sarek v3.1.2 -- error for ascat

[PyGuan]

I have encountered the following error. I have tried both with out without --save_output_as_bam. It didn't help. I also ensured sufficient memory (256G) is available.

Thank you!

_-[nf-core/sarek] Pipeline completed with errors-
[27/e13d07] NOTE: Process `NFCORE_SAREK:SAREK:BAM_VARIANT_CALLING_SOMATIC_ALL:BAM_VARIANT_CALLING_SOMATIC_ASCAT:ASCAT (CGMH-FH-RCC1T_vs_CGMH-FH-RCC1N)` terminated with an error exit status (1) -- Execution is retried (7)
Error executing process > 'NFCORE_SAREK:SAREK:BAM_VARIANT_CALLING_SOMATIC_ALL:BAM_VARIANT_CALLING_SOMATIC_ASCAT:ASCAT (CGMH-FH-RCC1T_vs_CGMH-FH-RCC1N)'

Caused by:
  Process `NFCORE_SAREK:SAREK:BAM_VARIANT_CALLING_SOMATIC_ALL:BAM_VARIANT_CALLING_SOMATIC_ASCAT:ASCAT (CGMH-FH-RCC1T_vs_CGMH-FH-RCC1N)` terminated with an error exit status (1)

Command executed:

  #!/usr/bin/env Rscript
  library(RColorBrewer)
  library(ASCAT)
  options(bitmapType='cairo')
  
  #build prefixes: <abspath_to_files/prefix_chr>
  allele_path = normalizePath("G1000_alleles_hg19")
  allele_prefix = paste0(allele_path, "/", "G1000_alleles_hg19", "_chr")
  
  loci_path = normalizePath("G1000_loci_hg19")
  loci_prefix = paste0(loci_path, "/", "G1000_loci_hg19", "_chr")
  
  #prepare from BAM files
  ascat.prepareHTS(
      tumourseqfile = "T.converted.cram",
      normalseqfile = "N.converted.cram",
      tumourname = paste0("T_vs_N", ".tumour"),
      normalname = paste0("T_vs_N", ".normal"),
      allelecounter_exe = "alleleCounter",
      alleles.prefix = allele_prefix,
      loci.prefix = loci_prefix,
      gender = "XY",
      genomeVersion = "hg19",
      nthreads = 16
      ,minCounts = 10
      ,BED_file = 'wgs_calling_regions_Sarek.list'
      ,chrom_names = c(1:22, 'X', 'Y')
      ,min_base_qual = 20
      ,min_map_qual = 35
      ,ref.fasta = 'human_g1k_v37_decoy.fasta'
  
  
  )
  
  
  #Load the data
  ascat.bc = ascat.loadData(
      Tumor_LogR_file = paste0("T_vs_N", ".tumour_tumourLogR.txt"),
      Tumor_BAF_file = paste0("T_vs_N", ".tumour_tumourBAF.txt"),
      Germline_LogR_file = paste0("T_vs_N", ".tumour_normalLogR.txt"),
      Germline_BAF_file = paste0("T_vs_N", ".tumour_normalBAF.txt"),
      genomeVersion = "hg19",
      gender = "XY"
  )
  
  #Plot the raw data
  ascat.plotRawData(ascat.bc, img.prefix = paste0("T_vs_N", ".before_correction."))
  
  # optional LogRCorrection
  if("GC_G1000_hg19" != "NULL") {
      gc_input = paste0(normalizePath("GC_G1000_hg19"), "/", "GC_G1000_hg19", ".txt")
  
      if("RT_G1000_hg19" != "NULL"){
          rt_input = paste0(normalizePath("RT_G1000_hg19"), "/", "RT_G1000_hg19", ".txt")
          ascat.bc = ascat.correctLogR(ascat.bc, GCcontentfile = gc_input, replictimingfile = rt_input)
          #Plot raw data after correction
          ascat.plotRawData(ascat.bc, img.prefix = paste0("T_vs_N", ".after_correction_gc_rt."))
      }
      else {
          ascat.bc = ascat.correctLogR(ascat.bc, GCcontentfile = gc_input, replictimingfile = RT_G1000_hg19)
          #Plot raw data after correction
          ascat.plotRawData(ascat.bc, img.prefix = paste0("T_vs_N", ".after_correction_gc."))
      }
  }
  
