Themisto version 3.2.2 is out, fixing a bug where the output was sometimes not fully flushed to disk when using gzipped sorted output. See release notes for details and pre-compiled Linux binaries.
Themisto is a succinct colored k-mer index supporting pseudo-alignment against a database of reference sequences similar to the tool Kallisto, Bifrost and Metagraph. For more information, see the preprint. This software is currently developed by the Compressed Data Structures group at the University of Helsinki.
Precompiled binaries are available for
- Linux x86_64
- macOS arm64
- macOS x86_64
Visit the Releases page to download a binary.
We currently support only Linux and macOS. For compilation, you will need a C++20 compliant compiler with OpenMP support, CMake v3.1 or newer, and Rust 1.77. If compiling with g++, make sure that the version is at least g++-10, or you might run into compilation errors with the standard library <filesystem> header.
These instructions have been tested to work on Ubuntu 18.04 with the aforementioned dependencies installed. To build the software, enter the Themisto directory and run
git submodule update --init --recursive
cd build
cmake .. -DMAX_KMER_LENGTH=31
make
If there is a linking error at the very end, try runnning make again. Where 31 is the maximum k-mer length (node length) to support, up to 255. The larger the k-mer length, the more time and memory the index construction takes. Values that are one less than a multiple of 32 work the best. This will create the binary atbuild/bin/themisto.
Troubleshooting: If you run into problems involving the <filesystem> header, you probably need to update your compiler. The compiler g++-10 should be sufficient. Install a new compiler and direct CMake to use it with the -DCMAKE_CXX_COMPILER option. For example, to set the compiler to g++-10, run CMake with the option -DCMAKE_CXX_COMPILER=g++-10.
The MacOS build only works with the gcc and g++ compilers. Install those compilers on the system and make sure that CXX environment variable is set to the g++ compiler. Also make sure that the g++ executable is actually the GCC g++ compiler and not just a link to Clang.
git submodule update --init --recursive
cd build
cmake -DCMAKE_C_COMPILER=$(which gcc) -DCMAKE_CXX_COMPILER=$(which g++) -DMAX_KMER_LENGTH=31 ..
make
Here 31 is the maximum k-mer length to support, up to 255. The larger the k-mer length, the more time and memory the index construction takes. If you run into problems involving zlib, add -DCMAKE_BUILD_ZLIB=1 into the cmake command.
Note that macOS has a very small limit for the number of concurrently opened files. Themisto can use temporary files to conserve RAM, and may run into this limit. To increase the limit, run the command
ulimit -n 2048
To build the Themisto index for a set of genomes, you need to pass in a text file that contains the paths to the FASTA files of the genomes, one file per line. Each FASTA file is given a different color 0,1,2,3... in the same order as they appear in the list. There are three example genomes of E. coli in example_input and a file at example_input/coli_file_list.txt listing the file names. To build the index for this data, run the following command:
./build/bin/themisto build -k 31 -i example_input/coli_file_list.txt --index-prefix my_index --temp-dir temp --mem-gigas 2 --n-threads 4 --file-colors
This builds an index with k = 31, such that the index files are written to my_index.tdbg and my_index.tcolors, using the directory temp as temporary storage, using four threads and up to 2GB of memory. We recommend to use a fast SSD drive for the temporary directory.
To align the four sequences in example_input/queries.fna against the index we just built, writing output to out.txt run:
./build/bin/themisto pseudoalign --query-file example_input/queries.fna --index-prefix my_index --temp-dir temp --out-file out.txt --n-threads 4 --threshold 0.7
This reports all colors such that at least a fraction 0.7 of the k-mers of the query are in the reference genome of the color, ignoring k-mers that are not found in any reference.
This should produce the following output file:
0 0 2
1 0 1 2
2 2
3 2
There is one line for each query sequence. The lines may appear in a different order if parallelism was used. The first integer on a line is the 0-based rank of a query sequence in the query file, and the rest of the integers are the colors that are pseudoaligned with the query. For example, here the query with rank 1 (i.e. the second sequence in the query file) pseudoaligns to colors 0, 1 and 2.
Build the Themisto index:
Usage:
build [OPTION...]
Basic options:
-k, --node-length arg The k of the k-mers. (default: 0)
-i, --input-file arg The input sequences in FASTA or FASTQ format. The
format is inferred from the file extension. If
the extension is .txt, the file is interpreted as
a list of filenames, one per line
-o, --index-prefix arg The de Bruijn graph will be written to
[prefix].tdbg and the color structure to
[prefix].tcolors.
--temp-dir arg Directory for temporary files. This directory
should have fast I/O operations and should have
as much space as possible.
