slow5lib

slow5lib is a software library for reading & writing SLOW5 files. slow5lib is designed to facilitate use of data in SLOW5 format by third-party software packages. Existing packages that read/write data in FAST5 or POD5 format can be easily modified to support SLOW5.

About SLOW5 format: SLOW5 is a new file format for storing signal data from Oxford Nanopore Technologies (ONT) devices. SLOW5 was developed to overcome inherent limitations in the standard FAST5 signal data format that prevent efficient, scalable analysis and cause many headaches for developers (and upcoming headaches with ONT’s latest POD5 format). SLOW5 can be encoded in human-readable ASCII format, or a more compact and efficient binary format (BLOW5) - this is analogous to the seminal SAM/BAM format for storing DNA sequence alignments. The BLOW5 binary format supports zlib (DEFLATE) compression, or other compression methods, thereby minimising the data storage footprint while still permitting efficient parallel access. Detailed benchmarking experiments have shown that SLOW5 format is an order of magnitude faster and significantly smaller than FAST5.

Publication: https://www.nature.com/articles/s41587-021-01147-4
SLOW5 specification: https://hasindu2008.github.io/slow5specs
slow5tools: https://github.com/hasindu2008/slow5tools
SLOW5 ecosystem: https://hasindu2008.github.io/slow5

Please cite the following in your publications when using slow5lib/pyslow5:

Gamaarachchi, H., Samarakoon, H., Jenner, S.P. et al. Fast nanopore sequencing data analysis with SLOW5. Nat Biotechnol 40, 1026-1029 (2022). https://doi.org/10.1038/s41587-021-01147-4

@article{gamaarachchi2022fast,
  title={Fast nanopore sequencing data analysis with SLOW5},
  author={Gamaarachchi, Hasindu and Samarakoon, Hiruna and Jenner, Sasha P and Ferguson, James M and Amos, Timothy G and Hammond, Jillian M and Saadat, Hassaan and Smith, Martin A and Parameswaran, Sri and Deveson, Ira W},
  journal={Nature biotechnology},
  pages={1--4},
  year={2022},
  publisher={Nature Publishing Group}
}

Building

Building slow5lib requires a compiler that supports C99 standard (with X/Open 7 POSIX 2008 extensions), which is widely available. To build the C/C++ library :

sudo apt-get install zlib1g-dev   #install zlib development libraries
git clone https://github.com/hasindu2008/slow5lib
cd slow5lib
make

This will generate lib/libslow5.a for static linking and libslow5.so for dynamic linking.

The commands to zlib development libraries on some popular distributions :

On Debian/Ubuntu : sudo apt-get install zlib1g-dev
On Fedora/CentOS : sudo dnf/yum install zlib-devel
On OS X : brew install zlib

Optional zstd compression

You can optionally enable zstd compression support when building slow5lib by invoking make zstd=1. This requires zstd 1.3 or higher development libraries installed on your system:

On Debian/Ubuntu : sudo apt-get libzstd1-dev # libzstd-dev on newer distributions if libzstd1-dev is unavailable
On Fedora/CentOS : sudo yum libzstd-devel
On OS X : brew install zstd

SLOW5 files compressed with zstd offer smaller file size and better performance compared to the default zlib. However, zlib runtime library is available by default on almost all distributions unlike zstd and thus files compressed with zlib will be more ‘portable’.

Without SIMD

slow5lib from version 0.3.0 onwards uses code from StreamVByte and by default requires vector instructions (SSSE3 or higher for Intel/AMD and neon for ARM). If your processor is an ancient processor with no such vector instructions, invoke make as make no_simd=1.

