Error in Germline

[parth2608]

Hi, I am trying to run Germline on the example provided in the repository. After successfully running preProcess, I run the following command:

python src/Monopogen.py germline -a apps/ -t 1 -r test/region.lst -p example/ -g example/chr20_2Mb.hg38.fa -s all -o out

But I get errors and the following is the output:

[2024-01-09 11:42:35,227] INFO Monopogen.py Performing germline variant calling... [2024-01-09 11:42:35,227] INFO germline.py Parameters in effect: [2024-01-09 11:42:35,227] INFO germline.py --subcommand = [germline] [2024-01-09 11:42:35,227] INFO germline.py --region = [test/region.lst] [2024-01-09 11:42:35,227] INFO germline.py --step = [all] [2024-01-09 11:42:35,227] INFO germline.py --out = [out] [2024-01-09 11:42:35,227] INFO germline.py --reference = [example/chr20_2Mb.hg38.fa] [2024-01-09 11:42:35,228] INFO germline.py --imputation_panel = [example/] [2024-01-09 11:42:35,228] INFO germline.py --max_softClipped = [1] [2024-01-09 11:42:35,228] INFO germline.py --app_path = [apps/] [2024-01-09 11:42:35,228] INFO germline.py --nthreads = [1] [2024-01-09 11:42:35,228] INFO germline.py --norun = [FALSE] [2024-01-09 11:42:35,228] INFO Monopogen.py Checking existence of essenstial resource files... [2024-01-09 11:42:35,236] INFO Monopogen.py Checking dependencies... ['bash out/Script/runGermline_chr20.sh'] /rsrch5/home/tdccct/ppshah/shared/pipelines/Monopogen/apps/bcftools: error while loading shared libraries: libcrypto.so.1.0.0: cannot open shared object file: No such file or directory /rsrch5/home/tdccct/ppshah/shared/pipelines/Monopogen/apps/bcftools: error while loading shared libraries: libcrypto.so.1.0.0: cannot open shared object file: No such file or directory [mpileup] 2 samples in 2 input files (mpileup) Max depth is above 1M. Potential memory hog! beagle.27Jul16.86a.jar (version 4.1) Copyright (C) 2014-2015 Brian L. Browning Enter "java -jar beagle.27Jul16.86a.jar" for a summary of command line arguments. Start time: 11:44 AM CST on 09 Jan 2024

Command line: java -Xmx20480m -jar beagle.jar gl=out/germline/chr20.gl.vcf.gz ref=example/CCDG_14151_B01_GRM_WGS_2020-08-05_chr20.filtered.shapeit2-duohmm-phased.vcf.gz chrom=chr20 out=out/germline/chr20.gp impute=false modelscale=2 nthreads=24 gprobs=true niterations=0

No genetic map is specified: using 1 cM = 1 Mb Exception in thread "main" java.lang.IllegalArgumentException: Missing line (#CHROM ...) after meta-information lines File source: out/germline/chr20.gl.vcf.gz null at vcf.VcfHeader.checkHeaderLine(VcfHeader.java:135) at vcf.VcfHeader.(VcfHeader.java:119) at vcf.VcfIt.(VcfIt.java:190) at vcf.VcfIt.create(VcfIt.java:175) at vcf.VcfIt.create(VcfIt.java:150) at main.Main.allData(Main.java:303) at main.Main.main(Main.java:111) gzip: out/germline/chr20.gp.vcf.gz: No such file or directory out/germline/chr20.gp.vcf.gz: No such file or directory beagle.27Jul16.86a.jar (version 4.1) Copyright (C) 2014-2015 Brian L. Browning Enter "java -jar beagle.27Jul16.86a.jar" for a summary of command line arguments. Start time: 11:44 AM CST on 09 Jan 2024

Command line: java -Xmx20480m -jar beagle.jar gt=out/germline/chr20.germline.vcf ref=example/CCDG_14151_B01_GRM_WGS_2020-08-05_chr20.filtered.shapeit2-duohmm-phased.vcf.gz chrom=chr20 out=out/germline/chr20.phased impute=false modelscale=2 nthreads=24 gprobs=true niterations=0

No genetic map is specified: using 1 cM = 1 Mb Exception in thread "main" java.lang.IllegalArgumentException: Missing line (#CHROM ...) after meta-information lines File source: out/germline/chr20.germline.vcf null at vcf.VcfHeader.checkHeaderLine(VcfHeader.java:135) at vcf.VcfHeader.(VcfHeader.java:119) at vcf.VcfIt.(VcfIt.java:190) at vcf.VcfIt.create(VcfIt.java:175) at vcf.VcfIt.create(VcfIt.java:150) at main.Main.allData(Main.java:297) at main.Main.main(Main.java:111) [2024-01-09 11:44:08,145] INFO Monopogen.py Success! See instructions above.

