xqtl-protocol icon indicating copy to clipboard operation
xqtl-protocol copied to clipboard
Published 1 month ago • verified publisher icon StatFunGen

Picard QC in star_output step

Open jaempawi opened this issue 3 years ago • 0 comments

34/150 of my samples do not complete the picard_qc step and I need help with those. This is my code: #!/bin/bash -l #$ -l h_rt=24:00:00 #$ -N star_align1 #$ -j y #$ -P casa module load miniconda conda activate sos sos run /restricted/projectnb/casa/xqtl-pipeline/pipeline/RNA_calling.ipynb STAR_output \ --cwd /restricted/projectnb/casa/jaempawi/data/ROSMAP_blood_rnaseq/star_align1 \ --STAR-index /restricted/projectnb/casa/jaempawi/reference_data/STAR_Index \ --gtf /restricted/projectnb/casa/jaempawi/reference_data/Homo_sapiens.GRCh38.103.chr.reformatted.gene.ERCC.gtf \ --samples /restricted/projectnb/casa/jaempawi/data/ROSMAP_blood_rnaseq/fastq/ROSMAP_blood_metadata5.list \ --data-dir /restricted/projectnb/casa/jaempawi/data/ROSMAP_blood_rnaseq/fastq \ --reference-fasta /restricted/projectnb/casa/jaempawi/reference_data/GRCh38_full_analysis_set_plus_decoy_hla.noALT_noHLA_noDecoy.fasta \ --container /restricted/projectnb/casa/jaempawi/rna_quantification.sif \ --ref-flat /restricted/projectnb/casa/jaempawi/reference_data/Homo_sapiens.GRCh38.103.chr.reformated.ERCC.gtf.ref.flat \ --walltime 24h \ --mem 40G -s build -J 50 -c csg.yml -q neurology

