KeyError: 'sequence_length_template' when basecalling is turned off

[lerminin]

Hi there,

I'm trying to run duplex basecalling data from a kit14/R10.4.1 Mk1B run. During run setup, we set basecalling to OFF in MinKNOW as we are basecalling with Guppy on a separate server after the run is complete.

When I run duplex_tools pairs_from_summary using the output sequencing_summary.txt file, I get the following KeyError:

[12:15:18 - FindPairs] Duplex tools version: 0.3.1
[12:15:18 - FindPairs] Loading sequencing summary.
[12:15:30 - FindPairs] Calculating metrics.
Traceback (most recent call last):
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/pandas/core/indexes/base.py", line 3802, in get_loc
    return self._engine.get_loc(casted_key)
  File "pandas/_libs/index.pyx", line 138, in pandas._libs.index.IndexEngine.get_loc
  File "pandas/_libs/index.pyx", line 165, in pandas._libs.index.IndexEngine.get_loc
  File "pandas/_libs/hashtable_class_helper.pxi", line 5745, in pandas._libs.hashtable.PyObjectHashTable.get_item
  File "pandas/_libs/hashtable_class_helper.pxi", line 5753, in pandas._libs.hashtable.PyObjectHashTable.get_item
KeyError: 'sequence_length_template'

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "conda_envs/duplex_tools_v0.3.1/bin/duplex_tools", line 8, in <module>
    sys.exit(main())
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/duplex_tools/__init__.py", line 39, in main
    args.func(args)
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/duplex_tools/pairs_from_summary.py", line 367, in main
    find_pairs(
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/duplex_tools/pairs_from_summary.py", line 86, in find_pairs
    seqsummary = calculate_metrics_for_next_strand(seqsummary)
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/duplex_tools/pairs_from_summary.py", line 267, in calculate_metrics_for_next_strand
    seqsummary["sequence_length_template"].shift(-1)
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/pandas/core/frame.py", line 3807, in __getitem__
    indexer = self.columns.get_loc(key)
  File "conda_envs/duplex_tools_v0.3.1/lib/python3.10/site-packages/pandas/core/indexes/base.py", line 3804, in get_loc
    raise KeyError(key) from err
KeyError: 'sequence_length_template'

I realized this is because our sequencing_summary.txt file only has these headers, and is missing the sequence_length_template header (among others):

filename_fastq filename_fast5 filename_pod5 parent_read_id read_id run_id channel mux minknow_events start_time duration pore_type experiment_id sample_id end_reason

This only seems happens for runs where we have explicitly turned off basecalling; for the runs where basecalling in MinKNOW is enabled, all required headers in the sequencing_summary.txt file are present.

Is there a way to recover the sequence_length_template information somehow to get duplex_tools to run?

[cjw85]

Hi @lerminin,

You need to provide the sequencing_summary.txt file produced by Guppy during basecalling.

[lerminin]

whoops silly oversight on my part - working now with the proper file, thanks!