Currently, the reference-based indexing code piggybacks off of the existing build command. To build the full reference index you will need the cuttlefish reduced GFA1 output. We'll provide an example below, assuming we start with a reference genome sequence in a fasta file called ref.fa. Further, we will assume for the time being that ref.fa contains no invalid characters (in practice this is achieved by running a genome through the fixFasta functionality of pufferfish).
First we need to build the compacted reference de Bruijn Graph. We do this using cuttlefish.
$ cuttlefish build -s ref.fa -k 31 -t 16 -o ref_dbg -m 8 -f 3
This creates 2 files called ref_dbg.cf_seq and ref_dbg.cf_seg. Next we build the reference index:
$ ./build ref_dbg 31 20 --canonical-parsing -o ref_idx
This will output 3 files, all with the prefix ref_idx. The files will be named ref_idx.sshash, ref_idx.ctab, and ref_index.refinfo. Together, these comprise the reference index over the original reference sequences in ref.fa.
The reference index loader assumed these specific suffixes. Finally, you can test out the index with:
$ ./test_load ref_idx ref.fa
This will iterate over all of the positions of ref.fa and ensure that the observed k-mer has an entry in the reference index that points back to the current reference sequence, at the current position, in the provided (forward) orientation.
This is a compressed dictionary data structure for k-mers (strings of length k over the DNA alphabet {A,C,G,T}), based on Sparse and Skew Hashing.
A (pre-print) paper describing the data structure can be found here.
For a dictionary of n k-mers, two basic queries are supported:
- i = Lookup(g), where i is in [0,n) if the k-mer g is found in the dictionary or i = -1 otherwise;
- g = Access(i), where g is the k-mer associated to the identifier i.
If also the abundances of the k-mers (their frequency counts) are stored in the dictionary, then the dictionary is said to be weighted and it also supports:
- c = Abundance(i), where i is a given k-mer identifier.
A membership query (determine if a given k-mer is present in the dictionary or not) is, therefore, supported by means of the lookup query. The dictionary can also stream through all k-mers of a given DNA file (.fasta or .fastq formats) to determine their membership to the dictionary.
NOTE: The Lookup query assumes that two k-mers being the reverse complement of each other are the same.
- Compiling the Code
- Dependencies
- Build a Dictionary
- Examples
- Input Files
- Large-scale Benchmark
- Author
The code is tested on Linux with gcc and on Mac with clang.
To build the code, CMake is required.
Clone the repository with
git clone --recursive https://github.com/jermp/sshash.git
If you have cloned the repository without --recursive, you will need to perform the following commands before
compiling:
git submodule init
git submodule update
To compile the code for a release environment (see file CMakeLists.txt for the used compilation flags), it is sufficient to do the following:
mkdir build
cd build
cmake ..
make -j
For a testing environment, use the following instead:
mkdir debug_build
cd debug_build
cmake .. -D CMAKE_BUILD_TYPE=Debug -D SSHASH_USE_SANITIZERS=On
make
The repository has minimal dependencies: it only uses the PTHash library (for minimal perfect hashing), and zlib to read gzip-compressed streams.
To automatically pull the PTHash dependency, just clone the repo with
--recursive as explained in Compiling the Code.
If you do not have zlib installed, you can do
sudo apt-get install zlib1g
if you are on Linux/Ubuntu, or
brew install zlib
if you have a Mac.
The driver program
called build can be used to build a dictionary.
From within the directory where the code was compiled (see the section Compiling the Code), run the command:
./build --help
to show the usage of the driver program (reported below for convenience).
Usage: ./build [-h,--help] input_filename k m [-s seed] [-l l] [-c c] [--canonical-parsing] [--abundances] [-o output_filename] [--check] [--bench] [--verbose]
input_filename
Must be a FASTA file (.fa/fasta extension) compressed with gzip (.gz) or not:
- without duplicate nor invalid kmers
- one DNA sequence per line.
For example, it could be the de Bruijn graph topology output by BCALM.
k
K-mer length (must be <= 31).
m
Minimizer length (must be < k).
[-s seed]
Seed for construction (default is 1).
[-l l]
A (integer) constant that controls the space/time trade-off of the dictionary. A reasonable values lies between 2 and 12 (default is 6).
[-c c]
A (floating point) constant that trades construction speed for space effectiveness of minimal perfect hashing. A reasonable value lies between 3.0 and 10.0 (default is 3.000000).
[--canonical-parsing]
Canonical parsing of k-mers. This option changes the parsing and results in a trade-off between index space and lookup time.
[--abundances]
Also store the abundances in compressed format.
[-o output_filename]
Output file name where the data structure will be serialized.
[--check]
Check correctness after construction.
[--bench]
Run benchmark after construction.
[--verbose]
Verbose output during construction.
[-h,--help]
Print this help text and silently exits.
