200 records.

capture

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

text

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

fromtext

Converts a plaintext tab-separated representation of a BUS file to a binary BUS file.

> bustools text
Usage: bustools text [options] bus-files

Options: 
-o, --output          File for text output

compress

Compresses a sorted BUS file.

> bustools compress
Usage: bustools compress [options] sorted-bus-file
Note: BUS file should be sorted by barcode-umi-ec

Options: 
-N, --chunk-size    Number of rows to compress as a single block
-o, --output        File to write compressed output
-p, --pipe          Write to standard output.

Reference for bustools compression:

Einarsson, P and Melsted, Páll BUSZ: compressed BUS files, Bioinformatics, 2023.

decompress

Decompresses (inflates) a compressed BUS file.

> bustools compress
Usage: bustools compress [options] sorted-bus-file
Note: BUS file should be sorted by barcode-umi-ec

Options: 
-o, --output        File to write decompressed output
-p, --pipe          Write to standard output.