This is the log file error I got for one of the incomplete samples: INFO 2022-09-15 11:13:05 RExecutor Executing R script via command: Rscript /tmp/script11928283018690918300.R INFO 2022-09-15 11:13:05 ProcessExecutor [1] "Checking if R is installed" 11:13:05.746 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/opt/picard-tools/picard.jar!/com/intel/gkl/native/libgkl_compression.so [Thu Sep 15 11:13:05 UTC 2022] CollectMultipleMetrics --INPUT /restricted/projectnb/casa/jaempawi/data/ROSMAP_blood_rnaseq/star_align1/Sample_145.Aligned.sortedByCoord.out.bam --OUTPUT /restricted/projectnb/casa/jaempawi/data/ROSMAP_blood_rnaseq/star_align1/Sample_145 --PROGRAM CollectAlignmentSummaryMetrics --PROGRAM CollectBaseDistributionByCycle --PROGRAM CollectInsertSizeMetrics --PROGRAM MeanQualityByCycle --PROGRAM QualityScoreDistribution --PROGRAM CollectGcBiasMetrics --VALIDATION_STRINGENCY STRICT --REFERENCE_SEQUENCE /restricted/projectnb/casa/jaempawi/reference_data/GRCh38_full_analysis_set_plus_decoy_hla.noALT_noHLA_noDecoy.fasta --ASSUME_SORTED true --STOP_AFTER 0 --METRIC_ACCUMULATION_LEVEL ALL_READS --INCLUDE_UNPAIRED false --VERBOSITY INFO --QUIET false --COMPRESSION_LEVEL 5 --MAX_RECORDS_IN_RAM 500000 --CREATE_INDEX false --CREATE_MD5_FILE false --GA4GH_CLIENT_SECRETS client_secrets.json --help false --version false --showHidden false --USE_JDK_DEFLATER false --USE_JDK_INFLATER false [Thu Sep 15 11:13:05 UTC 2022] Executing as jaempawi@scc-gi2 on Linux 3.10.0-1160.71.1.el7.x86_64 amd64; OpenJDK 64-Bit Server VM 11.0.15+10-Ubuntu-0ubuntu0.20.04.1; Deflater: Intel; Inflater: Intel; Provider GCS is not available; Picard version: Version:2.25.0 INFO 2022-09-15 11:14:16 SinglePassSamProgram Processed 1,000,000 records. Elapsed time: 00:00:10s. Time for last 1,000,000: 5s. Last read position: chr1:153,390,499 INFO 2022-09-15 11:14:27 SinglePassSamProgram Processed 2,000,000 records. Elapsed time: 00:00:21s. Time for last 1,000,000: 11s. Last read position: chr2:181,495,887 INFO 2022-09-15 11:14:41 SinglePassSamProgram Processed 3,000,000 records. Elapsed time: 00:00:35s. Time for last 1,000,000: 13s. Last read position: chr4:49,110,498 INFO 2022-09-15 11:14:50 SinglePassSamProgram Processed 4,000,000 records. Elapsed time: 00:00:45s. Time for last 1,000,000: 9s. Last read position: chr5:169,995,372 INFO 2022-09-15 11:15:02 SinglePassSamProgram Processed 5,000,000 records. Elapsed time: 00:00:57s. Time for last 1,000,000: 12s. Last read position: chr7:27,897,786 INFO 2022-09-15 11:15:13 SinglePassSamProgram Processed 6,000,000 records. Elapsed time: 00:01:08s. Time for last 1,000,000: 11s. Last read position: chr9:35,706,899 INFO 2022-09-15 11:15:24 SinglePassSamProgram Processed 7,000,000 records. Elapsed time: 00:01:18s. Time for last 1,000,000: 10s. Last read position: chr11:1,753,600 INFO 2022-09-15 11:15:32 SinglePassSamProgram Processed 8,000,000 records. Elapsed time: 00:01:27s. Time for last 1,000,000: 8s. Last read position: chr12:64,641,989 INFO 2022-09-15 11:15:43 SinglePassSamProgram Processed 9,000,000 records. Elapsed time: 00:01:37s. Time for last 1,000,000: 10s. Last read position: chr14:86,898,942 INFO 2022-09-15 11:15:52 SinglePassSamProgram Processed 10,000,000 records. Elapsed time: 00:01:47s. Time for last 1,000,000: 9s. Last read position: chr16:89,406,348 INFO 2022-09-15 11:16:03 SinglePassSamProgram Processed 11,000,000 records. Elapsed time: 00:01:58s. Time for last 1,000,000: 11s. Last read position: chr19:13,153,214 INFO 2022-09-15 11:16:10 SinglePassSamProgram Processed 12,000,000 records. Elapsed time: 00:02:05s. Time for last 1,000,000: 7s. Last read position: chr21:8,209,749 INFO 2022-09-15 11:16:13 SinglePassSamProgram Processed 13,000,000 records. Elapsed time: 00:02:07s. Time for last 1,000,000: 2s. Last read position: chr21:8,210,089 INFO 2022-09-15 11:16:15 SinglePassSamProgram Processed 14,000,000 records. Elapsed time: 00:02:09s. Time for last 1,000,000: 2s. Last read position: chr21:8,210,378 INFO 2022-09-15 11:16:17 SinglePassSamProgram Processed 15,000,000 records. Elapsed time: 00:02:12s. Time for last 1,000,000: 2s. Last read position: chr21:8,210,644 INFO 2022-09-15 11:16:19 SinglePassSamProgram Processed 16,000,000 records. Elapsed time: 00:02:14s. Time for last 1,000,000: 2s. Last read position: chr21:8,211,044 INFO 2022-09-15 11:16:24 SinglePassSamProgram Processed 17,000,000 records. Elapsed time: 00:02:19s. Time for last 1,000,000: 4s. Last read position: chr21:8,254,160 INFO 2022-09-15 11:16:30 SinglePassSamProgram Processed 18,000,000 records. Elapsed time: 00:02:25s. Time for last 1,000,000: 6s. Last read position: chr21:8,254,698 INFO 2022-09-15 11:16:35 SinglePassSamProgram Processed 19,000,000 records. Elapsed time: 00:02:29s. Time for last 1,000,000: 4s. Last read position: chr21:8,255,055 INFO 2022-09-15 11:16:39 SinglePassSamProgram Processed 20,000,000 records. Elapsed time: 00:02:34s. Time for last 1,000,000: 4s. Last read position: chr21:8,392,835 INFO 2022-09-15 11:16:42 SinglePassSamProgram Processed 21,000,000 records. Elapsed time: 00:02:36s. Time for last 1,000,000: 2s. Last read position: chr21:8,393,167 INFO 2022-09-15 11:16:44 SinglePassSamProgram Processed 22,000,000 records. Elapsed time: 00:02:38s. Time for last 1,000,000: 2s. Last read position: chr21:8,393,466 INFO 2022-09-15 11:16:46 SinglePassSamProgram