Troubles in inferring LOH
Giuseppe1995 opened this issue · 4 comments
Hi,
I am trying to compute Loss of Heterozygosity (LOH) using freec (version=11.6), but I get the following error while building pileup file
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/15
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/11
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/6
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/2
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/8
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/16
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/14
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/7
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/3
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
[opened FIFO for writing] /tmp/sambamba-pid849194-iyhf/12
[E::hts_open_format] Failed to open file "1" : No such file or directory
[mpileup] failed to open 1: No such file or directory
sambamba-pileup: Broken pipe
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/10" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/10: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/4" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/4: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/5" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/5: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/1" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/1: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/13" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/13: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/9" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/9: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/17" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/17: No such file or directory
[E::hts_open_format] Failed to open file "/tmp/sambamba-pid849194-iyhf/18" : No such file or directory
[mpileup] failed to open /tmp/sambamba-pid849194-iyhf/18: No such file or directory
Here follows the configuration file...
[general]
BedGraphOutput = TRUE
breakPointThreshold = 0.8
breakPointType = 4
chrFiles = /srv/ngsdata/dalteriog/reference_genomes/hs/chromosomes/
chrLenFile = /srv/ngsdata/dalteriog/reference_genomes/hs/No_M.hg19.len
coefficientOfVariation = 0.05
contamination = 0
contaminationAdjustment = FALSE
forceGCcontentNormalization = 0
gemMappabilityFile = /srv/ngsdata/dalteriog/reference_genomes/hs/out76_hg19.gem
minCNAlength = 1
minExpectedGC = 0.35
maxExpectedGC = 0.55
minimalSubclonePresence = 20
maxThreads = 18
outputDir = CNVDir/LOH_CNV/
ploidy = 2
readCountThreshold = 10
sambamba = /srv/ngsdata/dalteriog/Tools/miniconda3/envs/ngspipe/bin/sambamba
samtools = /srv/ngsdata/dalteriog/Tools/miniconda3/envs/ngspipe/bin/samtools
sex = XX
window = 50000
step = 10000[sample]
mateFile = SP_1_T/SP_1_T.finalSorted.bam
inputFormat = BAM
mateOrientation = FR[control]
mateFile = SP_1_N/SP_1_N.finalSorted.bam
inputFormat = BAM
mateOrientation = FR[BAF]
makePileup = /srv/ngs/analysis/dalteriog/reference_genomes/hs/hg19_snp142.SingleDiNucl.1based.bed
fastaFile = /srv/ngs/analysis/dalteriog/reference_genomes/hs/hg19.fa
SNPfile = /srv/ngs/analysis/dalteriog/reference_genomes/hs/hg19_snp142.SingleDiNucl.1based.txt.gz
... and the command I run
freec -conf config_LOH_CNV -sample SP_1_T/SP_1_T.finalSorted.bam -control SP_1_N/SP_1_N.finalSorted.bam
I checked the matching of chromosome nomenclature among the various genomic files, and it seems to be all correct
there is only a question that puzzles me, and is that samtools mpileup has been deprecated, although I don't think this is the problem.
Thank you in advance,
Giuseppe
Dear Giuseppe,
what happens if you try to read you BAM file with samtools? e.g., when you remove or comment
sambamba = /srv/ngsdata/dalteriog/Tools/miniconda3/envs/ngspipe/bin/sambamba?
I did! and despite it's apparently running, it is stuck for give or take 30 hours with this standard error:
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 18 threads
..consider the sample being female
..Breakpoint threshold for segmentation of copy number profiles is 0.8
..telocenromeric set to 50000
..FREEC is not going to adjust profiles for a possible contamination by normal cells
..Note, the Coefficient Of Variation won't be used since "window" = 50000 was set
..Step: 10000
..Output directory: CNVDir/LOH_CNV/
..Directory with files containing chromosome sequences: /srv/ngsdata/dalteriog/reference_genomes/hs/chromosomes/
..Sample file: SP_1_T/SP_1_T.finalSorted.bam
..Sample input format: BAM
..will use this instance of samtools: '/srv/ngsdata/dalteriog/Tools/miniconda3/envs/ngspipe/bin/samtools' to read BAM files
..Control file: SP_1_N/SP_1_N.finalSorted.bam
..Input format for the control file: BAM
FREEC will create a pileup to compute BAF profile!
...File with SNPs : /srv/ngs/analysis/dalteriog/reference_genomes/hs/hg19_snp142.SingleDiNucl.1based.bed
..Polynomial degree for "Sample ReadCount ~ Control ReadCount" normalization is 1
..Minimal CNA length (in windows) is 1
..File with chromosome lengths: /srv/ngsdata/dalteriog/reference_genomes/hs/No_M.hg19.len
..Mappability file/srv/ngsdata/dalteriog/reference_genomes/hs/out76_hg19.gem be used: all low mappability positions will be discarded
..uniqueMatch = FALSE
..average ploidy set to 2
..break-point type set to 4
..noisyData set to 0
..minimal number of reads per window in the control sample is set to 10
..Control-FREEC will look for subclones present in at least 20% of cell population
Creating Pileup file to compute BAF profile...
..File /srv/ngsdata/dalteriog/reference_genomes/hs/No_M.hg19.len was read to create a miniPileup
[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
and nothing seems to happen
Were any files created? If yes, were they empty?
Hi, sorry for the late reply!
Actually, after 3 days it finished giving as output the final "BAF" required file.