The enduring advantages of the SLOW5 file format for raw nanopore sequencing data
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Abstract
Nanopore sequencing is a widespread and important method in genomics science. The raw electrical current signal data from a typical nanopore sequencing experiment is large and complex. This can be stored in two alternative file formats that are presently supported: POD5 is a signal data file format used by default on instruments from Oxford Nanopore Technologies (ONT); SLOW5 is an open-source file format originally developed as an alternative to ONT’s previous file format, which was known as FAST5. The choice of format may have important implications for the cost, speed and simplicity of nanopore signal data analysis, management and storage. To inform this choice, we present a comparative evaluation of POD5 vs SLOW5. We conducted benchmarking experiments assessing file size, analysis performance and usability on a variety of different computer architectures. SLOW5 showed superior performance during sequential and non-sequential (random access) file reading on most systems, manifesting in faster, cheaper basecalling and other analysis, and we could find no instance in which POD5 file reading was significantly faster than SLOW5. We demonstrate that SLOW5 file writing is highly parallelisable, thereby meeting the demands of data acquisition on ONT instruments. Our analysis also identified differences in the complexity and stability of the software libraries for SLOW5 (slow5lib) and POD5 (pod5), including a large discrepancy in the number of underlying software dependencies, which may complicate the pod5 compilation process. In summary, many of the advantages originally conceived for SLOW5 remain relevant today, despite the replacement of FAST5 with POD5 as ONT’s core file format.
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ABSTRACTNanopore sequencing is a widespread and important method in genomics science. The raw electrical current signal data from a typical nanopore sequencing experiment is large and complex. This can be stored in two alternative file formats that are presently supported: POD5 is a signal data file format used by default on instruments from Oxford Nanopore Technologies (ONT); SLOW5 is an open-source file format originally developed as an alternative to ONT’s previous file format, which was known as FAST5. The choice of format may have important implications for the cost, speed and simplicity of nanopore signal data analysis, management and storage. To inform this choice, we present a comparative evaluation of POD5 vs SLOW5. We conducted benchmarking experiments assessing file size, analysis performance and usability on a variety of …
ABSTRACTNanopore sequencing is a widespread and important method in genomics science. The raw electrical current signal data from a typical nanopore sequencing experiment is large and complex. This can be stored in two alternative file formats that are presently supported: POD5 is a signal data file format used by default on instruments from Oxford Nanopore Technologies (ONT); SLOW5 is an open-source file format originally developed as an alternative to ONT’s previous file format, which was known as FAST5. The choice of format may have important implications for the cost, speed and simplicity of nanopore signal data analysis, management and storage. To inform this choice, we present a comparative evaluation of POD5 vs SLOW5. We conducted benchmarking experiments assessing file size, analysis performance and usability on a variety of different computer architectures. SLOW5 showed superior performance during sequential and non-sequential (random access) file reading on most systems, manifesting in faster, cheaper basecalling and other analysis, and we could find no instance in which POD5 file reading was significantly faster than SLOW5. We demonstrate that SLOW5 file writing is highly parallelisable, thereby meeting the demands of data acquisition on ONT instruments. Our analysis also identified differences in the complexity and stability of the software libraries for SLOW5 (slow5lib) and POD5 (pod5), including a large discrepancy in the number of underlying software dependencies, which may complicate the pod5 compilation process. In summary, many of the advantages originally conceived for SLOW5 remain relevant today, despite the replacement of FAST5 with POD5 as ONT’s core file format.
This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf118), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:
Reviewer 2: Jan Voges
Comments to Author: Synopsis: The manuscript builds on the authors' previous work introducing the SLOW5 format for Oxford Nanopore signal data as an improvement over the FAST5 format. Since then, Oxford Nanopore Technologies (ONT) has introduced its own new format, POD5. This paper directly compares SLOW5 and POD5. The authors claim that SLOW5 provides higher reading speeds for both sequential and random access, writing speeds sufficient to keep pace with data acquisition in sequencing machines, comparable file sizes with no significant storage penalty, a simpler implementation with fewer dependencies. The paper is clearly written, includes extensive supplementary information, and references the source code for all tools used in the experiments. Comments:
- Sequential access performance: To me it is unclear whether SLOW5's advantage in sequential access originates from its file layout or from the use of mmap I/O versus traditional I/O. A small ablation study, forcing both SLOW5 and POD5 tools to use the same I/O method on platforms with currently large performance differences, would clarify where the performance gain originates from.