-v, --verbose More verbose progress reporting into stderr.
Coloring (give only one) options:
-f, --file-colors Default if the input has multiple sequence
files. Creates a distinct color 0,1,2,... for
each file in the input file list, in the order
the files appear in the list
-e, --sequence-colors Default if the input has just a single sequence
file. Creates a distinct color 0,1,2,... for
each sequence in the input.
-c, --manual-colors arg A file containing one integer color per
sequence, one color per line. Colors may be
repeated. If there are multiple sequence files,
then this file should be a text file containing
the corresponding color filename for each
sequence file, one filename per line.
--no-colors Build only the de Bruijn graph without colors.
Can be loaded later with --load-dbg (see
--help-advanced)
Computational resources options:
--mem-gigas arg Number of gigabytes allowed for external memory
algorithms (must be at least 2). (default: 2)
-t, --n-threads arg Number of parallel exectuion threads. Default: 1
(default: 1)
Advanced options:
--forward-strand-only Do not add reverse complements of sequences
to the index
--load-dbg If given, loads a precomputed de Bruijn
graph from the index prefix. If this is
given, the value of parameter -k is ignored
because the order k is defined by the
precomputed de Bruijn graph.
--randomize-non-ACGT Replace non-ACGT letters with random
nucleotides. If this option is not given,
k-mers containing a non-ACGT character are
deleted instead.
-d, --colorset-pointer-tradeoff arg
This option controls a time-space tradeoff
for storing and querying color sets. If
given a value d, we store color set
pointers only for every d nodes on every
unitig. The higher the value of d, the
smaller then index, but the slower the
queries. The savings might be significant
if the number of distinct color sets is
small and the graph is large and has long
unitigs. (default: 1)
-s, --coloring-structure-type arg
Type of coloring structure to build
("sdsl-hybrid", "roaring"). (default:
sdsl-hybrid)
--from-index arg Take as input a pre-built Themisto index.
Builds a new index in the format specified
by --coloring-structure-type. This is
currently implemented by decompressing the
distinct color sets in memory before
re-encoding them, so this might take a lot
of RAM.
--silent Print as little as possible to stderr (only
errors).
Help options:
-h, --help Print usage instructions for commonly used options.
--help-advanced Print advanced options usage.
Usage example:
./build/bin/themisto build -k 31 -i example_input/coli_file_list.txt --index-prefix my_index --temp-dir temp --mem-gigas 2 --n-threads 4 --file-colors
This program aligns query sequences against an index that has been built previously. The output is one line per input read. Each line consists of a space-separated list of integers. The first integer specifies the rank of the read in the input file, and the rest of the integers are the identifiers of the colors of the sequences that the read pseudoaligns with. If the program is ran with more than one thread, the output lines are not necessarily in the same order as the reads in the input file. This can be fixed with the option --sort-output, but this will slow down the program.
The query can be given as one file, or as a file with a list of files. In the former case, we must specify one output file with the options --out-file, and in the latter case, we must give a file that lists one output filename per line using the option --out-file-list.
The query file(s) should be in fasta of fastq format. The format is inferred from the file extension. Recognized file extensions for fasta are: .fasta, .fna, .ffn, .faa and .frn . Recognized extensions for fastq are: .fastq and .fq. Gzipped sequence files with the extension .gz are also supported.
Usage:
pseudoalign [OPTION...]
Basic options:
-q, --query-file arg Input file of the query sequences (default:
"")
--query-file-list arg A list of query filenames, one line per
filename (default: "")
-o, --out-file arg Output filename. Print results if no output
filename is given. (default: "")
--out-file-list arg A file containing a list of output filenames,
one per line. (default: "")
-i, --index-prefix arg The index prefix that was given to the build
command.
--temp-dir arg Directory for temporary files.
--gzip-output Compress the output files with gzip.
--sort-output Sort the lines of the out files by sequence
rank in the input files.
-v, --verbose More verbose progress reporting into stderr.
Algorithm options:
--threshold arg Fraction of k-mer matches required to report
a color. If this is equal to 1, the
algorithm is implemented with a specialized
set intersection method. (default: 0.7)
--include-unknown-kmers Include all k-mers in the pseudoalignment,
even those which do not occur in the index.
--report-relevant-kmer-count
Appends to each output line a semicolon
followed by a space and then the number of
k-mers of the query that had at least 1
color.
