Playback issue

[alexomics]

Hi @alexomics,

I have same RUN ERROR issue on my demo run with in minutes of loading MinION data using playback option in MinKnow UI. I have a MinKnow with Dorado and running in a UBUNTU 22

Not sure where should I start? I currently struck at this step - https://github.com/LooseLab/readfish?tab=readme-ov-file#configuring-bulk-fast5-file-playback

Wondering, what are permission error I should be checking.
I do have full permission on 5555

$ ll /tmp/.guppy/
srwxrwxrwx 1 minknow minknow 0 Mar 28 09:04 5555

Thanks for your help,
sam

Originally posted by @bioteksampath in #337 (comment)

[alexomics]

@bioteksampath

Hi Sam,

I’ve moved this to a new issue for now. Which playback file are you using?

[bioteksampath]

Hi @alexomics
Thanks for your prompt response, i'm using the following file from the demo.
GXB02001_20230509_1250_FAW79338_X3_sequencing_run_NA12878_B1_19382aa5_ef4362cd.fast5

[alexomics]

Okay, that’s the right file. Can you find the error message from the control server log? It should be at:

/var/log/minknow/MS00000/control_server_log_0.txt

[bioteksampath]

i do have mutiple log files but this is the latest one

`base) sap223@gifs-c36qc14:/home/.gifs/sap223/Desktop/readfish_demo$ cat /var/log/minknow/MS00000/control_server_log-0.txt
2024-04-09 10:19:57.300601 INFO: starting_up (control)
hostname: gifs-c36qc14.usask.ca
system: ubuntu 20.04
Distribution: 23.11.7 (STABLE)
MinKNOW Core: 5.8.6
Bream: 7.8.2
Protocol configuration: 5.8.6
Dorado (build): 0.0.0.19120+441f78764
Dorado (connected): 7.2.13+fba8e8925

