The tutorial provides a short introduction to Fast5 files used to store raw data output of Oxford Nanopore Technologies' sequencing devices. The tutorial aims to provide background information for why users may have cause to interact with Fast5 files and show how to perform common manipulations.
Methods used in this tutorial include:
ont_fast5_api for manipulating read information within Fast5 files.The computational requirements for this tutorial are:
⚠️ Warning: This notebook has been saved with its outputs for demostration purposed. It is recommeded to select
Edit > Clear all outputsbefore using the notebook to analyse your own data.
This tutorial aims to elucidate the information stored within a Fast5 file, and how such files can be read, or parsed, within the Python programming language and on the command line.
The goals from this tutorial include:
ont_fast5_api,The tutorial includes a sample Fast5 dataset from a metagenomic sample.
Before anything else we will create and set a working directory:
from epi2melabs import ping
tutorial_name = "fast5_tutorial"
pinger = ping.Pingu()
pinger.send_notebook_ping('start', tutorial_name)
# create a work directory and move into it
working_dir = '/epi2melabs/{}/'.format(tutorial_name)
!mkdir -p "$working_dir"
%cd "$working_dir"
/epi2melabs/fast5_tutorial
This tutorial uses the ont_fast5_api software; this is not installed in the default EPI2ME Labs environment. We will install this now in an isolated manner so as to not interfere with the existing environment.
Please note that the software installed is not persistent and this step will need to be re-run if you stop and restart the EPI2ME Labs server.
# create a conda environment and install ont_fast5_api into it
!conda remove -y --name ont_fast5_api --all
!conda create -q -y -n ont_fast5_api python==3.6 pip 2>/dev/null
!. /opt/conda/etc/profile.d/conda.sh \
&& conda activate ont_fast5_api \
&& which pip \
&& pip install "ont_fast5_api>=3.1.6"
In order to provide a concrete example of handling a Fast5 files this tutorial is provided with an example dataset sampled from a MinION sequencing run: the dataset is not a full MinION run in order to reduced the download size.
To download the sample file we run the linux command wget. To execute the command click on the cell and then press Command/Ctrl-Enter, or click the Play symbol to the left-hand side.
bucket = "ont-exd-int-s3-euwst1-epi2me-labs"
domain = "s3-eu-west-1.amazonaws.com"
site = "https://{}.{}".format(bucket, domain)
site = "https://ont-exd-int-s3-euwst1-epi2me-labs.s3-eu-west-1.amazonaws.com"
!rm -rf sample_fast5
!wget -O sample_fast5.tar $site/fast5_tutorial/sample_fast5.tar
!tar -xvf sample_fast5.tar
!wget -O fast5_sample.bam $site/fast5_tutorial/fast5_sample.bam
!wget -O fast5_sample.bam.bai $site/fast5_tutorial/fast5_sample.bam.bai
Having downloaded the sample data we need to provide the filepaths as input to the notebook.
The form can be used to enter the filenames of your inputs.
input_folder = None
output_folder = None
def process_form(inputs):
global input_folder
global output_folder
input_folder = inputs.input_folder
output_folder = inputs.output_folder
# run a command to concatenate all the files together
!cecho ok "Making output folder"
!mkdir -p "$output_folder"
!test -d "$input_folder" \
&& cecho success "Found input folder." \
|| cecho error "Input folder does not exist."
!echo " - Found "$(find "$input_folder" -name "*.fast5" | wc -l)" fast5 files"
from epi2melabs.notebook import InputForm, InputSpec
input_form = InputForm(
InputSpec('input_folder', 'Input folder', '/epi2melabs/fast5_tutorial/sample_fast5'),
InputSpec('output_folder', 'Output folder', 'analysis'))
input_form.add_process_button(process_form)
input_form.display()
VBox(children=(HBox(children=(Label(value='Input folder', layout=Layout(width='150px')), interactive(children=…
Executing the above form will have checked the input folder attempted to find Fast5 files located in the folder.