  #Segment the data
  ascat.bc = ascat.aspcf(ascat.bc)
  
  #Plot the segmented data
  ascat.plotSegmentedData(ascat.bc)
  
  #Run ASCAT to fit every tumor to a model, inferring ploidy, normal cell contamination, and discrete copy numbers
  #If psi and rho are manually set:
  if (!is.null(NULL) && !is.null(NULL)){
      ascat.output <- ascat.runAscat(ascat.bc, gamma=1, rho_manual=NULL, psi_manual=NULL)
  } else if(!is.null(NULL) && is.null(NULL)){
      ascat.output <- ascat.runAscat(ascat.bc, gamma=1, rho_manual=NULL)
  } else if(!is.null(NULL) && is.null(NULL)){
      ascat.output <- ascat.runAscat(ascat.bc, gamma=1, psi_manual=NULL)
  } else {
      ascat.output <- ascat.runAscat(ascat.bc, gamma=1)
  }
  
  #Extract metrics from ASCAT profiles
  QC = ascat.metrics(ascat.bc,ascat.output)
  
  #Write out segmented regions (including regions with one copy of each allele)
  write.table(ascat.output[["segments"]], file=paste0("T_vs_N", ".segments.txt"), sep=" ", quote=F, row.names=F)
  
  #Write out CNVs in bed format
  cnvs=ascat.output[["segments"]][2:6]
  write.table(cnvs, file=paste0("T_vs_N",".cnvs.txt"), sep="    ", quote=F, row.names=F, col.names=T)
  
  #Write out purity and ploidy info
  summary <- tryCatch({
          matrix(c(ascat.output[["aberrantcellfraction"]], ascat.output[["ploidy"]]), ncol=2, byrow=TRUE)}, error = function(err) {
              # error handler picks up where error was generated
              print(paste("Could not find optimal solution:  ",err))
              return(matrix(c(0,0),nrow=1,ncol=2,byrow = TRUE))
      }
  )
  colnames(summary) <- c("AberrantCellFraction","Ploidy")
  write.table(summary, file=paste0("T_vs_N",".purityploidy.txt"), sep=" ", quote=F, row.names=F, col.names=T)
  
  write.table(QC, file=paste0("T_vs_N", ".metrics.txt"), sep="  ", quote=F, row.names=F)
  
  # version export
  f <- file("versions.yml","w")
  alleleCounter_version = system(paste("alleleCounter --version"), intern = T)
  ascat_version = sessionInfo()$otherPkgs$ASCAT$Version
  writeLines(paste0('"', "NFCORE_SAREK:SAREK:BAM_VARIANT_CALLING_SOMATIC_ALL:BAM_VARIANT_CALLING_SOMATIC_ASCAT:ASCAT", '"', ":"), f)
  writeLines(paste("    alleleCounter:", alleleCounter_version), f)
  writeLines(paste("    ascat:", ascat_version), f)
  close(f)

Command exit status:
  1

Command output:
  (empty)

Command error:
  Done reading locis
  Multi pos start:
  Reading locis
  [E::sam_itr_next] Null iterator
  [ERROR] (src/bam_access.c: bam_access_get_multi_position_base_counts:379 errno: No such file or directory) Error detected (-2) when trying to iterate through region
  [ERROR] (./src/alleleCounter.c: main:432 errno: None) Error scanning through bam file for loci list with dense snps.
  munmap_chunk(): invalid pointer
  Done reading locis
  Multi pos start:
  [E::sam_itr_next] Null iterator
  [ERROR] (src/bam_access.c: bam_access_get_multi_position_base_counts:379 errno: No such file or directory) Error detected (-2) when trying to iterate through region
  [ERROR] (./src/alleleCounter.c: main:432 errno: None) Error scanning through bam file for loci list with dense snps.
  Reading locis
  Done reading locis
  Multi pos start:
  munmap_chunk(): invalid pointer
  [E::sam_itr_next] Null iterator
  [ERROR] (src/bam_access.c: bam_access_get_multi_position_base_counts:379 errno: No such file or directory) Error   <<..............>>

Work dir:
  <<..............>>

Tip: you can try to figure out what's wrong by changing to the process work dir and showing the script file named `.command.sh`_

[PyGuan]

I ran it with GRCh37 and other default settings.