2024-04-09 10:19:57.302232 WARNING: partially_raised_file_limits (control_server)
actual: 1024
target: 4096
2024-04-09 10:19:57.302321 INFO: auth_guest_mode (rpc)
value: local_only
2024-04-09 10:19:57.303249 INFO: external_offload_service_not_configured (script)
auth_token_file:
external_service_port: 0
2024-04-09 10:19:57.304133 INFO: flow_cell_position_instantiated (mgmt)
device_id: MS00000
hardware_type: MINION_USB
os_identifier:
simulated: true
2024-04-09 10:19:57.304176 INFO: active_device_set (mgmt)
identifier: 0x7f16a40023b0
2024-04-09 10:19:57.304204 INFO: successfully_read_flow_cell_data (mgmt)
attempt: 1
flow_cell_data:
2024-04-09 10:19:57.306726 INFO: manager_notified_new_auth_token (util)
expires_in: 7510692439548057758ns
token_type: internal
2024-04-09 10:19:57.306747 INFO: manager_notified_new_auth_token (util)
expires_in: 88442360630897ns
token_type: local
2024-04-09 10:19:57.354926 INFO: device_discovered (engine)
device_id: MS00000
2024-04-09 10:19:58.349872 INFO: read_eeprom_id (mgmt)
eeprom_id: 4275878400
2024-04-09 10:19:58.355176 INFO: flowcell_discovered (engine)
asic_id: 131070
asic_id_eeprom: 4275878400
flow_cell_id:
2024-04-09 10:19:58.805690 INFO: firmware_component (rpc)
component_name: MinION FPGA
serial_number:
version: 1.0.1
2024-04-09 10:19:58.805765 INFO: firmware_component (rpc)
component_name: USB
serial_number:
version: 1.0.8
2024-04-09 10:21:43.734719 INFO: create_request_for_disabling_OS_standby (util)
2024-04-09 10:21:43.734899 INFO: sending_telemetry_message (ping)
data: {"monitor":"protocol_start","protocol_output_dir":"/var/lib/minknow/data/./t2/t2s2/20240409_1021_MS00000_MS00000_c5218bcb","script_arguments":["--f...
2024-04-09 10:21:43.736149 INFO: protocol_started (script)
flowcell_id: MS00000
host_serial_number:
identity: ['']
library_id: t2s2
output_path: /var/lib/minknow/data/./t2/t2s2/20240409_1021_MS00000_MS00000_c5218bcb
output_reads_dir: /var/lib/minknow/data/.
position_id: MS00000
protocol_group_id: t2
protocol_output_pattern: {protocol_group_id}/{sample_id}/{start_time}{device_id}{flow_cell_id}_{short_protocol_run_id}
run_id: c5218bcb-02b3-43a0-ada8-9ac255709794
sample_id_to_barcode: []
script_args: -s conf/package/utility/protocol_selector.py start sequencing/sequencing_MIN106_DNA:FLO-MIN106:SQK-LSK109 --fast5=on --pod5=off --fastq=on --bam=off --generate_bulk_file=off --active_channel_selection=on --base_calling=on --fast5_reads_per_file=4000 --fast5_data vbz_compress --fastq_reads_per_file=4000 --fastq_data compress --mux_scan_period=1.5 --pore_reserve=on --guppy_filename=dna_r9.4.1_450bps_fast.cfg --read_filtering min_qscore=8 --read_splitting enable=off --min_read_length=200 --simulation=/home/sap223/gifs/Desktop/readfish_demo/GXB02001_20230509_1250_FAW79338_X3_sequencing_run_NA12878_B1_19382aa5_ef4362cd.fast5
script_path: sequencing/sequencing_MIN106_DNA:FLO-MIN106:SQK-LSK109
2024-04-09 10:21:43.736346 INFO: disk_space_info_user (rpc)
filesystem_id: /
space_remaining: 163
unit: GB
2024-04-09 10:21:44.618448 INFO: waiting_for_device_ready_for_acquisition (mgmt)
2024-04-09 10:21:45.854335 INFO: switching_to_basecaller_config (basecalling)
new_filename: dna_r9.4.1_450bps_fast.cfg
2024-04-09 10:21:45.855110 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: preparing_connection (1)
previous_state: disconnected (0)
2024-04-09 10:21:45.855255 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: loading_config (2)
previous_state: preparing_connection (1)
2024-04-09 10:21:45.856787 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: loading_alignment_index (3)
previous_state: loading_config (2)
2024-04-09 10:21:45.856832 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: loading_bed_file (4)
previous_state: loading_alignment_index (3)
2024-04-09 10:21:45.856842 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: connecting (5)
previous_state: loading_bed_file (4)
2024-04-09 10:21:45.858650 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: connected (6)
previous_state: connecting (5)
2024-04-09 10:21:45.941750 INFO: protocol_phase_control_started (rpc)
2024-04-09 10:21:45.942007 INFO: base_device.wait_for_temperature (user_messages)
target: 34.0
timeout: 300
2024-04-09 10:21:45.942325 INFO: protocol_phase_changed (script)
phase: PHASE_INITIALISING
2024-04-09 10:21:46.855043 INFO: basecaller_client_created (basecalling)
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
2024-04-09 10:21:46.855078 INFO: successfully_created_basecall_client_with_config (basecalling)
alignment_options:
barcoding_options:
basecaller_config_name: dna_r9.4.1_450bps_fast.cfg
lamp_options:
2024-04-09 10:21:46.856067 INFO: basecaller_connection_changed_state (basecalling)
basecaller_error_string: [no_error]
client_name: MS00000_config_preload_203ff53a-6796-48b1-986c-643acaef5f73
next_state: disconnected (0)
previous_state: connected (6)
2024-04-09 10:22:01.962247 INFO: wait_for_temperature_complete (rpc)
elapsed_time: 16.0189
has_secondary_temp_limits: true
primary_temperature_begin: 35
primary_temperature_end: 34
primary_temperature_tolerance: 0.1
result: ReachedTemperature
secondary_temperature_begin: 35.0431
secondary_temperature_end: 34.0329
target_primary_temperature: 34
unstable_time: 1.00093
2024-04-09 10:22:03.066579 INFO: sending_telemetry_message (ping)
data: {"bulk_enabled":false,"fastq_enabled":true,"monitor":"exp_start","multi_fast5_enabled":true}
2024-04-09 10:22:03.098119 ERROR: exception_during_observer_callback (state_control)
callback_mode: transitioning
cause: Dynamic exception type: boost::filesystem::filesystem_error
std::exception::what: boost::filesystem::status: Permission denied [system:13]: "/home/sap223/gifs/Desktop/readfish_demo/GXB02001_20230509_1250_FAW79338_X3_sequencing_run_NA12878_B1_19382aa5_ef4362cd.fast5"