Usage

Simply include <slow5/slow5.h> in your C program and call the API functions. To compile your program and statically link against slow5lib:

gcc [OPTIONS] -I path/to/slow5lib/include your_program.c path/to/slow5lib/lib/libslow5.a -lm -lz

path/to/slow5lib/ is the absolute or relative path to the slow5lib repository cloned above. To dynamically link:

gcc [OPTIONS] -I path/to/slow5lib/include your_program.c -L path/to/slow5lib/lib/ -lslow5 -lm -lz

If you compiled slow5lib with zstd support enabled, make sure you append -lzstd to the above two commands.

For the documentation of the C API visit here and for the Python API visit here.

Examples

A public template repository is available at [https://github.com/hasindu2008/slow5-template] which you can directly use to setup your own repository that uses slow5lib to build a tool. Check the instructions and comments there.

Examples are provided under examples.

• sequential_read.c demonstrates how to read a slow5/blow5 file, sequentially from start to end.
• random_read.c demonstrates how to fetch a given read ID from a slow5/blow5 file.
• header_attribute.c demonstrates how to fetch a header data attribute from a slow5/blow5 file.
• auxiliary_field.c demonstrates how to fetch a auxiliary field from a slow5/blow5 file.
• random_read_pthreads.c demonstrates how to fetch given read IDs in parallel from a slow5/blow5 file using pthreads.
• random_read_openmp.c demonstrates how to fetch given read IDs in parallel from a slow5/blow5 file using openMP.
• write.c demonstrate how to write a new slow5/blow5 file.
• append.c demonstrates how to append to an existing slow5/blow5 file.

Some advanced examples are provided here. Following examples will be added upon request. If you are interested, open a GitHub issue rather than spending your time trying to figure out - will try to provide within a day or two.

• accessing header attributes when multiple read groups are present.
• an efficient input, processing, output pipeline for processing raw signals using slow5lib
• any other use case.

You can invoke examples/build.sh to compile the example programmes. Have a look at the script to see the commands used for compiling and linking. If you compiled slow5lib with zstd support enabled, make sure you append -lzstd to the compilation commands.

pyslow5

Python wrapper for slow5lib or pyslow5 can be installed using conda as conda install pyslow5 -c bioconda -c conda-forge or pypi as pip install pyslow5. The instructions to build pyslow5 and the usage instructions are here.

Other languages

A slow5 library for RUST programming language developed by @bsaintjo is available here. A slow5 library in GO language developed by @Koeng101 is available here. We are highly grateful to these community efforts. If anyone is interested in a library from another language, feel free to open an issue.

Current limitations & future work

slow5lib is a reference implementation for SLOW5 format. Depending on the interest from the community, the following limitations could be overcome and more performance optimisations can be performed. Open a GitHub issue if you are interested. Contributions are welcome.

• No native windows support: slow5lib works well on Windows through WSL, in fact, this is my primary development environment. I am not aware of anyone using native Windows for nanopore bioinformatics. However, if needed, methods used for minimap2 can be adopted.
• Does not support big-endian systems: Big-endian systems are rare nowadays and I do not have access to one to test. If necessary, it is a matter of writing a layer that swaps the bytes before/after writing to disk. Note: Not to be confused with big.LITTLE architecture which is something else on which slow5lib already works.
• When running with >64 threads, malloc() calls could reduce the thread efficiency. If that is the case, frequent mallocs could be replaced with kalloc in klib.
• Aggressive compiler optimisations (e.g., -O3) and architecture-specific compiler optimisations (e.g., -march=native) are not used in the makefile. These flags will improve performance at the cost of limited portability. These could be provided in a separate make target.

Notes

slow5lib from version 0.3.0 onwards has built in StreamVByte compression support to enable even smaller file sizes, which is applied to the raw signal by default when producing BLOW5 files. zlib compression is then applied by default to each SLOW5 record. If zstd is used instead of zlib on top of StreamVByte, it is similar to ONT’s latest vbz compression. BLOW5 files with zstd+StreamVByte are still about 25% smaller than vbz compressed FAST5 files.

Acknowledgement

slow5lib uses klib and StreamVByte. Some code snippets have been taken from Minimap2 and Samtools.