Fast5 files are used by the MinKNOW instrument software and the Guppy basecalling software to store the primary sequencing data from Oxford Nanopore Technologies' sequencing devices and the results of primary and secondary analyses such as basecalling information and modified-base detection.
Before discussing how to read and manipulate Fast5 files in Python we will first review their internal structure.
Files output by the MinKNOW instrument software and the Guppy basecalling software using the .fast5 file extension are a container file using the HDF5 format. As such they are a self-describing file with all the necessary information to correctly interpret the data they contain.
A Fast5 file differs from a generic HDF5 file in containing only a fixed, defined structure of data. This structure is elucidated in the ont_h5_validator repository on Github, specifically in the file multi_read_fast5.yaml.
Users are referred to the YAML schemas to gain an understanding of all the data contained in Fast5 files. Users are encouraged to raise Issues on the ont_h5_validator project if the schemas are unclear. The rest of this tutorial will be mostly practical in nature.
The schema file describes how the internal structure of a Fast5 file is laid out. There are three core concepts to understand:
An appreciation of these concepts is required for using the data contained within Fast5 files, though as we will see for common manipulations of Fast5 files users need only an awareness of these ideas.
Historically there have been two flavours of Fast5 files:
The internal layout, in terms of groups and datasets, of these two flavours of Fast5 are very similar. In essence a multi-read file embeds the group hierarchy of multiple single-read files within one HDF5 container.
Single-read files are deprecated and no longer used by MinKNOW or Guppy. We recommend that any single-read files are converted to multi-read files before further use or storage, how to do this is demonstrated later in this tutorial.
As noted above the ont_h5_validator project contains a full description of the expected contents of a Fast5 file. Here we will briefly highlight the key groups and datasets stored within a Fast5 file.
Using the dataset provided in above let's enumerate the contents of the first file using the h5ls program:
# i) find and list all .fast5 files
# ii) take the first file
# iii) use `h5ls` to list the file's contents
# iv) truncate the output to the first 19 lines
!find "$input_folder" -name "*.fast5" \
| head -n 1 \
| xargs h5ls -r \
| head -n 19
The search term refers to an legacy archive, often associated with a third-party utility designed to retrieve or bypass passwords on Siemens SIMATIC S7-200 Go to product viewer dialog for this item. and Go to product viewer dialog for this item. PLCs by reading the Micro Memory Card (MMC). Key Features and Functionality
MMC Image Reading: The tool typically functions by creating a raw image of the Siemens MMC card using standard hex editing software (like WinHex). Password Retrieval
: It identifies and extracts the password hash or cleartext from specific memory offsets within the MMC image file.
Support for Pre-2009 Hardware: These tools are primarily effective against older versions (e.g., pre-2009) where security was less robust.
Direct Unlock: Unlike a factory reset, which deletes the entire program, these utilities aim to provide the password so you can access and upload the existing logic from the PLC. Common Use Cases
Legacy Maintenance: Accessing programs from machines where the original manufacturer is no longer in business and the documentation is lost.
Password Recovery: Retrieving a forgotten password to allow program modifications or backups without wiping the device. Standard Alternatives The search term refers to an legacy archive,
For modern systems or cases where third-party tools are not used, the standard Siemens procedures are: Default Passwords: Older versions sometimes use a default password like Basisk.
