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If you are anything like us (Matt), reading a README is the last thing you do when running code. PLEASE DON'T DO THAT FOR READFISH. This will effect changes to your sequencing and - if you use it incorrectly - cost you money. We have added a list of GOTCHAs at the end of this README. We have almost certainly missed some... so - if something goes wrong, let us know so we can add you to the GOTCHA hall of fame!

Note

We also have more detailed documentation for your perusal at https://looselab.github.io/readfish

Note

Now also see our cool FAQ.

readfish is a Python package that integrates with the Read Until API.

The Read Until API provides a mechanism for an application to connect to a MinKNOW server to obtain read data in real-time. The data can be analysed in the way most fit for purpose, and a return call can be made to the server to unblock the read in progress and so direct sequencing capacity towards reads of interest.

This implementation of readfish requires Guppy version >= 6.0.0 and MinKNOW version core >= 5.0.0 . It will not work on earlier versions.

The code here has been tested with Guppy in GPU mode using GridION Mk1 and NVIDIA RTX2080 on live sequencing runs and an NVIDIA GTX1080 using playback on a simulated run (see below for how to test this). This code is run at your own risk as it DOES affect sequencing output. You are strongly advised to test your setup prior to running (see below for example tests).

Supported Sequencing Platforms

The following platforms are supported:

  • PromethION Big Boy
  • GridION Box
  • MinION Smol Boy

Warning

PromethION support is currently only available using the Mappy-rs plugin only. See here for more information.

Supported OS's

The following OSs are supported:

  • Linux yay
  • MacOS boo

Note

Note - MacOS supports is on MinKNOW 5.7 and greater using Dorado basecaller only.

Citation

The paper is available at nature biotechnology and bioRxiv

If you use this software please cite: 10.1038/s41587-020-00746-x

Readfish enables targeted nanopore sequencing of gigabase-sized genomes Alexander Payne, Nadine Holmes, Thomas Clarke, Rory Munro, Bisrat Debebe, Matthew Loose Nat Biotechnol (2020); doi: https://doi.org/10.1038/s41587-020-00746-x

Other works

An update preprint is available at bioRxiv

Barcode aware adaptive sampling for Oxford Nanopore sequencers Alexander Payne, Rory Munro, Nadine Holmes, Christopher Moore, Matt Carlile, Matthew Loose bioRxiv (2021); doi: https://doi.org/10.1101/2021.12.01.470722

Installation

Our preferred installation method is via conda.

The environment is specified as:

name: readfish
channels:
  - bioconda
  - conda-forge
  - defaults
dependencies:
  - python=3.10
  - pip
  - pip:
    - readfish[all]

Saving the snippet above as readfish_env.yml and running the following commands will create the environment.

conda env create -f readfish_env.yml
conda activate readfish

Installing with development dependencies

A conda yaml file is available for installing with dev dependencies - development.yml

curl -LO https://raw.githubusercontent.com/LooseLab/readfish/e30f1fa8ac7a37bb39e9d8b49251426fe1674c98/docs/development.yml?token=GHSAT0AAAAAACBZL42IS3QVM4ZGPPW4SHB6ZE67V6Q
conda env create -f development.yml
conda activate readfish_dev

‼️ Important!

The listed ont-pyguppy-client-lib version will probably not match the version installed on your system. To fix this, Please see this issue

ONT's Guppy GPU should be installed and running as a server.

Alternatively, readfish can be installed into a python virtual-environment

 
# Make a virtual environment
python3 -m venv readfish
. ./readfish/bin/activate
pip install --upgrade pip

# Install our readfish Software
pip install readfish[all]

# Install ont_pyguppy_client_lib that matches your guppy server version. E.G.
pip install ont_pyguppy_client_lib==6.3.8

Usage

 
usage: readfish [-h] [--version]  ...

positional arguments:
                   Sub-commands
    targets        Run targeted sequencing
    barcode-targets
                   Run targeted sequencing
    unblock-all    Unblock all reads
    validate       readfish TOML Validator

options:
  -h, --help       show this help message and exit
  --version        show program's version number and exit

See '<command> --help' to read about a specific sub-command.

