tombo plot error: Encountered invalid genome sequence request

[xiezhq]

I am trying to use Tombo to detect RNA modification in a RNA sequence (197 bases) because I need to reproduce published result using the same version of Tombo. I use the RNA sequence as the reference in a FASTA file. When I ran 'tombo plot per_read' command, it always reported error message (see screen shot below):

tombo plot per_read --genome-locations ref_rna:0 ref_rna:197 --per-read-statistics-filename Aclm100mM-1.vs.DMSO-1.per-read-stat.tombo.per_read_stats --genome-fasta ../mapping/ref.fa --fast5-basedirs single_fast5/Aclm100mM-1 --num-reads 2179 --num-bases 197 --pdf-filename results/plots/Aclm100mM-1.vs.DMSO-1.per-read-stat.pdf
[10:04:45] Parsing genome locations.
Traceback (most recent call last):
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/bin/tombo", line 11, in <module>
    sys.exit(main())
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/lib/python3.7/site-packages/tombo/__main__.py", line 279, in main
    _plot_commands.plot_main(args)
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/lib/python3.7/site-packages/tombo/_plot_commands.py", line 2363, in plot_main
    plot_per_read_mods_genome_location(*base_args, **kwargs)
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/lib/python3.7/site-packages/tombo/_plot_commands.py", line 1785, in plot_per_read_mods_genome_location
    int_data.add_seq(genome_index)
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/lib/python3.7/site-packages/tombo/tombo_helper.py", line 1936, in add_seq
    self.chrm, self.start, self.end, error_end=error_end))
  File "/mnt/depts/dept04/compbio/users/zxie2/mambaforge/envs/ont-tombo/lib/python3.7/site-packages/tombo/tombo_helper.py", line 834, in get_seq
    'Encountered invalid genome sequence request.')
tombo.tombo_helper.TomboError: Encountered invalid genome sequence request.

The content in FASTA file (RNA reference sequence) is as follows:

>ref_rna
GGCCUUCGGGCCAAAUCUAUAUAGCUAUUUUUUUUAACUUCCUUUAUUUUCCUUACAGGGUUUUAGACAAAAUCAAAAAGAAGGAAGGUGCUCACAUUCCUUAAAUUAAGGAGUAAGUCUGCCAGCAUUAUGAAAGUGAAUCUUACUUUUGUAAUCGAUCCGGUUCGCCGGAUCCAAAUCGGGCUUCGGUCCGGUUC

How can I solve this problem?

The commands I ran before command 'tombo plot per_read' are below (listed in order):

multi_to_single_fast5 --input_path /edgehpc/dept/compbio/instruments/ONT/TST12183/Aclm100mM-1/20230727_1759_MC-111751_FAW95962_3de906a4/fast5 --save_path Aclm100mM-1 --recursive -t 4

(I modified sequencing_summary.txt to change the single read file names to match the IDs in sequencing_summary.txt file)

tombo preprocess annotate_raw_with_fastqs --fast5-basedir single_fast5/Aclm100mM-1 --fastq-filenames fastq/Aclm100mM-1.fastq --overwrite --processes 4 --sequencing-summary-filenames sequencing_summary/Aclm100mM-1/sequencing_summary.modified.txt

tombo resquiggle --rna --overwrite --processes 4 --num-most-common-errors 5 --include-event-stdev --ignore-read-locks single_fast5/Aclm100mM-1/ ../mapping/ref.fa

tombo detect_modifications model_sample_compare --fast5-basedirs single_fast5/Aclm100mM-1/ --control-fast5-basedirs single_fast5/DMSO-1/ --statistics-file-basename Aclm100mM-1.vs.DMSO-1 --minimum-test-reads 100 --multiprocess-region-size 250 --processes 4

Thanks,
Zhiqun Xie