Factory Reset: If the password is unknown and the program is not needed, you can perform a memory reset (MRES) using the physical switch on the CPU to wipe the MMC and clear the password. Wipeout Utility : For
systems, a specific "Wipeout.exe" utility can be used to reset the CPU to factory defaults. S7-300 Password unlocking | PLCtalk - Interactive Q & A
In the mid-2000s, the Simatic S7-200 and S7-300 series were the workhorses of global industrial automation, controlling everything from factory assembly lines to critical infrastructure. The "unlock" RAR files from 2006 represent a turning point in industrial cybersecurity, marking the era when the proprietary "security by obscurity" of Programmable Logic Controllers (PLCs) began to crumble. The 2006 "Unlock" Artifact
The specific RAR files referenced (often titled S7_Unlock or S7ImgRd) were tools developed by independent researchers and enthusiasts to bypass Siemens' protection mechanisms. At the time, if an engineer lost the password to a PLC, there was no "official" recovery—the only choice was a factory reset that wiped the proprietary logic. These tools exploited two main vulnerabilities:
The MMC Image Hack: For the S7-300, the password wasn't just in the CPU; it was stored on the Micro Memory Card (MMC). Hackers realized they could use standard card readers and software like WinHex to create a raw image of the MMC. Send a special "stop" command to the CPU
Binary Extraction: Tools like S7ImgRd1.exe would scan the raw binary image of the card, locate the specific hex offset where the password was stored, and translate it back into plain text. Why This Mattered
Intellectual Property Theft: These files allowed competitors or curious parties to upload and decompile the "Know-How Protected" code blocks that companies spent years developing.
Legacy Maintenance: Ironically, these "hacking tools" became essential for maintenance teams at aging plants where the original programmers had disappeared, leaving behind locked, undocumented systems.
A Pre-Stuxnet Warning: This 2006 era of password-cracking tools was the precursor to much more sophisticated attacks, like the 2010 Stuxnet worm, which specifically targeted Siemens S7 systems by exploiting similar industrial protocols. Modern Safety Measures
Today, Siemens has largely moved away from these vulnerabilities. Newer models like the S7-1200 and S7-1500 use advanced encryption and digital certificates within the TIA Portal environment to prevent simple binary extraction. S7-300 MMC Password Recovery Guide | PDF - Scribd
The Simatic S7 series by Siemens is a line of programmable logic controllers (PLCs) widely used in industrial automation. The MMC cards are used for storing project data, recipes, and sometimes for logging. For Simatic S7 Devices:
If you're looking to unlock or access password-protected RAR files related to these devices, here are some general steps you can follow:
The S7-200 stores the password in the system block of its EEPROM. Unofficial unlockers use PC/PPI cable (RS-232 or USB) with a custom protocol:
Siemens SIMATIC S7 PLCs (S7-200, S7-300) often use MMC or similar memory modules to store user programs, data blocks, and configuration. Sometimes MMC contents are archived into RAR files for transport or backup. Password protection may be applied to protect projects and block contents. This post explains safe, legal approaches to recover access, extract archived RAR files, and restore PLC program access when you have proper authorization.
Many .rar files from 2006-2010 contain packed executables that modern antivirus flags as Trojan.PLC or Generic.Malware. Some are false positives (due to kernel-level USB access), but others are genuine keyloggers or ransomware. Always sandbox in a VM.
Rather than chasing a risky RAR from "2006-09-11", consider these legitimate approaches:
| Method | Applicability | Difficulty | Cost | |--------|--------------|------------|------| | Siemens Customer Support | S7-200 & S7-300 with proof of purchase | Medium | Free/Paid | | SIMATIC MMC Card Reader + S7IMGPRG (official) | S7-300 only – but erases data | Low | Official Siemens tool | | Third-party commercial unlockers (e.g., MMC PW Check, S7 Unlock Pro) | Both families – safe, documented | Medium | $100-500 USD | | Upload via MPI/DP with brute-force (using tools like S7Crack) | S7-300 only – very slow | High | Free (risky) |
The "2006-09-11.rar" method is essentially a relic. It is useful for historians or hobbyists running air-gapped Windows XP machines with legacy S7-200 CPUs. For a professional plant engineer, the risk of corrupting production code is simply too high.
The Fast5 files from a MinION run can become fairly sizeable, up to a few hundred gigabytes. Efficient and performant compression and indexing is therefore required.