For the examples, we are going to use some collections
of stitched unitigs from the directory ../data/unitigs_stitched.
These collections were built for k = 31, so dictionaries should be built with k = 31 as well to ensure correctness.
In the section Input Files, we explain how such collections of stitched unitigs can be obtained from raw FASTA files.
./build ../data/unitigs_stitched/salmonella_enterica_k31_ust.fa.gz 31 13 --check --bench -o salmonella_enterica.index
This example builds a dictionary for the k-mers read from the file ../data/unitigs_stitched/salmonella_enterica_k31_ust.fa.gz,
with k = 31 and m = 13. It also check the correctness of the dictionary (--check option), run a performance benchmark (--bench option), and serializes the index on disk to the file salmonella_enterica.index.
To run a performance benchmark after construction of the index, use:
./bench salmonella_enterica.index
To also store the abundances, use the option --abundances:
./build ../data/unitigs_stitched/with_abundances/salmonella_enterica_k31_ust.abundances.fa.gz 31 13 --abundances --check --verbose
./build ../data/unitigs_stitched/salmonella_100_k31_ust.fa.gz 31 15 -l 2 -o salmonella_100.index
This example builds a dictionary from the input file ../data/unitigs_stitched/salmonella_100_k31_ust.fa.gz (a pangenome consisting in 100 genomes of Salmonella Enterica), with k = 31, m = 15, and l = 2. It also serializes the index on disk to the file salmonella_100.index.
To perform some streaming membership queries, use:
./query salmonella_100.index ../data/queries/SRR5833294.10K.fastq.gz
if your queries are meant to be read from a FASTQ file, or
./query salmonella_100.index ../data/queries/salmonella_enterica.fasta.gz --multiline
if your queries are to be read from a (multi-line) FASTA file.
./build ../data/unitigs_stitched/salmonella_100_k31_ust.fa.gz 31 13 -l 4 -s 347692 --canonical-parsing -o salmonella_100.canon.index
This example builds a dictionary from the input file ../data/unitigs_stitched/salmonella_100_k31_ust.fa.gz (same used in Example 2), with k = 31, m = 13, l = 4, using a seed 347692 for construction (-s 347692), and with the canonical parsing modality (option --canonical-parsing). The dictionary is serialized on disk to the file salmonella_100.canon.index.
The "canonical" version of the dictionary offers more speed for only a little space increase (for a suitable choice of parameters m and l), especially under low-hit workloads -- when the majority of k-mers are not found in the dictionary. (For all details, refer to the paper.)
Below a comparison between the dictionary built in Example 2 (not canonical) and the one just built (Example 3, canonical).
./query salmonella_100.index ../data/queries/SRR5833294.10K.fastq.gz
index size: 10.3981 [MB] (6.36232 [bits/kmer])
==== query report:
num_kmers = 460000
num_valid_kmers = 459143 (99.8137% of kmers)
num_positive_kmers = 46 (0.0100187% of valid kmers)
num_searches = 42/46 (91.3043%)
num_extensions = 4/46 (8.69565%)
elapsed = 229.159 millisec / 0.229159 sec / 0.00381932 min / 498.172 ns/kmer
./query salmonella_100.canon.index ../data/queries/SRR5833294.10K.fastq.gz
index size: 11.0657 [MB] (6.77083 [bits/kmer])
==== query report:
num_kmers = 460000
num_valid_kmers = 459143 (99.8137% of kmers)
num_positive_kmers = 46 (0.0100187% of valid kmers)
num_searches = 42/46 (91.3043%)
num_extensions = 4/46 (8.69565%)
elapsed = 107.911 millisec / 0.107911 sec / 0.00179852 min / 234.589 ns/kmer
We see that the canonical dictionary is twice as fast as the regular dictionary for low-hit workloads, even on this tiny example, for only +0.4 bits/k-mer.
SSHash is meant to index k-mers from collections that do not contain duplicates nor invalid k-mers (strings containing symbols different from {A,C,G,T}). These collections can be obtained, for example, by extracting the maximal unitigs of a de Bruijn graph.
Doing so is easy to do using the tool BCALM2. This tool builds a compacted de Bruijn graph and outputs its maximal unitigs. From the output of BCALM2, we can then stitch (i.e., glue) some unitigs to reduce the number of nucleotides. The stitiching process is carried out using the UST tool.
Below we provide a complete example (assuming both BCALM2 and UST are installed correctly) that downloads the Human (GRCh38) Chromosome 13 and extracts the maximal stitiched unitigs for k = 31.
mkdir DNA_datasets
wget http://ftp.ensembl.org/pub/current_fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.13.fa.gz -O DNA_datasets/Homo_sapiens.GRCh38.dna.chromosome.13.fa.gz
~/bcalm/build/bcalm -in ~/DNA_datasets/Homo_sapiens.GRCh38.dna.chromosome.13.fa.gz -kmer-size 31 -abundance-min 1 -nb-cores 8
~/UST/ust -k 31 -i ~/Homo_sapiens.GRCh38.dna.chromosome.13.fa.unitigs.fa
gzip Homo_sapiens.GRCh38.dna.chromosome.13.fa.unitigs.fa.ust.fa
rm ~/Homo_sapiens.GRCh38.dna.chromosome.13.fa.unitigs.fa
See also the script scripts/download_and_preprocess_datasets.sh
for precise arguments.