Any guidance to resolve this would be helpful. I am working on HPC system.

Thank you!

[jinzhuangdou]

Hi the error shows "/rsrch5/home/tdccct/ppshah/shared/pipelines/Monopogen/apps/bcftools: error while loading shared libraries: ". Could you try to see whether it works (as mentioned in FAQs)?

bcftools: error while loading shared libraries: libbz2.so.1.0: not able to open shared object file: No such file or directly Adding the apps folder of Monopogen in your library environment export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:/xx/apps

[parth2608]

I did that, but I still get the following error

/rsrch5/home/tdccct/ppshah/shared/pipelines/Monopogen/apps/bcftools/rsrch5/home/tdccct/ppshah/shared/pipelines/Monopogen/apps/bcftools: : symbol lookup errorsymbol lookup error: : /lib64/libk5crypto.so.3/lib64/libk5crypto.so.3: : undefined symbol: EVP_KDF_ctrl, version OPENSSL_1_1_1bundefined symbol: EVP_KDF_ctrl, version OPENSSL_1_1_1b

[jinzhuangdou]

Are you able to compile bcftools at your end? The version required are samtools Version: 1.2 (using htslib 1.2.1) bcftools Version: 1.8 (using htslib 1.8) beagle.27Jul16.86a.jar (version 4.1) tabix Version: 1.9 bgzip Version: 1.9.

[Coaxecva]

Hi @jinzhuangdou,

I successfully ran preprocess, but when I run Monopogen germline, I added all autosome 1-22 (even with 1 chr22), I got warnings, and it seems the process took forever to process:

python Monopogen/src/Monopogen.py germline -a Monopogen/apps -r region.lst -p ./ -t 22 -g Homo_sapiens_assembly38.fasta -m 3 -s all -o out_SC399_9

CPU time :                                   86062.00 sec.
Max Memory :                                 7 GB
Average Memory :                             5.07 GB
Total Requested Memory :                     30.00 GB
Delta Memory :                               23.00 GB

[2024-11-12 16:08:10,587] INFO Monopogen.py Performing germline variant calling... [2024-11-12 16:08:10,587] INFO germline.py Parameters in effect: [2024-11-12 16:08:10,587] INFO germline.py --subcommand = [germline] [2024-11-12 16:08:10,588] INFO germline.py --region = [region.lst] [2024-11-12 16:08:10,588] INFO germline.py --step = [all] [2024-11-12 16:08:10,588] INFO germline.py --out = [out_SC399_9] [2024-11-12 16:08:10,588] INFO germline.py --reference = [Homo_sapiens_assembly38.fasta] [2024-11-12 16:08:10,588] INFO germline.py --imputation_panel = [./] [2024-11-12 16:08:10,588] INFO germline.py --max_softClipped = [3] [2024-11-12 16:08:10,588] INFO germline.py --app_path = [Monopogen/apps] [2024-11-12 16:08:10,588] INFO germline.py --nthreads = [22] [2024-11-12 16:08:10,588] INFO germline.py --norun = [FALSE] [2024-11-12 16:08:10,588] INFO Monopogen.py Checking existence of essenstial resource files... [2024-11-12 16:08:10,614] INFO Monopogen.py Checking dependencies... [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files [mpileup] 1 samples in 1 input files (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! (mpileup) Max depth is above 1M. Potential memory hog! Lines total/split/realigned/skipped: 36550225/124788/11454/0

[theheking]

I have the same error, but unable to compile due to the --app_path required flag, conflict of installations.

Are you able to compile bcftools at your end? The version required are samtools Version: 1.2 (using htslib 1.2.1) bcftools Version: 1.8 (using htslib 1.8) beagle.27Jul16.86a.jar (version 4.1) tabix Version: 1.9 bgzip Version: 1.9.

[katestan]

I am also getting the error /camp/home/stanlek/home/users/stanlek/Monopogen/apps/bcftools: symbol lookup error: /lib64/libk5crypto.so.3: undefined symbol: EVP_KDF_ctrl, version OPENSSL_1_1_1b

You can hardcode a path to your own compiled version of bcftools within Monopogen.py to get around the -app_path required flag, conflict of installations issue @theheking