[FriederikeHanssen]

Hey! This looks like an error with the ASCAT tool. Can you attach the .command.log, .command.out, and .command.errfor the process. You can find it in the linked work dir.

The parameter --save_output_bam doesn't do anything for the variant calling tools. It is used to convert the output of the preprocessing processes (markduplicates and applybqsr) to BAM in the results folder.

[PyGuan]

Here you go. Take note I edited the file to mask my actual working directory.

command.err.txt
command.log.txt
command.sh.txt
command.run.txt

[PyGuan]

Just for you information, I also encountered issues with msisensorpro and deepvariant, but only for GRCh37. It worked okay with GRCh38.
So, I am not sure whether it is related to the reference genome I am using. I downloaded the references from here: s3://ngi-igenomes/igenomes/Homo_sapiens/GATK/GRCh37/

[SusiJo]

Actually, this sounds more like an issue with cancerit/alleleCount than Ascat. There are some issues mentioned on their github repo. Can you check out if any of those are of help?

• Throw informative error when BAM index missing cancerit/alleleCount#14
• Null Iterator Error cancerit/alleleCount#69
• Confusing error when input is unmapped cancerit/alleleCount#71

[PyGuan]

Just an update, the error can be due to this "connection time out" error:

[W::find_file_url] [W::find_file_url] Failed to open reference "https://www.ebi.ac.uk/ena/cram/md5/96e414eace405d8c27a6d35ba19df56f": Connection timed outFailed to ope
n reference "https://www.ebi.ac.uk/ena/cram/md5/6c198acf68b5af7b9d676dfdd531b5de": Connection timed out

I'm not sure exactly what's going on. The connection timeout seems to be intermittent. I tried running the pipeline a few times, and each time some samples were cleared.

It could be that our server is blocking too-frequent access to external data, or EBI is denying my connection.

For now, I guess, when this happens, I'll just rerun the pipeline.

[PyGuan]

Just an update, the error can be due to this "connection time out" error:

[W::find_file_url] [W::find_file_url] Failed to open reference "https://www.ebi.ac.uk/ena/cram/md5/96e414eace405d8c27a6d35ba19df56f": Connection timed outFailed to ope n reference "https://www.ebi.ac.uk/ena/cram/md5/6c198acf68b5af7b9d676dfdd531b5de": Connection timed out

I'm not sure exactly what's going on. The connection timeout seems to be intermittent. I tried running the pipeline a few times, and each time some samples were cleared.

It could be that our server is blocking too-frequent access to external data, or EBI is denying my connection.

For now, I guess, when this happens, I'll just rerun the pipeline.

I was referring to GRCh38. For GRCh37, the issue persisted.

[oghzzang]

Dear user,

Hello, I resolved it!
This issue was related to the reference for ascat.

Check if the chromosomes in your BAM file have the "chr" prefix.
There's a high chance that the "chr" prefix is missing.

In that case, you need to modify the "ascat_loci" in the igenome.config file.
Currently, the default configuration in Sarek downloads "${params.igenomes_base}/Homo_sapiens/GATK/GRCh37/Annotation/ASCAT/G1000_loci_hg19.zip", which includes the "chr" prefix.
You can either download a version without "chr" for your local file or simply remove "chr" using a quick sed command.

Please refer to the bottom of the following page for information on "'chr'-based versus non 'chr'-based reference":
Reference: VanLoo-lab ascat ReferenceFiles for WGS