strobealign: A fast short-read aligner

Strobealign is a read mapper that is typically significantly faster than other read mappers while achieving comparable or better accuracy, see the performance evaluation.

Features

• Map single-end and paired-end reads
• Multithreading support
• Fast indexing (<1 minute for a human-sized reference genome using four cores)
• On-the-fly indexing by default. Optionally create an on-disk index.
• Output in standard SAM format or produce even faster results by writing PAF (without alignments)
• Strobealign is most suited for read lengths between 100 and 500 bp

Background

Strobealign achieves its speedup by using a dynamic seed size obtained from syncmer-thinned strobemers.

For details, refer to Strobealign: flexible seed size enables ultra-fast and accurate read alignment. The paper describes v0.7.1 of the program.

For an introduction, see also the 📺 RECOMB-Seq video from 2022: “Flexible seed size enables ultra-fast and accurate read alignment” (12 minutes). For a more detailed presentation of the underlying seeding mechanism in strobealign (strobemers) see 📺 “Efficient sequence similarity searches with strobemers” (73 minutes).

Table of contents

1. Installation
2. Usage
3. Command-line options
4. Index files
5. Changelog
6. Contributing
7. Evaluation
8. Citation
9. Version info
10. License

Installation

Conda

Strobealign is available from Bioconda.

1. Follow the Bioconda setup instructions
2. Install strobealign into a new Conda environment:

   conda create -n strobealign strobealign samtools
   

3. Activate the environment that was just created:

   conda activate strobealign
   

4. Run strobealign:

   strobealign --version
   

From source

To compile strobealign from sources, you need a somewhat recent Rust version, which you can obtain via Rustup. At the time of writing, the Rust versions included in current Debian and Ubuntu releases are too old.

When Rust is available, you can compile strobealign with

RUSTFLAGS='-C target-cpu=native' cargo build --release

The resulting binary will then be available at target/release/strobealign.

See the contributing instructions for how to compile strobealign as a developer.

Python bindings

Experimental Python bindings were available in the C++ version of strobealign (until version 0.17.0) and have not been ported to Rust. We may add them back, but until then, you will need to get strobealign 0.17.0, and can then install them with pip install .. The only documentation for the moment are the tests in tests/*.py.

Usage

To align paired-reads against a reference FASTA and produce a sorted BAM file, using eight threads:

strobealign -t 8 ref.fa reads.1.fastq.gz reads.2.fastq.gz | samtools sort -o sorted.bam

For single-end reads:

strobealign -t 8 ref.fa reads.fastq.gz | samtools sort -o sorted.bam

For mixed reads (the input file can contain both single and paired-end reads):

strobealign -t 8 ref.fa --interleaved reads.fastq.gz | samtools sort -o sorted.bam

In alignment mode, strobealign produces SAM output. By piping the output directly into samtools, the above commands avoid creating potentially large intermediate SAM files and also reduce disk I/O.

To produce unsorted BAM, use samtools view instead of samtools sort.

Mapping-only mode

The command-line option -x switches strobealign into mapping-only mode, in which it will output PAF files instead of SAM. For example:

strobealign -x -t 8 ref.fa reads.1.fastq.gz reads.2.fastq.gz | igzip > output.paf.gz

igzip is a faster version of gzip that is part of ISA-L. If it is not available, replace it with pigz or regular gzip in the command.

PAF output includes only mapped reads. Unmapped reads are omitted. This is also true for paired-end reads: If one of the reads is unmapped, only the mapped one is output.

Abundance estimation mode (for metagenomic binning)

The command-line option --aemb switches strobealign into abundance estimation mode, intended for metagenomic binning. In this mode, strobealign outputs a single table with abundance values in tab-separated value format instead of SAM or PAF.

Paired-end example:

strobealign -t 8 --aemb ref.fa reads.1.fastq.gz reads.2.fastq.gz > abundances.tsv

The output table contains one row for each contig of the reference. The first column is the reference/contig id and the second its abundance.

The abundance is the number of bases mapped to a contig, divided by the length of the contig. When a read maps to multiple locations, each of the n locations is weighted 1/n. Note that this is in contrast to alignment mode, where by default strobealign would output only a single alignment for each read (this can be changed with -N).

Further columns may be added to this table in future versions of strobealign.

Command-line options

Please run strobealign --help to see the most up-to-date list of command-line options. Some important ones are:

• -r: Mean read length. If given, this overrides the read length estimated from the input file(s). This is usually only required in combination with --create-index, see index files.
• -t N, --threads=N: Use N threads (both for mapping and indexing).
• --eqx: Emit = and X CIGAR operations instead of M.
• -x: Only map reads, do not do no base-level alignment. This switches the output format from SAM to PAF.
• --aemb: Output estimated abundance value of each contig, see section above.
• --rg-id=ID: Add RG tag to each SAM record.
• --rg=TAG:VALUE: Add read group metadata to the SAM header. This can be specified multiple times. Example: --rg-id=1 --rg=SM:mysamle --rg=LB:mylibrary.
• -N INT: Output up to INT secondary alignments. By default, no secondary alignments are output.
• -U: Suppress output of unmapped single-end reads and pairs in which both reads are unmapped.
• --use-index: Use a pre-generated index instead of generating a new one.
• --create-index, -i: Generate a strobemer index file (.sti) and write it to disk next to the input reference FASTA. Do not map reads. If read files are provided, they are used to estimate read length. See index files.

Index files

Background

Strobealign needs to build an index (strobemer index) of the reference before it can map reads to it. The optimal indexing parameters depend on the length of the input reads. There are pre-defined sets of parameters that are optimized for different read lengths. These canonical read lengths are 50, 75, 100, 125, 150, 250 and 400. When deciding which of the pre-defined indexing parameter sets to use, strobealign chooses one whose canonical read length is close to the average read length of the input.

The average read length of the input is normally estimated from the first 500 reads, but can also be explicitly set with -r.

Pre-computing an index (.sti)

By default, strobealign creates a new index every time the program is run. Depending on CPU, indexing a human-sized genome takes 1 to 2 minutes, which is not long compared to mapping many millions of reads. However, for repeatedly mapping small libraries, it is faster to pre-generate an index on disk and use that.

To create an index, use the --create-index option. Since strobealign needs to know the read length, either provide it with read file(s) as if you wanted to map them:

strobealign --create-index -t 8 ref.fa reads.1.fastq.gz reads.2.fastq.gz

Or set the read length explicitly with -r:

strobealign --create-index -t 8 ref.fa -r 150

This creates a file named ref.fa.rX.sti containing the strobemer index, where X is the canonical read length that the index is optimized for (see above). To use the index when mapping, provide option --use-index when doing the actual mapping:

strobealign --use-index -t 8 ref.fa reads.1.fastq.gz reads.2.fastq.gz | samtools ...

• Note that the .sti files are usually tied to a specific strobealign version. That is, when upgrading strobealign, the .sti files need to be regenerated. Strobealign detects whether the index was created with an incompatible version and refuses to load it.
• Index files are about four times as large as the reference.

Explanation

Multi-context seeds

Strobealign uses randstrobes as seeds, which in our case consist of two k-mers ("strobes") that are somewhat close to each other. When a seed is looked up in the index, it is only found if both strobes match. By changing the way in which the index is stored in v0.15.0, it became possible to support multi-context seeds. With those changes, strobealign falls back to looking up only one of the strobes (a "partial seed") if the full seed cannot be found. This results in better mapping rate and accuracy for read lengths of up to about 200 nt.

Usage of multi-context seeds is enabled by default in strobealign since v0.16.0. The strategy is to first search for