Summary report fails as multiqc doesn't generate SVG files.

[nagendraKU]

Description of the bug

Running the test profile fails at the Summary report stage because Multiqc doesn't generate SVG file for fastqc_per_sequence_quality_scores_plot.svg. I checked the results/multiqc/multiqc_data folder and the fastqc_per_sequence_quality_scores_plot.txt data file is present. The multiqc_plots folder is empty in all its subfolders (svg, pdf, png).

Run log summary:

NFCORE_AMPLISEQ:AMPLISEQ:METADATA_ALL (Metadata.tsv) COMPLETED  0
NFCORE_AMPLISEQ:AMPLISEQ:METADATA_PAIRWISE (Metadata.tsv) COMPLETED     0
NFCORE_AMPLISEQ:AMPLISEQ:RENAME_RAW_DATA_FILES (sampleID_1a) COMPLETED  0
NFCORE_AMPLISEQ:AMPLISEQ:RENAME_RAW_DATA_FILES (sampleID_2a) COMPLETED  0
NFCORE_AMPLISEQ:AMPLISEQ:RENAME_RAW_DATA_FILES (sampleID_1) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:RENAME_RAW_DATA_FILES (sampleID_2) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:FASTQC (sampleID_1a) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_BASIC (sampleID_1a) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_BASIC (sampleID_2a) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:FASTQC (sampleID_2a) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:FASTQC (sampleID_1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_BASIC (sampleID_1)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_BASIC (sampleID_2)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:FASTQC (sampleID_2) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_PREPTAX:FORMAT_TAXONOMY_QIIME   COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:FORMAT_TAXONOMY COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_SUMMARY_STD (cutadapt_standard)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_QUALITY1 (FW)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_QUALITY1 (RV)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:CUTADAPT_WORKFLOW:CUTADAPT_SUMMARY_MERGE (cutadapt_standard_summary.tsv)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:TRUNCLEN (FW) COMPLETED    0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:TRUNCLEN (RV) COMPLETED    0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_PREPTAX:QIIME2_EXTRACT (GTGYCAGCMGCCGCGGTAA-GGACTACNVGGGTWTCTAAT)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_TAXONOMY_WF:CUTADAPT_TAXONOMY (assignTaxonomy)   COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_FILTNTRIM (sampleID_1a)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_FILTNTRIM (sampleID_1)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_FILTNTRIM (sampleID_2)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_FILTNTRIM (sampleID_2a)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_PREPTAX:QIIME2_TRAIN (GTGYCAGCMGCCGCGGTAA-GGACTACNVGGGTWTCTAAT) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_QUALITY2 (RV)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_PREPROCESSING:DADA2_QUALITY2 (FW)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_ERR (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_DENOISING (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_RMCHIMERA (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_STATS (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_MERGE COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:MERGE_STATS_STD (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:VSEARCH_CLUSTER (ASV_post_clustering)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:FILTER_CLUSTERS (ASV_post_clustering)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:BARRNAP (ASV_post_clustering_filtered.fna)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:BARRNAPSUMMARY COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:FILTER_SSU (ASV_post_clustering_filtered.fna)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:FILTER_LEN_ASV (ASV_seqs.ssu.fasta) COMPLETED  0
NFCORE_AMPLISEQ:AMPLISEQ:MERGE_STATS_FILTERSSU (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:MERGE_STATS_FILTERLENASV (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_TAXONOMY:QIIME2_INSEQ (ASV_seqs.len.fasta)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_INASV (ASV_table.len.tsv) COMPLETED     0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_INSEQ (ASV_seqs.len.fasta) COMPLETED    0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_TAXONOMY_WF:DADA2_TAXONOMY (ASV_seqs.len.fasta,assignTaxonomy.trim.fastq.gz)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_TAXONOMY:QIIME2_CLASSIFY (rep-seqs.qza,GTGYCAGCMGCCGCGGTAA-GGACTACNVGGGTWTCTAAT-classifier.qza) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:DADA2_TAXONOMY_WF:DADA2_ADDSPECIES (ASV_tax.rds,addSpecies.fna)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:PHYLOSEQ_INTAX_QIIME2 (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_INTAX (ASV_tax_species.gtdb_R07-RS207.tsv)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:SBDIEXPORTREANNOTATE (ASV_tax_species.gtdb_R07-RS207.tsv)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:SBDIEXPORT (ASV_table.len.tsv,ASV_tax_species.gtdb_R07-RS207.tsv,Metadata.tsv) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_TABLEFILTERTAXA (taxa:mitochondria,chloroplast;min-freq:10;min-samples:2)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_EXPORT:QIIME2_EXPORT_RELASV COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:FILTER_STATS COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_EXPORT:QIIME2_EXPORT_RELTAX COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:PHYLOSEQ_WORKFLOW:PHYLOSEQ_INASV COMPLETED     0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_SEQFILTERTABLE (rep-seqs.qza-filter-by-filtered-table.qza)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:ANCOMBC_FORMULA_TAX (filtered-table-treatment1-2) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:ANCOMBC_FORMULA_TAX (filtered-table-treatment1-3) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_FILTERSAMPLES_ANCOM (mix8) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_FILTERSAMPLES_ANCOM (badpairwise10)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_FILTERSAMPLES_ANCOM (treatment1)   COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:ANCOMBC_FORMULA_TAX (filtered-table-treatment1-4) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:ANCOMBC_FORMULA_ASV (filtered-table-treatment1)   COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOT (1) COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:MERGE_STATS_FILTERTAXA (1) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOTAVG:QIIME2_INASV_BPAVG (rel-table-ASV.tsv)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:PHYLOSEQ_WORKFLOW:PHYLOSEQ (qiime2) COMPLETED  0
NFCORE_AMPLISEQ:AMPLISEQ:PHYLOSEQ_WORKFLOW:PHYLOSEQ (dada2) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_EXPORT:QIIME2_EXPORT_ABSOLUTE   COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (mix8--2)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_ASV (mix8-)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (mix8--4)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (mix8--3)      COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (badpairwise10--2)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (badpairwise10--4)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_ASV (badpairwise10-)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (badpairwise10--3)     COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (treatment1--3)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (treatment1--2)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_TAX (treatment1--4)        COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_ANCOM:QIIME2_ANCOMBC_ASV (treatment1-)  COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOTAVG:QIIME2_FEATURETABLE_GROUP (badpairwise10)    COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOTAVG:QIIME2_FEATURETABLE_GROUP (treatment1)       COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_EXPORT:COMBINE_TABLE_QIIME2 (1) COMPLETED       0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_EXPORT:COMBINE_TABLE_DADA2 COMPLETED    0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOTAVG:QIIME2_BPAVG (1) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:QIIME2_BARPLOTAVG:QIIME2_BPAVG (2) COMPLETED   0
NFCORE_AMPLISEQ:AMPLISEQ:MULTIQC COMPLETED 0
NFCORE_AMPLISEQ:AMPLISEQ:SUMMARY_REPORT (1) FAILED 1