TOML File

For information on the TOML files see TOML.md. There are several example TOMLS, with comments explaining what each field does, as well as the overall purpose of the TOML file here - https://github.com//LooseLab/readfish/tree/refactor/docs/_static/example_tomls.

Testing

 To test readfish on your configuration we recommend first running a playback experiment to test unblock speed and then selection.

The following steps should all happen with a configuration (test) flow cell inserted into the target device. A simulated device can also be created within MinKNOW, following these instructions. This assumes that you are runnning MinKNOW locally, using default ports. If this is not the case a developer API token is required on the commands as well, as well as setting the correct port.

  1. Linux

    In the readfish virtual environment we created earlier:

    • See help
    python -m minknow_api.examples.manage_simulated_devices --help
    • Add Minion position
    python -m minknow_api.examples.manage_simulated_devices --add MS00000
    • Add PromethION position
    python -m minknow_api.examples.manage_simulated_devices --prom --add S0

  2. Mac

    In the readfish virtual environment we created earlier:

    • See help
    python -m minknow_api.examples.manage_simulated_devices --help
    • Add Minion position
    python -m minknow_api.examples.manage_simulated_devices --add MS00000
    • Add PromethION position
    python -m minknow_api.examples.manage_simulated_devices --prom --add S0

As a back up it is possible to restart MinKNOW with a simulated device. This is done as follows:

  1. Stop minknow

    On Linux:

    cd /opt/ont/minknow/bin
sudo systemctl stop minknow

  2. Start MinKNOW with a simulated device

    On Linux

    sudo ./mk_manager_svc -c /opt/ont/minknow/conf --simulated-minion-devices=1 &

You may need to add the host 127.0.0.1 in the MinKNOW UI.

Configuring bulk FAST5 file Playback

Download an open access bulk FAST5 file. This file is 21Gb so make sure you have sufficient space.

Previously to set up Playback using a pre-recorded bulk FAST5 file, it was necessary to edit the sequencing configuration file that MinKNOW uses. This is no longer the case. The following steps are left after this section for reference only.

To start sequencing using playback, simply begin setting up the run in the MinKNOW UI as you would usually. Under Run Options you can select Simulated Playback and browse to the downloaded Bulk Fast5 file.

![Run Options Screenshot](./ _static/images/simulated_playback_run_options.png)

[!NOTE]
Note - The below instructions, whilst they will still work, are no longer required. They are left here for reference only. As of Minknow 5.7, it is possible to select a bulk FAST5 file for playback in the MinKNOW UI.

Old method Configuring bulk FAST5 file Playback

 To setup a simulation the sequencing configuration file that MinKNOW uses must be edited. Steps:

  1. Download an open access bulk FAST5 file. This file is 21Gb so make sure you have plenty of space. This file is a record of a sequencing run using R9.4.1 pores, is non-barcoded and the library was produced using DNA extracted from the NA12878 cell line.

  2. Copy a sequencing TOML file to the user_scripts folder:

    On Mac if your MinKNOW output directory is the default:

    mkdir -p /Library/MinKNOW/data/user_scripts/simulations
cp /Applications/MinKNOW.app/Contents/Resources/conf/package/sequencing/sequencing_MIN106_DNA.toml /Library/MinKNOW/data/user_scripts/simulations/sequencing_MIN106_DNA_sim.toml

    On Linux:

    sudo mkdir -p /opt/ont/minknow/conf/package/sequencing/simulations
cp /opt/ont/minknow/conf/package/sequencing/sequencing_MIN106_DNA.toml /opt/ont/minknow/conf/package/sequencing/simulations/sequencing_MIN106_DNA_sim.toml

  3. Edit the copied file to add the following line under the line that reads "[custom_settings]":

    simulation = "/full/path/to/your_bulk.FAST5"

    Change the text between the quotes to point to your downloaded bulk FAST5 file.

