Error: unable to open file or unable to determine types for file

[stroke1989]

Hi, I'm a new to FREEC. Thanks for this excellent tool. I tired to use FREEC to analyze CNV + LOH of my WES data. However, ERROR occurred: Control-FREEC v11.6 : a method for automatic detection of copy number alterations, subclones and for accurate estimation of contamination and main ploidy using deep-sequencing data Multi-threading mode using 6 threads ..Breakpoint threshold for segmentation of copy number profiles is 1.2 ..telocenromeric set to 50000 ..FREEC is not going to output normalized copy number profiles into a BedGraph file (for example, for visualization in the UCSC GB). Use "[general] BedGraphOutput=TRUE" if you want a BedGraph file ..FREEC is not going to adjust profiles for a possible contamination by normal cells ..Window = 0 was set ..Output directory: /home/ug0416/software/FREEC-11.6/data/output ..Directory with files containing chromosome sequences: /home/ug0416/ref_hg19/ucsc_hg19_chromosome ..will use a threshold of 5 read(s) per SNP position to calculate beta allel frequency (BAF) values ..Sample file: /home/ug0416/mydata/05.gatk/a.BQSR/2nd_BQSR/SCA_082_T.sorted.markdup.recal.bam ..Sample input format: BAM ..will use this instance of samtools: '/home/ug0416/.conda/envs/sambamba/bin/samtools' to read BAM files ..Control file: /home/ug0416/mydata/05.gatk/a.BQSR/2nd_BQSR/SCA_082_N.sorted.markdup.recal.bam ..Input format for the control file: BAM FREEC will create a pileup to compute BAF profile! ...File with SNPs : /home/ug0416/software/FREEC-11.6/data/hg19_snp142.SingleDiNucl.1based.bed ..forceGCcontentNormalization was set to 1: will use GC-content to normalize the read count data ..minimal expected GC-content (general parameter "minExpectedGC") was set to 0.35 ..maximal expected GC-content (general parameter "maxExpectedGC") was set to 0.55 ..Minimal CNA length (in windows) is 3 ..File with chromosome lengths: /home/ug0416/software/FREEC-11.6/data/hg19.len ..File /home/ug0416/software/FREEC-11.6/data/hg19.len was read ..Using the default minimal mappability value of 0.85 ..uniqueMatch = FALSE ..FREEC will try to guess the correct ploidy (for each ploidy specified in 'ploidy' parameter) ..It will try ploidies: 2 3 ..break-point type set to 4 ..noisyData set to 1 ..minimal number of reads per window in the control sample is set to 60 ..Control-FREEC will not look for subclones Creating Pileup file to compute BAF profile... ..will increase flanking regions by -1252 bp Error: unable to open file or unable to determine types for file /home/ug0416/software/FREEC-11.6/data/output/SCA_082_T.sorted.markdup.recal.bam_NewCaptureRegions.bed

• Please ensure that your file is TAB delimited (e.g., cat -t FILE).
• Also ensure that your file has integer chromosome coordinates in the expected columns (e.g., cols 2 and 3 for BED). [mpileup] 1 samples in 1 input files ..If you have got an error at this step and a mini-pileup file is empty, check that you are using samtools v1.1 or later and provide a corresponding path in your config file [mpileup] 1 samples in 1 input files ..If you have got an error at this step and a mini-pileup file is empty, check that you are using samtools v1.1 or later and provide a corresponding path in your config file ... -> Done!

  The ERROR indicated that "unable to open file or unable to determine types for file /home/ug0416/software/FREEC-11.6/data/output/SCA_082_T.sorted.markdup.recal.bam_NewCaptureRegions.bed"

  The following is my config file:

  For more options see: http://boevalab.com/FREEC/tutorial.html#CONFIG

[general] chrLenFile = /home/ug0416/software/FREEC-11.6/data/hg19.len window = 0 ploidy = 2,3 outputDir = /home/ug0416/software/FREEC-11.6/data/output bedtools = /pub/anaconda3/bin/bedtools samtools = /home/ug0416/.conda/envs/sambamba/bin/samtools

sex=XY

breakPointType=4 chrFiles = /home/ug0416/ref_hg19/ucsc_hg19_chromosome

maxThreads=6

breakPointThreshold=1.2 noisyData=TRUE printNA=TRUE

readCountThreshold=60

[sample]

mateFile = /home/ug0416/mydata/05.gatk/a.BQSR/2nd_BQSR/SCA_082_T.sorted.markdup.recal.bam inputFormat = BAM mateOrientation = FR

[control]

mateFile = /home/ug0416/mydata/05.gatk/a.BQSR/2nd_BQSR/SCA_082_N.sorted.markdup.recal.bam inputFormat = BAM mateOrientation = FR

[BAF]

SNPfile = /home/ug0416/software/FREEC-11.6/data/hg19_snp142.SingleDiNucl.1based.txt minimalCoveragePerPosition = 5 makePileup = /home/ug0416/software/FREEC-11.6/data/hg19_snp142.SingleDiNucl.1based.bed fastaFile = /home/ug0416/ref_hg19/ucsc.hg19.fasta

[target]

captureRegions = /home/ug0416/software/FREEC-11.6/data/IDTV2.bed

I have no idea how to solve this issue. Hope your response! Appreciate!

[valeu]

Does your output directory have writing rights? /home/ug0416/software/FREEC-11.6/data/output/

[dariober]

Maybe related: https://github.com/dariober/cnv_facets/issues/45

Long story short: the input reads have been mistakenly aligned using the first-in-pair fastq file twice. This is going to cause all sorts of issues for any program.