For the most part the self describing and indexed nature of the HDF5 format ensures that data within a file can be quickly retrieved. However for a MinION run multiple Fast5 files are created each with a subset of the sequencing reads produced by the sequencer. Therefore finding the information pertaining to a read of a known ID cannot be done without a supplementary index cross-referencing the reads contained within in file; the alternative is to open all the files in turn and enquire about their contents. *The sequencing_summary.txt file produced by both MinKNOW and Guppy provides an index of the reads contained within in each Fast5 file*. This index can of course be reconstructed if required (as in the case of nanopolish index), though we recommend always storing the sequencing summary with the Fast5 data files.
Due to the large volume of data created by nanopore sequencing devices Oxford Nanopore Technologies has developed a bespoke compression scheme for ionic current trace data known as VBZ. VBZ is a combination of two open compression algorithms and is itself open and freely available from the Github release page. Ordinarily it will not be necessary to install the VBZ compression library and HDF5 plugin to simply use MinKNOW and Guppy as these software applications include their own copy of VBZ. However if you wish to read Fast5 files using third party applications (such as h5py) you will need to install the VBZ plugin.
The section above has given an outline to the data contained within a Fast5 file and how the file is arranged. Again for a more fulsome description of the contents of files users are directed to the ont_h5_validator project. In this section we will highlight several methods for manipulating the data contained within Fast5 files.
Oxford Nanopore Technologies provides a Python-based software for accessing data stored within a set of Fast5 files: ont_fast5_api. For the most part this set of tools hides from the user the need to understand anything about the nature of Fast5 files. Here we will show how to perform some common tasks that might be required when dealing with Fast5 files. For a guide in using ont_fast5_api programmatically please see the documention.
Since some older programs have not been updated to use multi-read files it can sometimes be necessary to convert such files to the deprecated single-read flavour. To do this run:
!rm -rf $output_folder/single-reads
!run multi_to_single_fast5 \
--input_path $input_folder --save_path $output_folder/single-reads \
--recursive
The output of the above command is a set of folders each containing a subset of the sequencing reads, one read per file. The filename of each read corresponds to the read's unique identifier.
!ls $output_folder/single-reads/0 2>/dev/null | head -n 5
00058fe1-e555-4a64-a41b-7f58fb7d6d6b.fast5 000dd482-c0d5-4520-aa86-8ee8bb61fd58.fast5 00158d74-4b7f-445a-b0ac-e1606f6c09b7.fast5 004a0bd2-edcf-4c2c-89bc-009a232cdb6a.fast5 0057b9d1-e566-4518-8b81-f69b30c6da99.fast5
A similar program exists to convert single-read files to multi-read files. We recommend that all datasets are updated to multi-read files for longer term storage. Here we will convert the single-reads created above back to multi-read files:
!rm -rf $output_folder/multi-reads
!run single_to_multi_fast5 \
--input_path $output_folder/single-reads --save_path $output_folder/multi-reads \
--filename_base prefix --batch_size 8000 --recursive
| 3 of 3|####################################################|100% Time: 0:00:55
The output of this command is a single directory containing all multi-read files. The filenames are prefixed with prefix as taken by the --filename_base argument of the program. The --batch_size argument here controls the number of reads per file:
!ls $output_folder/multi-reads
filename_mapping.txt prefix_0.fast5 prefix_1.fast5 prefix_2.fast5
The filename_mapping.txt cross-references the data from the input files with the output files.