observer: MantaControl

2024-04-09 10:22:03.492513 INFO: data_acquisition_starting (engine)
acquisition_run_id: 9824c93cc5f3296457bbe0e5608f6b17889f26e1
options: allow_file_output=true, enable_analysis=true, generate_reports=true, send_basecalling_metrics=true, send_sequencing_read_metrics=true, generate_final_summary=true
2024-04-09 10:22:03.497570 ERROR: failed_to_finish_transition (state_control)
destination: CreateSharedData
observer: MantaControl
operation_begin: 2024-04-09T16:22:03.010772
operation_end: 2024-04-09T16:23:03.009772
source: Idle
stage: transition
2024-04-09 10:22:03.597656 INFO: data_acquisition_finished (engine)
acquisition_run_id: 9824c93cc5f3296457bbe0e5608f6b17889f26e1
2024-04-09 10:22:03.597688 INFO: stop_processing_unblocks_called (mgmt)
2024-04-09 10:22:03.597713 WARNING: called_stop_processing_unblocks_while_not_processing (mgmt)
2024-04-09 10:22:33.599492 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.599893 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.600049 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.600192 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.600319 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.600465 WARNING: forcibly_set_statistic_to_finished (pipeline_stats)
in_function:
stat_name:
2024-04-09 10:22:33.601490 INFO: sending_telemetry_message (ping)
data: {"monitor":"exp_stop","stop_reason":"INTERNAL_ERROR"}
2024-04-09 10:22:33.623661 ERROR: stopping_protocol_due_to_internal_error (control)
2024-04-09 10:22:33.623984 INFO: killing_protocol (script)
run_id: c5218bcb-02b3-43a0-ada8-9ac255709794
script_path: sequencing/sequencing_MIN106_DNA:FLO-MIN106:SQK-LSK109
2024-04-09 10:22:33.632163 INFO: protocol_phase_control_ended (rpc)
2024-04-09 10:22:33.656313 INFO: protocol_finished_internal_error (script)
run_id: c5218bcb-02b3-43a0-ada8-9ac255709794
2024-04-09 10:22:33.656511 INFO: sending_telemetry_message (ping)
data: {"acquisitions":[],"monitor":"protocol_end","script_name":"sequencing/sequencing_MIN106_DNA:FLO-MIN106:SQK-LSK109","stop_reason":"FINISHED_WITH_ERR...
2024-04-09 10:22:33.656771 INFO: clear_request_for_disabling_OS_standby (util)
2024-04-09 10:22:34.024178 INFO: rpc_error_occured (rpc)
details: {
"error_message": "Failed to start run: Error in observer 'MantaControl': boost::filesystem::status: Permission denied [system:13]: "/home/sap223/gifs/Desktop/readfish_demo/GXB02001_20230509_1250_FAW79338_X3_sequencing_run_NA12878_B1_19382aa5_ef4362cd.fast5"",
"exit_code": -1
}
path: /minknow_api.acquisition.AcquisitionService/start
status: UNKNOWN
user_identity: ['']
2024-04-09 10:32:14.897521 INFO: manager_notified_hw_state_change (util)
new_identifier:
new_state: unavailable
2024-04-09 10:32:14.897583 INFO: manager_requested_exit (util)
when: asap
2024-04-09 10:32:14.897826 INFO: active_device_detached (mgmt)
identifier: 0x7f16a40023b0
2024-04-09 10:32:14.900639 INFO: minion_disconnected (engine)
2024-04-09 10:32:15.658833 INFO: exiting (control_server)`

[alexomics]

So the error from that log is that MinKNOW cannot access the bulk file. I would try moving it to MinKNOW’s data folder at

/var/lib/minknow/data

make sure to update the simulation location and try again

[bioteksampath]

Hi @alexomics
Thanks for your response.
yes, Minknow recognise input.fast5 from minknow/data folder but not from other locations. strange.

