Sequencing Scan Troubleshooting

Scans not running

Check timestamps on the historical runs grid to see when it was last run.

Is the seqauto_single_worker OK on the Server Status page?

On the server you can:

Check the logs
Check if processes are running:

# Check if worker is running
ps aux | grep seqauto_single_worker
# Check if celery beat is running
ps aux | grep beat

Suggested Fixes: Start and stop workers. If these fail, check systemd logs, or you can probably recreate the same error by running python3 manage.py shell

Scans Failing

The first step is to work out what’s happening. Look at the details page for the most recently failed SeqAutoRun by clicking the “View SeqAuto Run” link in the left most column of grid on that row. Hopefully there should be a stack trace and it mentions the SequencingRun or directory that caused it to fail.

General tips for failed runs

Remember you can always disable a particular run using .variantgrid_skip_flowcell - this will allow you to keep scans going while you fix the particular run
If a sequencing scan fails during “create models” stage, it’s possible the state of the record being processed is bad, for instance the GOIs or patients not properly linked to samples as described in SampleSheet, but as timestamps and hash match it won’t be loaded and fixed next time. It’s probably a good idea to delete the SequencingRun (through webapp) before restarting the next scan
If a scan fails, maybe give it another go and it’ll work this time

Weird one-off database integrity errors, file not found errors etc

We scan files, then load existing ones, then process them for ~10-30 mins.

If someone moves or deletes a file on the disk, or deletes a SequencingRun on the web, we will expect the file/data to be there as our state is now out of date

Example:

seqauto.models.models_seqauto.SequencingSample.DoesNotExist: SequencingSample matching query does not exist.

Example:

django.db.utils.IntegrityError: insert or update on table "seqauto_unalignedreads" violates foreign key constraint "seqauto_unalignedrea_fastq_r1_id_bd7bd92c_fk_seqauto_f"
DETAIL:  Key (fastq_r1_id)=(476466) is not present in table "seqauto_fastq".

Example:

DETAIL:  Key (sequencing_run_id)=(X) is not present in table "seqauto_sequencingrun".

Example:

FileNotFoundError: [Errno 2] No such file or directory: 'X'

Fix: Run the scan again. For many strange errors, try re-running and hopefully it will have fixed itself.

Scan files permission error

Example:

Type: <class 'subprocess.CalledProcessError'>, Value: Command '['/opt/variantgrid/seqauto/scripts/tau/find_flowcells.sh', '/opt/variantgrid/media_root/scan_resources/seqauto_run_5493']' returned non-zero exit status 1

This is almost always due to a new directory appearing with permission access problems. Unfortunately I don’t keep the error logs here, so you will need to copy/paste the command run and execute it on the shell

/opt/variantgrid/seqauto/scripts/tau/find_flowcells.sh /opt/variantgrid/media_root/scan_resources/seqauto_run_5493

If you look at the output of the command, it should tell you which directory/file it had a permission error on

Fix: Alter permissions to ensure the file and all directories leading up to it are readable

Create models permission error

  File "/data/variantgrid/seqauto/models/models_seqauto.py", line 139, in get_file_last_modified
    return path.stat().st_mtime
  File "/usr/lib/python3.8/pathlib.py", line 1198, in stat
    return self._accessor.stat(self)

PermissionError: [Errno 1] Operation not permitted: 'X/4_QC/sequencing_stats/RunParameters.xml'

This is due to a previously accessible file becoming not visible due to permissions. At least we record the path this time

Fix: Alter permissions to ensure the file and directory are readable

Create models missing files:

Example:

  File "/data/variantgrid/seqauto/sequencing_files/create_resource_models.py", line 387, in process_sequencing_run
    instrument_name, experiment_name = get_run_parameters(run_parameters_dir)
  File "/data/variantgrid/seqauto/illumina/run_parameters.py", line 35, in get_run_parameters
    raise ValueError(msg)
ValueError: Couldn't find RunParameters.xml or runParameters.xml in SequencingRun/4_QC/sequencing_stats

Fix: Copy in file. If you don’t have it, disable SequencingRun dir via “.variantgrid_skip_flowcell”

SampleSheet.csv parsing error

  File "/data/variantgrid/seqauto/illumina/samplesheet.py", line 120, in convert_sheet_to_df
    df = pd.read_csv(csv_file, index_col=None, comment='#', skip_blank_lines=True, skipinitialspace=True, dtype=str)
pandas.errors.ParserError: Error tokenizing data. C error: Expected 31 fields in line 18, saw 32

This is likely due to someone editing a SampleSheet.csv by hand

Fix: Fix SampleSheet.csv, either by hand or open it in a spreadsheet, save and export as CSV. If can’t fix disable SequencingRun dir via “.variantgrid_skip_flowcell”

Code fails during create models

Example:

      File "/data/variantgrid/seqauto/sequencing_files/create_resource_models.py", line 66, in create_samplesheet_samples
        raise KeyError(key) from err
    KeyError: 'SampleID'

This was due to older SampleSheet.csv code not handling diff version of sheet that had “Sample_ID”, and we needed to modify SampleSheet parser.

Example:

File "/data/variantgrid/seqauto/illumina/samplesheet.py", line 163, in convert_sheet_to_df
if index2 := get_first_col(["Index2", 'index2']):
File "/usr/local/lib/python3.8/dist-packages/pandas/core/generic.py", line 1326, in __nonzero__
raise ValueError(
ValueError: The truth value of a Series is ambiguous. Use a.empty, a.bool(), a.item(), a.any() or a.all().

seqauto.sequencing_files.create_resource_models.SeqAutoRunError: Error processing "Sequencing Run X"

Fix: Fix code. Deploy upgrade, restart services.

If developer is away:

Raise an issue about it, so they can be informed, and verify fix when they return
This is likely new code, so check git logs, can possibly revert change to previous version.
If it’s something non-critical you can just delete the lines about getting and storing the barcode. Or catch the exception and continue on.

That way scans can continue to run in general, at the loss of a small amount of data (which we can fix upon developer returning)