!head $output_folder/multi-reads/filename_mapping.txt
26cb0f7d-8db2-4e2d-aa4e-9d273ccf1d66.fast5 analysis/multi-reads/prefix_0.fast5 b4441e24-a5d3-4357-bc24-4a169520d096.fast5 analysis/multi-reads/prefix_0.fast5 5d63b4ae-e9c7-43cb-b73c-7b3bc7facd57.fast5 analysis/multi-reads/prefix_0.fast5 5880c8b8-5c67-45cd-9082-2be09a7fc1d4.fast5 analysis/multi-reads/prefix_0.fast5 77d557c6-2154-4792-ad2d-49c9ca5f4bdd.fast5 analysis/multi-reads/prefix_0.fast5 afa10699-8648-4e7a-8bec-86118f202e8d.fast5 analysis/multi-reads/prefix_0.fast5 fb15566d-370c-478e-a190-d4221407e500.fast5 analysis/multi-reads/prefix_0.fast5 34465bd4-2335-4390-8675-daef5390ea79.fast5 analysis/multi-reads/prefix_0.fast5 67b3c07c-c4db-40e9-a18b-c10c8eeb70f5.fast5 analysis/multi-reads/prefix_0.fast5 133ac0a7-54d4-4681-8653-49b174fe6e7c.fast5 analysis/multi-reads/prefix_0.fast5
As mentioned in the discussion above it can be useful to have an index of which reads are contained within which multi-read files. Usually this indexing is provided by the sequencing_summary.txt file output by MinKNOW and Guppy. However if it is lost, here's a way to recover the information:
# build a script that will do the work
with open("build_read_index.sh", 'w') as fh:
fh.write(
'''
echo -e "filename\tread_id"
find $1 -name "*.fast5" \\
| parallel --tag h5ls -f -r \\
| grep "read_.\{8\}-.\{4\}-.\{4\}-.\{4\}-.\{12\} Group" \\
| sed "s# Group##" | sed "s#/read_##"
''')
# run the script
!bash build_read_index.sh $input_folder > read_index.txt
The read_index.txt output file contains the simple index we desire:
!head read_index.txt
filename read_id /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00085dbe-217a-40f2-90c0-3bb15669f32c /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00237911-92b3-49b4-9d13-2ea6a2ded996 /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0025338c-3ea8-4168-b999-fe7f7fd597ee /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00408494-e245-401e-8c9a-575ee491971b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00485ea4-a2fc-4b75-9969-9f1b1ab997da /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 004fbd46-3565-4505-8ade-bfa5bffa499b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0067fb48-9e65-415a-966a-fbf25c62e730 /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 0091aa27-0f2f-4e79-bb6e-6bfa1629326b /epi2melabs/fast5-tutorial/sample_fast5/workspace/FAK42335_2bf4f211a2e2d04662e50f27448cfd99dafbd7ee_400.fast5 00a52e30-a584-4ed8-97cf-074c601b0403
The program fast5_subset within ont_fast5_api can be used to create a new file set containing only a subset of reads.
The sample data contains data from a microbial mock community. Using the accompanying BAM alignment file lets find the reads with align to a single reference sequence:
!rm -rf read_list.txt
!echo "read_id" > read_list.txt
!samtools view fast5_sample.bam lfermentum \
| awk '{print $1}' \
| tee -a read_list.txt \
| echo "Found" $(wc -l) "reads"
Found 1100 reads
We can now use this file with the subsetting program:
!echo $input_folder
!rm -rf $output_folder/lfermentum
!run fast5_subset --input $input_folder --save_path $output_folder/lfermentum \
--read_id_list read_list.txt --batch_size 8000 --recursive
/epi2melabs/fast5_tutorial/sample_fast5 | 1105 of 1105|##############################################|100% Time: 0:00:02 INFO:Fast5Filter:1100 reads extracted
Analyses groups¶It can be the case that it is desirable to remove the Analyses groups from multi-read files. For example if live basecalling were performed during a run but these results are not wanted before data is archived.
To accomplish this task we will use the compress_fast5 program with the --sanitize option:
!rm -rf $output_folder/sanitized
!run compress_fast5 --input_path $input_folder --save_path $output_folder/sanitize \
--compression vbz --recursive --threads 8 --sanitize
| 5 of 5|####################################################|100% Time: 0:00:12
This achieves an approximate 3.5X reduction in filesize:
!du -sh $input_folder $output_folder/sanitize
2.4G /epi2melabs/fast5_tutorial/sample_fast5 682M analysis/sanitize
In this notebook we have introduced the Variant Call Format with an examplar file from the Medaka consensus and variant calling program. We have outlined the contents of such files and how they can be intepreted with a selection of common software packages.
The code tools presented here can be run on any dataset from an Oxford Nanopore Technologies' device. The code will run within the EPI2ME Labs notebook server environment.