However, i have couple of other issues, hope you can provide some feedback.
Since i have Dorado basecaller (not guppy) do i need to change anything in the .toml file, because i got below error.

readfish targets --toml Readfish_test_1600_N11.toml --device MS00000 --log-file Ap10test.log --experiment-name human_select_test
RuntimeError: The dna_r9.4.1_450bps_fast base-calling config listed in the readfish config TOML is not suitable for this flowcell and kit combination.
Please check the guppy_config value in the caller_settings.guppy section of your TOML file.
The following models are are given by ONT as suitable for this flow cell/kit combo:
dna_r10.4.1_e8.2_400bps_5khz_fast.cfg
dna_r10.4.1_e8.2_400bps_5khz_hac.cfg
dna_r10.4.1_e8.2_400bps_5khz_sup.cfg
dna_r10.4.1_e8.2_400bps_5khz_modbases_5hmc_5mc_cg_fast.cfg
dna_r10.4.1_e8.2_400bps_5khz_modbases_5hmc_5mc_cg_hac.cfg and dna_r10.4.1_e8.2_400bps_5khz_modbases_5hmc_5mc_cg_sup.cfg

`

log file.
Ap10test.log

human_chr_selection.toml.txt

[alexomics]

I can see from the control server log that you are using the R10 (5KHz) bulk file for playback. As such the dna_r9.4.1_450bps_fast config cannot be used for base calling. I would recommend using the dna_r10.4.1_400bps_5khz_fast config instead.
For the caller settings:

[caller_settings.guppy]
config = "dna_r10.4.1_e8.2_400bps_5khz_fast"
address = "ipc:///tmp/.guppy/5555"
debug_log = "live_reads.fq"

---

I can also see from the TOML file you uploaded that you've set the n_threads option for the aligner. To make use of multi-threaded alignment you will need to use the mappy_rs option and set the correct fn_idx_in path:

[mapper_settings.mappy]
fn_idx_in = "/path/to/hg38.mmi"
debug_log = "live_alignments.paf"
n_threads = 24

[bioteksampath]

Thanks @alexomics

I was able to run the demo successfully. I have some additional questions regarding my own research, and I'm hoping you could provide some feedback:

1. I want to genotype 500 structural variants (SVs) greater than 5kb across 50 lines. What would be the best approach for this?
2. Concerning target sizes in the target.bed file, I've heard that each target should be around 50kb to ensure good sequence coverage. Could you share your thoughts on the minimum size and maximum number of targets?
3. How many libraries should be maximally pooled or barcoded in a single MinION sequencing run for a genome size of 1Gb?
4. Could you recommend any downstream analysis tools for validation and visualization?

Apologies for the barrage of questions.

[mattloose]

HI @bioteksampath ,

The above questions aren't issues with readfish and also are not linked to the original issue (which is now resolved).

It is helpful to keep these things separate. I shall close this issue and reference your comment in a new discussion - #346

[bioteksampath]

Thanks, @mattloose and @alexomics,

I wanted to share the steps I took to address the issues we encountered with the Nanopore adaptive sequencing setup, I believe it will be useful for others.

1. Minknow File Access: I found that Minknow was unable to access the demo fast5 (R10 data) file from a directory other than /var/lib/minknow/data. To resolve this, I moved the file to this folder. Please note that writing fast5 files to /var/lib/minknow/data requires sudo permission.

2. Write Permission: To allow Minknow to write to /tmp/.guppy/5555, I used the command chmod 777 /tmp/.guppy/5555. However, obtaining write permission may require a request to the sudo user.