  4. Optional, If running GUPPY in GPU mode, set the parameter break_reads_after_seconds = 1.0 to break_reads_after_seconds = 0.4. This results in a smaller read chunk. For R10.4 this is not required but can be tried. For adaptive sampling on PromethION, this should be left at 1 second.

  5. In the MinKNOW GUI, right click on a sequencing position and select Reload Scripts. Your version of MinKNOW will now playback the bulkfile rather than live sequencing.

  6. Start a sequencing run as you would normally, selecting the corresponding flow cell type to the edited script (here FLO-MIN106) as the flow cell type.

Whichever instructions you followed, the run should start and immediately begin a mux scan. Let it run for around five minutes after which your read length histogram should look as below: alt text

Testing unblock response

Now we shall test unblocking by running readfish unblock-all which will simply eject every single read on the flow cell.

  1. To do this run: 
    readfish unblock-all --device <YOUR_DEVICE_ID> --experiment-name "Testing readfish Unblock All"
  2. Leave the run for a further 5 minutes and observe the read length histogram. If unblocks are happening correctly you will see something like the below: alt text A closeup of the unblock peak shows reads being unblocked quickly: alt text

If you are happy with the unblock response, move on to testing base-calling.

Testing base-calling and mapping

To test selective sequencing you must have access to a guppy basecall server (>=6.0.0) and configure a TOML file.

  1. First make a local copy of the example TOML file:

    curl -O https://raw.githubusercontent.com/LooseLab/readfish/master/docs/_static/example_tomls/human_chr_selection.toml

  2. If on PromethION, edit the mapper_settings.mappy section to read:

    [mapper_settings.mappy-rs]

  3. Modify the fn_idx_in field in the file to be the full path to a minimap2 index of the human genome.

  4. Modify the targets fields for each condition to reflect the naming convention used in your index. This is the sequence name only, up to but not including any whitespace. e.g. >chr1 human chromosome 1 would become chr1. If these names do not match, then target matching will fail.

We can now validate this TOML file to see if it will be loaded correctly.

readfish validate human_chr_selection.toml

Errors with the configuration will be written to the terminal along with a text description of the conditions for the experiment as below.

2023-10-05 15:29:18,934 readfish /home/adoni5/mambaforge/envs/readfish_dev/bin/readfish validate human_chr_selection.toml
2023-10-05 15:29:18,934 readfish command='validate'
2023-10-05 15:29:18,934 readfish log_file=None
2023-10-05 15:29:18,934 readfish log_format='%(asctime)s %(name)s %(message)s'
2023-10-05 15:29:18,934 readfish log_level='info'
2023-10-05 15:29:18,934 readfish no_check_plugins=False
2023-10-05 15:29:18,934 readfish no_describe=False
2023-10-05 15:29:18,934 readfish prom=False
2023-10-05 15:29:18,934 readfish toml='human_chr_selection.toml'
2023-10-05 15:29:18,934 readfish.validate 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
2023-10-05 15:29:18,937 readfish.validate Loaded TOML config without error
2023-10-05 15:29:18,937 readfish.validate Initialising Caller
2023-10-05 15:29:18,945 readfish.validate Caller initialised
2023-10-05 15:29:18,945 readfish.validate Initialising Aligner
2023-10-05 15:29:18,947 readfish.validate Aligner initialised
2023-10-05 15:29:18,948 readfish.validate Configuration description:
Region hum_test (control=False).
Region applies to section of flow cell (# = applied, . = not applied):

    ################################
    ################################
    ################################
    ################################
    ################################
    ################################
    ################################
    ################################

2023-10-05 15:29:18,948 readfish.validate Using the mappy plugin. Using reference: /home/adoni5/Documents/Bioinformatics/refs/hg38_no_alts.fa.gz.split/hg38_chr_M.mmi.