Processed 23,000,000 records. Elapsed time: 00:02:41s. Time for last 1,000,000: 2s. Last read position: chr21:8,393,767 INFO 2022-09-15 11:16:48 SinglePassSamProgram Processed 24,000,000 records. Elapsed time: 00:02:43s. Time for last 1,000,000: 2s. Last read position: chr21:8,394,225 INFO 2022-09-15 11:16:51 SinglePassSamProgram Processed 25,000,000 records. Elapsed time: 00:02:46s. Time for last 1,000,000: 3s. Last read position: chr21:8,437,235 INFO 2022-09-15 11:16:56 SinglePassSamProgram Processed 26,000,000 records. Elapsed time: 00:02:50s. Time for last 1,000,000: 4s. Last read position: chr21:8,437,523 INFO 2022-09-15 11:16:58 SinglePassSamProgram Processed 27,000,000 records. Elapsed time: 00:02:53s. Time for last 1,000,000: 2s. Last read position: chr21:8,437,852 INFO 2022-09-15 11:17:01 SinglePassSamProgram Processed 28,000,000 records. Elapsed time: 00:02:55s. Time for last 1,000,000: 2s. Last read position: chr21:8,438,277 INFO 2022-09-15 11:17:03 SinglePassSamProgram Processed 29,000,000 records. Elapsed time: 00:02:58s. Time for last 1,000,000: 2s. Last read position: chr21:8,988,397 INFO 2022-09-15 11:17:12 SinglePassSamProgram Processed 30,000,000 records. Elapsed time: 00:03:07s. Time for last 1,000,000: 9s. Last read position: chrX:83,186,897 INFO 2022-09-15 11:17:19 SinglePassSamProgram Processed 31,000,000 records. Elapsed time: 00:03:14s. Time for last 1,000,000: 6s. Last read position: chrM:2,022 INFO 2022-09-15 11:17:25 SinglePassSamProgram Processed 32,000,000 records. Elapsed time: 00:03:19s. Time for last 1,000,000: 5s. Last read position: chrM:13,255 INFO 2022-09-15 11:17:28 SinglePassSamProgram Processed 33,000,000 records. Elapsed time: 00:03:23s. Time for last 1,000,000: 3s. Last read position: chr22_KI270733v1_random:126,280 INFO 2022-09-15 11:17:30 SinglePassSamProgram Processed 34,000,000 records. Elapsed time: 00:03:25s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:126,578 INFO 2022-09-15 11:17:33 SinglePassSamProgram Processed 35,000,000 records. Elapsed time: 00:03:28s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:126,906 INFO 2022-09-15 11:17:35 SinglePassSamProgram Processed 36,000,000 records. Elapsed time: 00:03:30s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:127,328 INFO 2022-09-15 11:17:38 SinglePassSamProgram Processed 37,000,000 records. Elapsed time: 00:03:33s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:171,176 INFO 2022-09-15 11:17:40 SinglePassSamProgram Processed 38,000,000 records. Elapsed time: 00:03:35s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:171,521 INFO 2022-09-15 11:17:42 SinglePassSamProgram Processed 39,000,000 records. Elapsed time: 00:03:37s. Time for last 1,000,000: 2s. Last read position: chr22_KI270733v1_random:171,846 INFO 2022-09-15 11:17:44 SinglePassSamProgram Processed 40,000,000 records. Elapsed time: 00:03:39s. Time for last 1,000,000: 1s. Last read position: chr22_KI270733v1_random:172,219 INFO 2022-09-15 11:17:47 SinglePassSamProgram Processed 41,000,000 records. Elapsed time: 00:03:41s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:109,146 INFO 2022-09-15 11:17:50 SinglePassSamProgram Processed 42,000,000 records. Elapsed time: 00:03:44s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:109,539 INFO 2022-09-15 11:17:55 SinglePassSamProgram Processed 43,000,000 records. Elapsed time: 00:03:50s. Time for last 1,000,000: 5s. Last read position: chrUn_GL000220v1:109,860 INFO 2022-09-15 11:18:01 SinglePassSamProgram Processed 44,000,000 records. Elapsed time: 00:03:55s. Time for last 1,000,000: 5s. Last read position: chrUn_GL000220v1:110,174 INFO 2022-09-15 11:18:08 SinglePassSamProgram Processed 45,000,000 records. Elapsed time: 00:04:03s. Time for last 1,000,000: 7s. Last read position: chrUn_GL000220v1:110,649 INFO 2022-09-15 11:18:11 SinglePassSamProgram Processed 46,000,000 records. Elapsed time: 00:04:06s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:153,438 INFO 2022-09-15 11:18:13 SinglePassSamProgram Processed 47,000,000 records. Elapsed time: 00:04:08s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:153,752 INFO 2022-09-15 11:18:16 SinglePassSamProgram Processed 48,000,000 records. Elapsed time: 00:04:10s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:154,090 INFO 2022-09-15 11:18:19 SinglePassSamProgram Processed 49,000,000 records. Elapsed time: 00:04:13s. Time for last 1,000,000: 2s. Last read position: chrUn_GL000220v1:154,538 [Thu Sep 15 11:18:19 UTC 2022] picard.analysis.CollectMultipleMetrics done. Elapsed time: 5.24 minutes. Runtime.totalMemory()=2422210560 To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp Exception in thread "main" htsjdk.samtools.SAMException: Reference sequence (202) not found in /restricted/projectnb/casa/jaempawi/reference_data/GRCh38_full_analysis_set_plus_decoy_hla.noALT_noHLA_noDecoy.fasta at htsjdk.samtools.reference.ReferenceSequenceFileWalker.get(ReferenceSequenceFileWalker.java:104) at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:154) at picard.analysis.CollectMultipleMetrics.doWork(CollectMultipleMetrics.java:739) at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:308) at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103) at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113)

Thank you in advance!

jaempawi avatar Sep 15 '22 17:09 jaempawi