3. Configuration Update: I made changes to the .toml configuration information. It is compatible with Dorado caller. Here are the updated settings:

   For Guppy/Dorado:

   [caller_settings.guppy]
   config = "dna_r10.4.1_e8.2_400bps_5khz_fast"
   address = "ipc:///tmp/.guppy/5555"
   debug_log = "live_reads.fq"
   

   For Multi-Threads:

   [mapper_settings.mappy_rs]
   fn_idx_in = "/path/to/hg38.mmi"
   debug_log = "live_alignments.paf"
   n_threads = 24
   

   For Single Thread:

   [mapper_settings.mappy]
   fn_idx_in = "/path/to/hg38.mmi"
   debug_log = "live_alignments.paf"
   n_threads = 4
   

4. Additional Information: If using a targets bed file, ensure it is 6 cloumn bed 9tab separated) or 4 column comma-separated. Here's an example format:

   targets = ["/path/to/targets.csv"]
   Chr1
   Scf777,510021,510052,+
   Scf4546,108529,108529,+
   Chr11,1,58419410,+
   Chr12,1,5639592,+
   

OR .bed in 6 column

targets = ["/path/to/targets.bed"]
777	510021	510052	.	.	+
4546	108529	109529	.	.	+

These adjustments should help resolve the issues we encountered.

Hope this helps someone!
Sam

[mattloose]

Could you check your bed file format please?

The format you provide above is not a bed file format - the bed file format spcs are available here https://github.com/samtools/hts-specs/blob/master/BEDv1.pdf and are explicitly not comma separated - rather they are tab or whitespace separated, which readfish does support.

Could you share the file that you used which did not work?

[bioteksampath]

@mattloose thanks for pointing out.|

Strange is i can not validate the .bed format, but target csv

(rf_con) cxo314@gifs-c36qc14:/data/reference/canola$ head targets_315Svs.bed
777	510021	520052	+
4546	108529	109529	+
N11	1	58419410	+
N12	1	5639592	+
N13	1	5639592	+
N19	1	5639592	+
N2	68644	69237	+

(rf_con) cxo314@gifs-c36qc14:/data/reference/canola$ readfish validate canola_test_1600_N11_barcode1-4_bed.toml 
2024-04-15 12:55:49,862 readfish /home/cxo314/gifs/.conda/envs/rf_con/bin/readfish validate canola_test_1600_N11_barcode1-4_bed.toml
2024-04-15 12:55:49,862 readfish command='validate'
2024-04-15 12:55:49,862 readfish log_file=None
2024-04-15 12:55:49,862 readfish log_format='%(asctime)s %(name)s %(message)s'
2024-04-15 12:55:49,862 readfish log_level='info'
2024-04-15 12:55:49,862 readfish no_check_plugins=False
2024-04-15 12:55:49,862 readfish no_describe=False
2024-04-15 12:55:49,862 readfish prom=False
2024-04-15 12:55:49,862 readfish toml='canola_test_1600_N11_barcode1-4_bed.toml'
2024-04-15 12:55:49,862 readfish.validate .
.
.
.
2024-04-15 12:55:49,878 readfish.validate Loaded TOML config without error
2024-04-15 12:55:49,878 readfish.validate Initialising Caller
2024-04-15 12:55:49,919 readfish.validate Caller initialised
2024-04-15 12:55:49,920 readfish.validate Initialising Aligner
2024-04-15 12:55:51,695 readfish.validate Aligner initialised
2024-04-15 12:55:51,700 readfish.validate Configuration description:
Number of barcodes in the Conf (excluding unclassified and classified): 4
Barcode unclassified_reads (control=False), Barcode classified_reads (control=False), Barcode NAM00_300csv (control=False), Barcode NAM01_300csv (control=False), Barcode NAM04_300csv (control=False) and Barcode NAM05_300csv (control=False)

Region canola_test (control=False).
Region applies to section of flow cell (# = applied, . = not applied):

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


NOTE - The following 315 contigs are listed as targets but have not been found on the target reference:
 4546	108529	108529	+, 777	510021	510052	+, N11	1	58419410	+, N12	1	5639592	+, N13	1	5639592	+, N19	1	5639592	+, N2	10057805	10064396	+, N2	100

But my target in csv got validated

`(rf_con) cxo314@gifs-c36qc14:/data/reference/canola$ head adaONT_300SVcsv.txt
777,510021,510052,+
4546,108529,108529,+
N11,1,58419410,+
N12,1,5639592,+
N13,1,5639592,+
N19,1,5639592,+
N2,68644,69237,+
N2,190124,190157,+
N2,211196,213168,+
N2,310498,310546,+