Region hum_test has targets on 1 contig, with 1 found in the provided reference.
This region has 2 total targets (+ve and -ve strands), covering approximately 100.00% of the genome.

  1. If your toml file validates then run the following command:

  2. readfish targets --toml <PATH_TO_TOML> --device <YOUR_DEVICE_ID> --log-file test.log --experiment-name human_select_test

  3. In the terminal window you should see messages reporting the speed of mapping of the form:

    2023-10-05 15:24:03,910 readfish.targets MinKNOW is reporting PHASE_MUX_SCAN, waiting for PHASE_SEQUENCING to begin.
2023-10-05 15:25:48,150 readfish._read_until_client Protocol phase changed to PHASE_SEQUENCING
2023-10-05 15:25:48,724 readfish.targets 0494R/0.5713s; Avg: 0494R/0.5713s; Seq:0; Unb:494; Pro:0; Slow batches (>1.00s): 0/1
2023-10-05 15:25:52,132 readfish.targets 0004R/0.1831s; Avg: 0249R/0.3772s; Seq:0; Unb:498; Pro:0; Slow batches (>1.00s): 0/2
2023-10-05 15:25:52,600 readfish.targets 0122R/0.2494s; Avg: 0206R/0.3346s; Seq:0; Unb:620; Pro:0; Slow batches (>1.00s): 0/3
2023-10-05 15:25:52,967 readfish.targets 0072R/0.2144s; Avg: 0173R/0.3046s; Seq:0; Unb:692; Pro:0; Slow batches (>1.00s): 0/4
2023-10-05 15:25:53,349 readfish.targets 0043R/0.1932s; Avg: 0147R/0.2823s; Seq:0; Unb:735; Pro:0; Slow batches (>1.00s): 0/5
2023-10-05 15:25:53,759 readfish.targets 0048R/0.2011s; Avg: 0130R/0.2688s; Seq:0; Unb:783; Pro:0; Slow batches (>1.00s): 0/6
2023-10-05 15:25:54,206 readfish.targets 0126R/0.2458s; Avg: 0129R/0.2655s; Seq:0; Unb:909; Pro:0; Slow batches (>1.00s): 0/7
2023-10-05 15:25:54,580 readfish.targets 0082R/0.2180s; Avg: 0123R/0.2595s; Seq:0; Unb:991; Pro:0; Slow batches (>1.00s): 0/8
2023-10-05 15:25:54,975 readfish.targets 0053R/0.2110s; Avg: 0116R/0.2542s; Seq:0; Unb:1,044; Pro:0; Slow batches (>1.00s): 0/9
2023-10-05 15:25:55,372 readfish.targets 0057R/0.2051s; Avg: 0110R/0.2492s; Seq:0; Unb:1,101; Pro:0; Slow batches (>1.00s): 0/10
2023-10-05 15:25:55,817 readfish.targets 0135R/0.2467s; Avg: 0112R/0.2490s; Seq:0; Unb:1,236; Pro:0; Slow batches (>1.00s): 0/11
2023-10-05 15:25:56,192 readfish.targets 0086R/0.2206s; Avg: 0110R/0.2466s; Seq:0; Unb:1,322; Pro:0; Slow batches (>1.00s): 0/12
2023-10-05 15:25:56,588 readfish.targets 0060R/0.2138s; Avg: 0106R/0.2441s; Seq:0; Unb:1,382; Pro:0; Slow batches (>1.00s): 0/13
2023-10-05 15:25:56,989 readfish.targets 0060R/0.2123s; Avg: 0103R/0.2418s; Seq:0; Unb:1,442; Pro:0; Slow batches (>1.00s): 0/14
2023-10-05 15:25:57,429 readfish.targets 0133R/0.2502s; Avg: 0105R/0.2424s; Seq:0; Unb:1,575; Pro:0; Slow batches (>1.00s): 0/15
2023-10-05 15:25:57,809 readfish.targets 0089R/0.2280s; Avg: 0104R/0.2415s; Seq:0; Unb:1,664; Pro:0; Slow batches (>1.00s): 0/16
2023-10-05 15:25:58,210 readfish.targets 0059R/0.2247s; Avg: 0101R/0.2405s; Seq:0; Unb:1,723; Pro:0; Slow batches (>1.00s): 0/17
^C2023-10-05 15:25:58,238 readfish.targets Keyboard interrupt received, stopping readfish
WARNING
Note: if these times are longer than the number of seconds specified in the break read chunk in the sequencing TOML, you will have performance issues. Contact us via github issues for support.