Command used and terminal output

Command used:
$ nextflow run nf-core/ampliseq -r 2.12.0 -profile test,singularity --outdir testresults --skip_alpha_rarefaction --skip_diversity_indices

Output

Pipeline completed with errors-
ERROR ~ Error executing process > 'NFCORE_AMPLISEQ:AMPLISEQ:SUMMARY_REPORT (1)'

Caused by:
  Process `NFCORE_AMPLISEQ:AMPLISEQ:SUMMARY_REPORT (1)` terminated with an error exit status (1)


Command executed:

  \#!/usr/bin/env Rscript
  library(rmarkdown)

 \# Work around  https://github.com/rstudio/rmarkdown/issues/1508
  \# If the symbolic link is not replaced by a physical file
  \# output- and temporary files will be written to the original directory.
  file.copy("./report_template.Rmd", "./template.Rmd", overwrite = TRUE)

  rmarkdown::render("template.Rmd", output_file = "summary_report.html", params = list(css='nf-core_style.css',report_logo='nf-core-ampliseq_logo_light_long.png',workflow_manifest_version='2.12.0',workflow_scriptid='bf04a499cc',report_title='Summary of analysis results',metadata='Metadata.tsv',input_samplesheet='Samplesheet.tsv',mqc_plot='multiqc_plots/svg/fastqc_per_sequence_quality_scores_plot.svg',cutadapt_summary='cutadapt_summary.tsv',trunc_qmin=25,trunc_rmin=0.75,trunclenf='null',trunclenr='null',max_ee=2,dada_qc_f_path='FW_qual_stats.svg',dada_qc_r_path='RV_qual_stats.svg',dada_pp_qc_f_path='FW_preprocessed_qual_stats.svg',dada_pp_qc_r_path='RV_preprocessed_qual_stats.svg',dada_filtntrim_args='filterAndTrim.args.txt',dada_sample_inference='independent',dada_err_path='1_1.err.svg,1_2.err.svg',dada_err_run='1',asv_table_path='ASV_table.tsv',path_asv_fa='ASV_post_clustering_filtered.fna',path_dada2_tab='DADA2_table.tsv',dada_stats_path='DADA2_stats.tsv',vsearch_cluster='ASV_post_clustering_filtered.table.tsv',vsearch_cluster_id='0.97',path_barrnap_sum='summary.tsv',filter_ssu_stats='stats.ssu.tsv',filter_ssu_asv='ASV_seqs.ssu.fasta',filter_ssu='bac',filter_len_asv='stats.len.tsv',filter_len_asv_len_orig='ASV_len_orig.tsv',min_len_asv=0,max_len_asv=255,dada2_taxonomy='ASV_tax_species.gtdb_R07-RS207.tsv',dada2_ref_tax_title='GTDB - Genome Taxonomy Database - Release R07-RS207',dada2_ref_tax_file='[https://data.gtdb.ecogenomic.org/releases/release207/207.0/genomic_files_reps/bac120_ssu_reps_r207.tar.gz, https://data.gtdb.ecogenomic.org/releases/release207/207.0/genomic_files_reps/ar53_ssu_reps_r207.tar.gz]',dada2_ref_tax_citation='Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil PA, Hugenholtz P. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018 Nov;36(10):996-1004. doi: 10.1038/nbt.4229. Epub 2018 Aug 27. PMID: 30148503.',cut_dada_ref_taxonomy='assignTaxonomy.cutadapt.log',qiime2_taxonomy='taxonomy.tsv',qiime2_ref_tax_title='Greengenes 16S - Version 13_8 - clustered at 85% similarity - for testing purposes only',qiime2_ref_tax_file='[https://data.qiime2.org/2023.7/tutorials/training-feature-classifiers/85_otus.fasta, https://data.qiime2.org/2023.7/tutorials/training-feature-classifiers/85_otu_taxonomy.txt]',qiime2_ref_tax_citation='McDonald, D., Price, M., Goodrich, J. et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J 6, 610�618 (2012). https://doi.org/10.1038/ismej.2011.139',val_used_taxonomy='DADA2',filter_stats_tsv='count_table_filter_stats.tsv',qiime2_filtertaxa='245,42',exclude_taxa='mitochondria,chloroplast',min_frequency='10',min_samples='2',barplot=TRUE,metadata_category_barplot='treatment1,badpairwise10',abundance_tables=TRUE,ancombc='ancombc/da_barplot/Category-mix8-ASV,ancombc/da_barplot/Category-mix8-level-2,ancombc/da_barplot/Category-mix8-level-4,ancombc/da_barplot/Category-mix8-level-3,ancombc/da_barplot/Category-badpairwise10-level-2,ancombc/da_barplot/Category-badpairwise10-level-4,ancombc/da_barplot/Category-badpairwise10-ASV,ancombc/da_barplot/Category-badpairwise10-level-3,ancombc/da_barplot/Category-treatment1-level-3,ancombc/da_barplot/Category-treatment1-level-2,ancombc/da_barplot/Category-treatment1-level-4,ancombc/da_barplot/Category-treatment1-ASV',ancombc_formula='ancombc_formula/da_barplot/Category-treatment1-level-2,ancombc_formula/da_barplot/Category-treatment1-level-3,ancombc_formula/da_barplot/Category-treatment1-ASV,ancombc_formula/da_barplot/Category-treatment1-level-4',sbdi='sbdi/annotation.tsv,sbdi/asv-table.tsv,sbdi/dna.tsv,sbdi/emof.tsv,sbdi/event.tsv',phyloseq='phyloseq/qiime2_phyloseq.rds,phyloseq/dada2_phyloseq.rds'), envir = new.env())

  writeLines(c("\"NFCORE_AMPLISEQ:AMPLISEQ:SUMMARY_REPORT\":",
      paste0("    R: ", paste0(R.Version()[c("major","minor")], collapse = ".")),
      paste0("    rmarkdown: ", packageVersion("rmarkdown")),
      paste0("    knitr: ", packageVersion("knitr")) ),
      "versions.yml")

Command exit status:
  1

Command output:
  [1] TRUE
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  4/156 [setup]
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  20/156 [unnamed-chunk-8]

Command error:
  [1] TRUE


  processing file: template.Rmd
  1/156
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  4/156 [setup]
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  6/156 [unnamed-chunk-1]
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  Quitting from lines 245-246 [unnamed-chunk-8] (template.Rmd)
  Error in `knitr::include_graphics()`:
  ! Cannot find the file(s): "multiqc_plots/svg/fastqc_per_sequence_quality_scores_plot.svg"
  Backtrace:
   1. knitr::include_graphics(params$mqc_plot)
  Execution halted

Relevant files

nextflow.log

System information

Nextflow 24.10.3.5933
ampliseq 2.12.0
Singularity 3.8.7

[d4straub]

I run the pipeline with NXF_VER=24.10.3 nextflow run nf-core/ampliseq -r 2.12.0 -profile test,singularity --outdir results_test_2.12.0 --skip_alpha_rarefaction --skip_diversity_indices and it worked fine. So the problem seems to be not the pipeline.
I assume some problem with the file system or such. Could you please simply resume the pipeline, i.e. append a -resume to your command?

[nagendraKU]

Hi @d4straub, Thanks for the suggestion. I have tried -resume on the test profile (several times) and also processed another dataset. The pipeline still fails at the same step as noted above.

[d4straub]

There are some MultiQC versions with bugs that do not produce the expected figures. However, this pipeline uses the versions that produce figures or forces MultiQC to do so if possible. This is based on extensive testing of myself and it works for others as well, so I am sure thats not it, except if you have fiddled somehow with the MultiQC version.
Can you run the test on a different machine? I assume it has to do with your computational infrastructure if it isnt the MultiQC version.
If that also fails, either skip MultiQC (append --skip_multiqc) or skip the report generation (--skip_report) to make the pipeline pass all steps and finish successfully.

[nagendraKU]

Hi @d4straub, Thanks for the suggestions! I haven't done any mucking about with software versions :-)

I ran the pipeline to completion with --skip-report for now but will look into getting the entire pipeline to run at a later time. Thanks for all your help!