Hello nf-core team, I've encountered an issue while running the Sarek pipeline. Here are the details: 1. The Nextflow command used to launch the pipeline: nextflow run nf-core/sarek -r 3.4.2 -profile docker --input explicit_sample.csv --outdir /home/drseed/BAM2/final_result_40 --genome GATK.GRCh38 --dbsnp /home/drseed/BAM2/ref/known_indels/1000G_phase1.snps.high_confidence.hg38.vcf.gz --known_indels /home/drseed/BAM2/ref/known_indels/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz --max_cpus 8 --max_memory '120.GB' --tools mutect2,strelka,snpeff,vep --pon /home/drseed/BAM2/ref/pon/1000g_pon.hg38.vcf.gz --germline_resource /home/drseed/BAM2/ref/af-only-gnomad.hg38.vcf.gz --germline_resource_tbi /home/drseed/BAM2/ref/af-only-gnomad.hg38.vcf.gz.tbi --vep_cache --vep_cache /home/drseed/BAM2/vep_cache/homo_sapiens/112_GRCh38 --vep_species homo_sapiens --vep_genome GRCh38 --vep_cache_version 112 --snpeff_db 106 --snpeff_genome GRCh38 --igenomes_base s3://ngi-igenomes/igenomes/ -c customed.config -with-dag flowchart_39.png Custom Nextflow config file (customed.config): params { monochromeLogs = false max_cpus = 8 max_memory = '120.GB' } process { withName: 'NFCORE_SAREKsarekFASTP' { cpus = { check_max( 8 * task.attempt, 'cpus' ) } memory = { check_max( 16.GB * task.attempt, 'memory' ) } } withName: 'NFCORE_SAREKsarek(BWA|MARKDUPLICATES|BASERECALIBRATOR|APPLYBQSR)' { memory = { check_max( 32.GB * task.attempt, 'memory' ) } } withName: 'NFCORE_SAREKsarek(MUTECT2|FREEBAYES|STRELKA_SOMATIC|SNPEFF|VEP)' { memory = { check_max( 64.GB * task.attempt, 'memory' ) } } withName: 'NFCORE_SAREKPREPARE GENOMEUNZIP_GC' { ext.httpRetry = 3 ext.httpRetryWait = 60 } withName: 'NFCORE_SAREKsarekVEP' { memory = { check_max( 80.GB * task.attempt, 'memory' ) } } errorStrategy = { task.exitStatus in [143,137,104,134,139] ? 'retry' : 'finish' } maxRetries = 3 maxErrors = '-1' } def check_max(obj, type) { // ... (function definition as in your config) } process.errorStrategy = { task.exitStatus in [143,137,104,134,139] ? 'retry' : 'finish' } process.maxRetries = 3 dag { overwrite = true } params { dbsnp ='/home/drseed/BAM2/folder/1000G_phase1.snps.high_confidence.hg38.vcf.gz' dbsnp_tbi ='/home/drseed/BAM2/folder/1000G_phase1.snps.high_confidence.hg38.vcf.gz.tbi' known_indels ='/home/drseed/BAM2/folder/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz' known_indels_tbi ='/home/drseed/BAM2/folder/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz.tbi' pon ='/home/drseed/BAM2/ref/pon/1000g_pon.hg38.vcf.gz' pon_tbi ='/home/drseed/BAM2/ref/pon/1000g_pon.hg38.vcf.gz.tbi' } The full error messages: The sample-sheet only contains normal-samples, but the following tools, which were requested with "--tools", expect at least one tumor-sample : mutect2 This path is not available within annotation-cache. Please check https://annotation-cache.github.io/ to create a request for it.

.nextflow.log

file Aug-31 063809.646 [Actor Thread 189] ERROR nextflow.extension.OperatorImpl - @unknown nextflow.exception.WorkflowScriptErrorException: The sample-sheet only contains normal-samples, but the following tools, which were requested with "--tools", expect at least one tumor-sample : mutect2 Aug-31 063809.678 [main] ERROR nextflow.cli.Launcher - @unknown nextflow.exception.WorkflowScriptErrorException: This path is not available within annotation-cache. Please check https://annotation-cache.github.io/ to create a request for it. I've verified that my

explicit_sample.csv

file contains both normal (status 0) and tumor (status 1) samples. I've checked for and removed any trailing spaces in the CSV file. The pipeline works when I use a simplified sample sheet with just one pair of normal and tumor samples. I'm unsure why the pipeline is not recognizing the tumor samples in my full sample sheet. The annotation-cache error seems to be related to the VEP cache, but I'm not sure how to resolve this. Any assistance in resolving these issues would be greatly appreciated. Thank you!

Hi Saheed, If you have double-checked the sample sheet and don't see any mistakes, there is a good chance that your error is actually unrelated to that -- I think this sometimes happens with the validation plugin when there is a problem with another parameter, e.g. a missing/invalid reference file, such as the annotation cache issue you are also seeing. So I suggest cleaning up your other parameters first: • You are using

--genome

, which is supposed to pull in the iGenomes reference files (which work well for sarek; I would not suggest deviating much from those unless you really know what you are doing), but then you are using a whole bunch of parameters to override what is in the

igenomes.config

of the pipeline. This does not make sense. ◦ You are setting

--igenomes_base

to the default (S3 bucket), but then you are overriding that pretty much every single file with a local path (

/home/drseed/BAM2/folder/...

). If you have you downloaded the iGenomes reference files (at least for

GATK.GRCh38

) and preserved the direcotory structure, you can jut set

--igenomes_base

to that location (the parent directory of

Homo_sapiens/

) and remove all other parameters pointing to individuals file (1000-genomes, dbSNP, germline resource, etc.). ◦ BTW, some you these individual file parameters were specified twice, in the config file and the command line. Note that because of the way the config file is read, parameters there are often overwritten later, so you should not have

params{}

in a config file used with

-c

(although that would be fine in a profile). Instead, you can use the parameters on the command line or put them in a JSON or YAML file and use that with

-params-file

. • In your command line, you have

--vep_cache

twice, and only the second time with a directory as argument. If that directory contains the downloaded VEP cache, then just removing the extraneous

--vep_cache

should be enough. 🤞 Pro tip: Typing ````` will open a window to paste your code or error messages, which separates them from the rest of the text and makes them more readable.

Thank you @Thomas Danhorn you for your detailed explanation