This log is a little dense at first. Moving from left to right, we have:

[Date Time] [Logger Name] [Batch Stats]; [Average Batch Stats]; [Count commands sent]; [Slow Batch Info]

Using the provided log as an example:

On 2023-10-05 at 15:25:56,989, the Readfish targets command logged a batch of read signal:

- It saw 60 reads in the current batch.
- The batch took 0.2123 seconds.
- On average, batches are 103 reads, which are processed in 0.2418 seconds.
- Since the start, 0 reads were sequenced, 1,442 reads were unblocked, and 0 reads were asked to proceed.
- Out of 14 total batches processed, 0 were considered slow (took more than 1 second).

The important thing to note here is that the average batch time is less than the break read chunk time in the sequencing TOML. The slow batch section will show the number of batches that were slower than break reads. If the average is lower, or the slow batch count is high, you will have performance issues. Contact us via github issues for support.

If you are happy with the speed of mapping, move on to testing a selection.

Testing expected results from a selection experiment.

The only way to test readfish on a playback run is to look at changes in read length for rejected vs accepted reads. To do this:

  1. Start a fresh simulation run using the bulkfile provided above.
  2. Restart the readfish command (as above): 
    readfish targets --toml <PATH_TO_TOML> --device <YOUR_DEVICE_ID> --log-file test.log --experiment-name human_select_test
  3. Allow the run to proceed for at least 15 minutes (making sure you are writing out read data!).
  4. After 15 minutes it should look something like this: alt text Zoomed in on the unblocks: alt text

Common Gotcha's

These may or may not (!) be mistakes we have made already...

  1. If the previous run has not fully completed - i.e is still basecalling or processing raw data,you may connect to the wrong instance and see nothing happening. Always check the previous run has finished completely.
  2. If you have forgotten to remove your simultation line from your sequencing toml you will forever be trapped in an inception like resequencing of old data... Don't do this!
  3. If basecalling doesn't seem to be working check:
    • Check your basecalling server is running.
    • Check the ip of your server is correct.
    • Check the port of your server is correct.
  4. If you are expecting reads to unblock but they do not - check that you have set control=false in your readfish toml file. control=true will prevent any unblocks but does otherwise run the full analysis pipeline.
  5. Oh no - every single read is being unblocked - I have nothing on target!
    • Double check your reference file is in the correct location.
    • Double check your targets exist in that reference file.
    • Double check your targets are correctly formatted with contig name matching the record names in your reference (Exclude description - i.e the contig name up to the first whitespace).
  6. Where has my reference gone? If you are using a _live TOML file - e.g running iter_align or iter_cent, the previous reference MMI file is deleted when a new one is added. This obviously saves on disk space use(!) but can lead to unfortunate side effects - i.e you delete your MMI file. These can of course be recreated but user beware.

Happy readfish-ing!

Acknowledgements

We're really grateful to lots of people for help and support. Here's a few of them...

From the lab: Teri Evans, Sam Holt, Lewis Gallagher, Chris Alder, Thomas Clarke

From ONT: Stu Reid, Chris Wright, Rosemary Dokos, Chris Seymour, Clive Brown, George Pimm, Jon Pugh

From the Nanopore World: Nick Loman, Josh Quick, John Tyson, Jared Simpson, Ewan Birney, Alexander Senf, Nick Goldman, Miten Jain, Lukas Weilguny

And for our Awesome Logo please checkout out @tim_bassford from @TurbineCreative!