Pinus Taeda ("pine", GCA_000404065.3) and Ambystoma Mexicanum ("axolotl", GCA_002915635.2) are some of the largest genome assemblies, respectively counting 10,508,232,575 and 17,987,935,180 distinct k-mers for k = 31.
After running BCALM2 and UST, we build the indexes as follows.
./build ~/DNA_datasets.larger/GCA_000404065.3_Ptaeda2.0_genomic.ust_k31.fa.gz 31 20 -l 6 -c 7 -o pinus.m20.index
./build ~/DNA_datasets.larger/GCA_000404065.3_Ptaeda2.0_genomic.ust_k31.fa.gz 31 19 -l 6 -c 7 --canonical-parsing -o pinus.m19.canon.index
./build ~/DNA_datasets.larger/GCA_002915635.3_AmbMex60DD_genomic.ust_k31.fa.gz 31 21 -l 6 -c 7 -o axolotl.m21.index
./build ~/DNA_datasets.larger/GCA_002915635.3_AmbMex60DD_genomic.ust_k31.fa.gz 31 20 -l 6 -c 7 --canonical-parsing -o axolotl.m20.canon.index
The following table summarizes the space of the dictionaries.
| Dictionary | Pine | Axolotl | ||
|---|---|---|---|---|
| GB | bits/k-mer | GB | bits/k-mer | |
| SSHash, regular | 13.21 | 10.06 | 22.28 | 9.91 |
| SSHash, canonical | 14.94 | 11.37 | 25.03 | 11.13 |
To query the dictionaries, we use SRR17023415 fastq reads (23,891,117 reads, each of 150 bases) for the pine, and GSM5747680 multi-line fasta (15,548,160 lines) for the axolotl.
Timings have been collected on an Intel Xeon Platinum 8276L CPU @ 2.20GHz, using a single thread.
| Dictionary | Pine | Axolotl | ||
|---|---|---|---|---|
| (>75% hits) | (>86% hits) | |||
| tot (min) | avg (ns/k-mer) | tot (min) | avg (ns/k-mer) | |
| SSHash, regular | 19.2 | 400 | 4.2 | 269 |
| SSHash, canonical | 14.8 | 310 | 3.2 | 208 |
Below the complete query reports.
./query pinus.m20.index ~/DNA_datasets.larger/queries/SRR17023415_1.fastq.gz
==== query report:
num_kmers = 2866934040
num_valid_kmers = 2866783488 (99.9947% of kmers)
num_positive_kmers = 2151937575 (75.0645% of valid kmers)
num_searches = 418897117/2151937575 (19.466%)
num_extensions = 1733040458/2151937575 (80.534%)
elapsed = 1146.58 sec / 19.1097 min / 399.933 ns/kmer
./query pinus.m19.canon.index ~/DNA_datasets.larger/queries/SRR17023415_1.fastq.gz
==== query report:
num_kmers = 2866934040
num_valid_kmers = 2866783488 (99.9947% of kmers)
num_positive_kmers = 2151937575 (75.0645% of valid kmers)
num_searches = 359426304/2151937575 (16.7025%)
num_extensions = 1792511271/2151937575 (83.2975%)
elapsed = 889.779 sec / 14.8297 min / 310.359 ns/kmer
./query axolotl.m21.index ~/DNA_datasets.larger/queries/Axolotl.Trinity.CellReports2017.fasta.gz --multiline
==== query report:
num_kmers = 931366757
num_valid_kmers = 748445346 (80.3599% of kmers)
num_positive_kmers = 650467884 (86.9092% of valid kmers)
num_searches = 124008258/650467884 (19.0645%)
num_extensions = 526459626/650467884 (80.9355%)
elapsed = 250.173 sec / 4.16955 min / 268.608 ns/kmer
./query axolotl.m20.canon.index ~/DNA_datasets.larger/queries/Axolotl.Trinity.CellReports2017.fasta.gz --multiline
==== query report:
num_kmers = 931366757
num_valid_kmers = 748445346 (80.3599% of kmers)
num_positive_kmers = 650467884 (86.9092% of valid kmers)
num_searches = 106220473/650467884 (16.3299%)
num_extensions = 544247411/650467884 (83.6701%)
elapsed = 193.871 sec / 3.23119 min / 208.158 ns/kmer
Giulio Ermanno Pibiri - [email protected]