Trouble connecting to Guppy basecall server

[diego-ds-souza]

Hello,

I'm encountering connection errors with ReadBouncer 1.2.2 when trying to connect to Guppy basecall server. I am getting the following error messages, depending on the host I provide my config.toml file:

When I set the server address as 127.0.0.1:5555, I receive the this error:

...
Trying to connect to MinKNOW
Host : 127.0.0.1
Port : 9502
Connecting to MinKNOW using the token: ff5278d7-0850-4c90-b8cf-af0b3cc09cb5
Connection successfully established!
Waiting for device to start sequencing!
Please start the sequencing run now!
Sequencing has begun. Starting live signal processing!
Connecting to Guppy basecall server on address : 127.0.0.1:5555
[guppy/error] basecall_service::BasecallClient::worker_loop: Connection error. [bad_reply] Could not interpret message from server for request: LOAD_CONFIG. Reply: INVALID_PROTOCOL
[bad_reply] Could not interpret message from server for request: LOAD_CONFIG. Reply: INVALID_PROTOCOL

And when I use any other different server address (e.g, localhost or ipc:///home/nanopore/5555), I receive this timeout error:

...
Trying to connect to MinKNOW
Host : 127.0.0.1
Port : 9502
Connecting to MinKNOW using the token: ff5278d7-0850-4c90-b8cf-af0b3cc09cb5
Connection successfully established!
Waiting for device to start sequencing!
Please start the sequencing run now!
Sequencing has begun. Starting live signal processing!
Connecting to Guppy basecall server on address : ipc:///home/nanopore/5555
[guppy/error] basecall_service::BasecallClient::worker_loop: Connection error. [timed_out] Timeout waiting for reply to request: LOAD_CONFIG
[timed_out] Timeout waiting for reply to request: LOAD_CONFIG

I have tried to resolve these issues by following recommendations from the ReadBouncer issues tracker Timeout waiting for reply to request: LOAD_CONFIG #40 and 1.2.0 - getting core dump on classify test #51, but without success.

I am currently using ReadBouncer version 1.2.2, and I saw this version fixed the incompatibility with Guppy versions 6.2, 6.3 and 6.4.. However, my Guppy basecall server is version 6.5.7, so I'm wondering if these connection problems might be due to a version incompatibility.

Could you provide guidance or suggestions on how to resolve these problems? Any insights into whether this is a version compatibility problem or something else would be greatly appreciated.

For reference, here are the commands I am using to run ReadBouncer and my config.toml file:

$ /home/nanopore/ReadBouncer/build/ReadBouncer-1.2.2-Linux/bin/ReadBouncer --config /home/nanopore/Desktop/ReadBouncer/config_test1.toml

  usage               = "target"
  output_directory    = '/home/nanopore/Desktop/ReadBouncer/output_test1'
  log_directory       = '/home/nanopore/Desktop/ReadBouncer/logs_test1'
  
  [IBF]
  
  kmer_size           = 13
  fragment_size       = 100000
  target_files        = ['/home/nanopore/Desktop/Readfish/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta']
  exp_seq_error_rate  = 0.1
  threads             = 3
  
  [MinKNOW]
  
  host                = "127.0.0.1"
  port                = "9502"
  flowcell            = "MN44660"
  token_path          = "test/tmp/minknow-auth-token.json"
   
  [Basecaller]
  
  caller              = "Guppy"
  host                = "127.0.0.1"
  port                = 5555
  config              = "dna_r9.4.1_450bps_fast"

Thank you!

[JensUweUlrich]

Hi,

sorry for not getting back to you earlier. Unfortunately, ONT withdraw my access to its guppy core libraries, which I need for the integration of the GuppyClient in ReadBouncer. Thus Guppy 6.4 is the latest and last Guppy version I can support with ReadBouncer. So you could try to start a separate Guppy_6.4 server instance and connect ReadBouncer with the server, which is what I did two months ago for a sequencing project.

I actually have plans for the development of ReadBouncer, but no time to realize them after starting a PostDoc in a new lab.

Best
Jens

[sayangoswami]

Hi Jens,

Thanks for taking the time to respond to the queries raised by the community. I am facing this same version-incompatibility problem and would like to get it running using Guppy_6.4. However, I can't seem to find a link to download Guppy_6.4. If you could offer any suggestions, I would be very grateful.

Regards,

Sayan

[JensUweUlrich]

Hi Sayan,

Do you need a link to GuppyGPU_6.4 for Windows OS or Linux Ubuntu 20?

Best
Jens

[sayangoswami]

Linux Ubuntu 20. Thank you so much.

[JensUweUlrich]

You can download the debian package for version 6.4.6 here