--relevant-kmers-fraction arg
Accept a pseudoalignment only if at least
this fraction of k-mers of the read had at
least 1 color. (default: 0.0)
Computational resources options:
-t, --n-threads arg Number of parallel execution threads. Default: 1
(default: 1)
Advanced options:
--rc Include reverse complement matches in the
pseudoalignment. This option only makes
sense if the index was built with
--forward-strand-only. Otherwise this option
has no effect except to slow down the query.
--buffer-size-megas arg Size of the input buffer in megabytes in
each thread. If this is larger than the
number of nucleotides in the input divided
by the number of threads, then some threads
will be idle. So if your input files are
really small and you have a lot of threads,
consider using a small buffer. (default:
8.0)
--silent Print as little as possible to stderr (only
errors).
Help options:
-h, --help Print usage instructions for commonly used options.
--help-advanced Print advanced usage instructions.
Usage example:
pseudoalign pseudoalign --query-file example_input/queries.fna --index-prefix my_index --temp-dir temp --out-file out.txt --n-threads 4 --threshold 0.7
Examples:
Pseudoalign example_input/queries.fna against an index and print results:
./build/bin/themisto pseudoalign --query-file example_input/queries.fna --index-prefix my_index --temp-dir temp
Pseudoalign example_input/queries.fna against an index and write results to out.txt:
./build/bin/themisto pseudoalign --query-file example_input/queries.fna --index-prefix my_index --temp-dir temp --out-file out.txt
Pseudoalign a list of fasta files in input_list.txt into output filenames in output_list.txt:
./build/bin/themisto pseudoalign --query-file-list input_list.txt --index-prefix my_index --temp-dir temp --out-file-list output_list.txt
This command dumps the unitigs and optionally their colors out of an existing Themisto index.
Usage:
extract-unitigs [OPTION...]
-i, --index-prefix arg The index prefix that was given to the build
command.
--fasta-out arg Output filename for the unitigs in FASTA format
(optional). (default: "")
--gfa-out arg Output the unitig graph in GFA1 format
(optional). (default: "")
--colors-out arg Output filename for the unitig colors (optional).
If this option is not given, the colors are not
computed. Note that giving this option affects
the unitigs written to unitigs-out: if a unitig
has nodes with different color sets, the unitig
is split into maximal segments of nodes that have
equal color sets. The file format of the color
file is as follows: there is one line for each
unitig. The lines contain space-separated
strings. The first string on a line is the FASTA
header of a unitig (without the '>'), and the
following strings on the line are the integer
color labels of the colors of that unitig. The
unitigs appear in the same order as in the FASTA
file. (default: "")
--min-colors arg Extract maximal unitigs with at least (>=)
min-colors in each node. Can't be used with
--colors-out. (optional) (default: 0)
-v, --verbose More verbose progress reporting into stderr.
-h, --help Print usage
This command prints various statistics about the k-mers and colors in an existing index.
Usage:
stats [OPTION...]
-i, --index-prefix arg The index prefix that was given to the build
command.
--unitigs Also compute statistics on unitigs. This takes a
while and requires the temporary directory to be
set.
--temp-dir arg Directory for temporary files.
-h, --help Print usage
This command prints a file where each line corresponds to a k-mer in the index. The line starts with the k-mer, followed by space, followed by the color set of that k-mer. If --sparse is given, the color set is printed as a space-separated list of integers. Otherwise, the color set is printed as a string of zeroes and ones such that the i-th character is '1' iff color i is present in the color set.
Example:
./build/bin/themisto dump-color-matrix -i my_index -o dump.txt --sparse
Full instructions:
Usage:
dump-color-matrix [OPTION...]
-i, --index-prefix arg The index prefix that was given to the build
command.
-o, --output-file arg The output file for the dump.
-v, --verbose More verbose progress reporting into stderr.
--silent Print as little as possible to stderr (only
errors).
--sparse Print only the indices of non-zero entries.
-h, --help Print usage
git submodule init
git submodule update
cd googletest
mkdir build
cd build
cmake ..
make
cd ../../build
cmake .. -DCMAKE_BUILD_TYPE=Debug -DBUILD_THEMISTO_TESTS=1
make
This builds the tests to build/bin/themisto_tests. The test executable must be ran at the root of the repository, or otherwise it wont find the test input files at example_input.
To build release binaries for Linux, use a machine with as old of a libc as possible for maximum compatibility. It's also important to disable architecture-specific optimizations in Roaring, so use the following cmake command:
cmake .. -DCMAKE_BUILD_ZLIB=1 -DCMAKE_BUILD_BZIP2=1 -DROARING_DISABLE_NATIVE=ON -DCMAKE_BUILD_TYPE=Release
See the Wiki in Github for instructions on how to set up the build environment.
This software is licensed under GPLv2. See LICENSE.txt.