(rf_con) cxo314@gifs-c36qc14:/data/reference/canola$ readfish validate canola_test_1600_N11_barcode1-4_csv.toml 
2024-04-15 13:10:53,713 readfish /home/cxo314/gifs/.conda/envs/rf_con/bin/readfish validate canola_test_1600_N11_barcode1-4_csv.toml
2024-04-15 13:10:53,714 readfish command='validate'
2024-04-15 13:10:53,714 readfish log_file=None
2024-04-15 13:10:53,714 readfish log_format='%(asctime)s %(name)s %(message)s'
2024-04-15 13:10:53,714 readfish log_level='info'
2024-04-15 13:10:53,714 readfish no_check_plugins=False
2024-04-15 13:10:53,714 readfish no_describe=False
2024-04-15 13:10:53,714 readfish prom=False
2024-04-15 13:10:53,714 readfish toml='canola_test_1600_N11_barcode1-4_csv.toml'
2024-04-15 13:10:53,714 readfish.validate eJztWE1v3DYQvetXEPIlRtfaXccGEgM5OCkSBEjsIHF6MVyBK1Er1hKpiJQ/.
.
.
.
2024-04-15 13:10:53,727 readfish.validate Loaded TOML config without error
2024-04-15 13:10:53,727 readfish.validate Initialising Caller
2024-04-15 13:10:53,770 readfish.validate Caller initialised
2024-04-15 13:10:53,770 readfish.validate Initialising Aligner
2024-04-15 13:10:55,572 readfish.validate Aligner initialised
2024-04-15 13:10:55,573 readfish.validate Configuration description:
Number of barcodes in the Conf (excluding unclassified and classified): 4
Barcode unclassified_reads (control=False), Barcode classified_reads (control=False), Barcode NAM00_300csv (control=False), Barcode NAM01_300csv (control=False), Barcode NAM04_300csv (control=False) and Barcode NAM05_300csv (control=False)

Region canola_test (control=False).
Region applies to section of flow cell (# = applied, . = not applied):

2024-04-15 13:10:57,011 readfish.validate Using the mappy_rs plugin. Using reference: /data/reference/canola/N99_hifi.mmi.

Region canola_test has targets on 8 contigs, with 8 found in the provided reference.
This region has 314 total targets (+ve and -ve strands), covering approximately 3.76% of the genome.

Barcode unclassified_reads has targets on 0 contigs, with 0 found in the provided reference.
This barcode has 0 total targets (+ve and -ve strands), covering approximately 0.00% of the genome.

Barcode classified_reads has targets on 0 contigs, with 0 found in the provided reference.
This barcode has 0 total targets (+ve and -ve strands), covering approximately 0.00% of the genome.

Barcode NAM00_300csv has targets on 8 contigs, with 8 found in the provided reference.
This barcode has 314 total targets (+ve and -ve strands), covering approximately 3.76% of the genome.

Barcode NAM01_300csv has targets on 8 contigs, with 8 found in the provided reference.
This barcode has 314 total targets (+ve and -ve strands), covering approximately 3.76% of the genome.

Barcode NAM04_300csv has targets on 8 contigs, with 8 found in the provided reference.
This barcode has 314 total targets (+ve and -ve strands), covering approximately 3.76% of the genome.

Barcode NAM05_300csv has targets on 8 contigs, with 8 found in the provided reference.
This barcode has 314 total targets (+ve and -ve strands), covering approximately 3.76% of the genome.
`

[mattloose]

Hi,

The issue is that your BED file is not the correct format. If you look in the docs here - https://looselab.github.io/readfish/toml.html#bed-or-csv-targets

we specify that for a BED file it needs to be in the 6 column format:

chrom chromStart chromEnd name score strand

name and score can be left as a "." if no information is being provided. However, the four column format you provided above does not match the 6 column format.

You would need:

777 510021 520052 . . +

For the CSV format we do take the four columns.

Hope that helps.

[bioteksampath]

Thanks @mattloose the 6 cloumn .bed worked. (seems files name should end with .bed, if bed.txt not works)