- Figure 4: While POD5's dependency structure is indeed more complex than that of slow5lib, the current tree representation exaggerates this complexity. Many common packages (e.g., Python, zlib) appear multiple times as dependency of multiple other packages. A dependency graph where each package appears only once would be a more informative representation.
- Figure 5: POD5 versions prior to 0.1.0 appear to be preview releases (and are even marked as such on GitHub). Breaking changes during early previews are normal, so including them in the same visual space as stable versions risks being misleading.
- Figure 5: Breaking change at version 0.1.12: The timeline indicates a breaking change at POD5 version 0.1.12 which seems particularly relevant as the latest breaking change after version 0.1.0. However, this change is not reflected in the POD5 compatibility matrix on the right. An explanation of what type of breaking change occurred would clarify its impact and help readers assess compatibility risk.
- Random access "walker strategy": A brief explanation comparing it to SLOW5's index-file approach would improve accessibility without requiring readers to consult external documentation.
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ABSTRACTNanopore sequencing is a widespread and important method in genomics science. The raw electrical current signal data from a typical nanopore sequencing experiment is large and complex. This can be stored in two alternative file formats that are presently supported: POD5 is a signal data file format used by default on instruments from Oxford Nanopore Technologies (ONT); SLOW5 is an open-source file format originally developed as an alternative to ONT’s previous file format, which was known as FAST5. The choice of format may have important implications for the cost, speed and simplicity of nanopore signal data analysis, management and storage. To inform this choice, we present a comparative evaluation of POD5 vs SLOW5. We conducted benchmarking experiments assessing file size, analysis performance and usability on a variety of …
ABSTRACTNanopore sequencing is a widespread and important method in genomics science. The raw electrical current signal data from a typical nanopore sequencing experiment is large and complex. This can be stored in two alternative file formats that are presently supported: POD5 is a signal data file format used by default on instruments from Oxford Nanopore Technologies (ONT); SLOW5 is an open-source file format originally developed as an alternative to ONT’s previous file format, which was known as FAST5. The choice of format may have important implications for the cost, speed and simplicity of nanopore signal data analysis, management and storage. To inform this choice, we present a comparative evaluation of POD5 vs SLOW5. We conducted benchmarking experiments assessing file size, analysis performance and usability on a variety of different computer architectures. SLOW5 showed superior performance during sequential and non-sequential (random access) file reading on most systems, manifesting in faster, cheaper basecalling and other analysis, and we could find no instance in which POD5 file reading was significantly faster than SLOW5. We demonstrate that SLOW5 file writing is highly parallelisable, thereby meeting the demands of data acquisition on ONT instruments. Our analysis also identified differences in the complexity and stability of the software libraries for SLOW5 (slow5lib) and POD5 (pod5), including a large discrepancy in the number of underlying software dependencies, which may complicate the pod5 compilation process. In summary, many of the advantages originally conceived for SLOW5 remain relevant today, despite the replacement of FAST5 with POD5 as ONT’s core file format.
This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf118), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:
Reviewer 1: Wouter De Coster
The authors describe the SLOW5 format and its benefits compared to the standard POD5 format for storing raw sequencing data from nanopore sequencers. The paper is well written and easy to understand. The advantages of the SLOW5 format are clear, and the comparison is adequately executed and described. However, the developers seem unable to persuade others to adopt it widely, and change might need to come from ONT themselves, who may be most concerned about disrupting their existing workflows, especially for parallel writing during sequencing. Nevertheless, the authors seem to have also addressed that issue, as demonstrated with a simulation experiment.
Please find my specific suggestions below.
Sincerely, Wouter De Coster
Major: While I understand that the software name SLOW5 was an initial variation of the FAST5 format, I don't think that the words 'slow' or the number '5' are particularly appropriate descriptions or helpful in making a case for using the file format, as it is neither slow nor related to HDF5. However, once a name is chosen, I understand the reluctance to change it. Additionally, it seems the evaluations are conducted using the binary BLOW5 format. Wouldn't it then make more sense to emphasize BLOW5 in the text and title?
Minor: I would italicize the 'make' tool for users unfamiliar with build tools in the Usability section, as it is a rather strange sentence if reading 'make' as a verb, not a tool. Perhaps the same could be applied to other dependencies in that section for consistency. Then again, the primary target audience will probably understand what 'make' means in this context.
There is a typo in the benchmarking procedure section: 'confoudning'.
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