Missing amplicon ID error

[dnhare]

Hi there,

I've recently installed ncov-tools but the snakemake script repeatedly fails to execute due to the following error. I'd greatly appreciate any advice as to how to correct this error.

Many thanks.

Dan

Error: At least one layer must contain all faceting variables: amplicon_id.

• Plot is missing amplicon_id
• Layer 1 is missing amplicon_id Backtrace: █
  1. └─global::plot_fraction_covered_by_amplicon(df, args$output)
  2. └─ggplot2::ggsave(outname, width = 15, height = 10)
  3. ├─grid::grid.draw(plot)
  4. └─ggplot2:::grid.draw.ggplot(plot)
  5. ├─base::print(x)
  6. └─ggplot2:::print.ggplot(x)
  7. ├─ggplot2::ggplot_build(x)
  8. └─ggplot2:::ggplot_build.ggplot(x)
  9. └─layout$setup(data, plot$data, plot$plot_env)
  10. └─ggplot2:::f(..., self = self)
  11. └─self$facet$compute_layout(data, self$facet_params)
  12. └─ggplot2:::f(...)
  13. ├─ggplot2:::unrowname(...)
  14. │ └─base::is.data.frame(x)
  15. └─ggplot2::combine_vars(data, params$plot_env, vars, drop = params$drop) Execution halted [Thu Aug 5 13:01:21 2021] Error in rule make_qc_plot_fraction_covered_by_amplicon: jobid: 1 output: plots/None_amplicon_covered_fraction.pdf shell: Rscript /workflow/rules/../scripts/plot/plot_qc_sequencing.R -t amplicon_covered_fraction -o plots/None_amplicon_covered_fraction.pdf (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!)

[rdeborja]

@dnhare Can you tell me which primer scheme you're using? The script that parses the primer file expects the ARTIC primer BED name field format (i.e. nCoV-2019_1_LEFT, nCoV-2019_1_RIGHT).

The input file used to generate the failing plot is qc_sequencing/<samplename>.amplicon_coverage.bed. Can you check the following:

• a qc_sequencing/.amplicon_coverage.bed file exists for each sample
• each of the .amplicon_coverage.bed files have an amplicon_id column filled with an identifier and not an empty field

Could you copy and paste the first few lines of a qc_sequencing/*.amplicon_coverage.bed file?

[dnhare]

Thank you very much for your prompt response.

We're using ARTIC V3 primer scheme. The path to the primer_bed in the config.yaml file is: ~/artic-ncov2019/primer_schemes/nCoV-2019/V3/nCoV-2019.primer.bed

Unfortunately, the issue seems to be that we are not generating a qc_sequencing directory, or .amplicon.coverage.bed files.

Are these supposed to be outputs after executing the snakemake script, or outputs from the artic pipeline?

[jts]

This error can occur when the pipeline fails to find any samples. Can you double-check that your bam files are named using the pattern defined in your config.yaml?

On Aug 5, 2021, at 12:01 PM, dnhare @.***> wrote:

 Thank you very much for your prompt response.

We're using ARTIC V3 primer scheme. The path to the primer_bed in the config.yaml file is: ~/artic-ncov2019/primer_schemes/nCoV-2019/V3/nCoV-2019.primer.bed

Unfortunately, the issue seems to be that we are not generating a qc_sequencing directory, or .amplicon.coverage.bed files.

Are these supposed to be outputs after executing the snakemake script, or outputs from the artic pipeline?

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[dnhare]

Thank you @jts

As far as I can tell my bam files are in the pattern specified in my config.yaml (I haven't changed the default pattern of "{data_root}/{sample}.sorted.bam")

The bam files are named in the data_root directory as numbered files e.g. "18.sorted.bam"

There are also corresponding .bam.bai files in that directory e.g. "18.bam.bai" etc.

Do you think this naming structure could be preventing the pipeline from finding any samples?

I've tried renaming some of the bam files and adjusting the config.yaml file accordingly to see if that makes a difference, but I get the same error.

Thanks for your help!

[dnhare]

@jts @rdeborja This was solved by specifying the absolute path to the sample directory in the config.yaml file, and removing '~'

Thanks for your help with this.

[rdeborja]

Thanks for letting us know @dnhare. Great tip in case anyone else encounters the same issue.