I am trying to map MiSeq reads to a reference genome and extract mutations using MToolBox, which implements gsnap, GATK, Picard, and other tools. When running the tool with example data, there were no errors, so I believe the tool is installed correctly. However, when I used the data sequenced with MiSeq for this run, I encountered the following error. Specifically, the OUTPUT file (OUT.sam.bam) from SORTING OUT.sam FILES WITH PICARDTOOLS was not generated. How should I address this issue? Could it be a problem with the sequence data? Could someone please help me? Best regards, >##### EXECUTING READ MAPPING WITH MAPEXOME... >mapExome for sample pc-mtGXL, files found: pc-mtGXL.R1.fastq.gz pc-mtGXL.R2.fastq.gz >Mapping onto mtDNA... /lustre7/home/User/MToolBox-1.2.1/MToolBox/bin/gmap/bin/gsnap -D /lustre7/home/User/MToolBox-1.2.1/MToolBox/gmapdb/ --gunzip -d chrM -A sam --nofails --pairmax-dna=500 --query-unk-mismatch=1 --read-group-id=sample --read-group-name=sample --read-group-library=sample --read-group-platform=sample -n 1 -Q -O -t 8 pc-mtGXL.R1.fastq.gz pc-mtGXL.R2.fastq.gz > /home/User/MTDNA-prospective/pc_GXL/out/OUT_pc-mtGXL/outmt.sam 2> /home/User/MTDNA-prospective/pc_GXL/out/OUT_pc-mtGXL/logmt.txt Extracting FASTQ from SAM... Mapping onto complete human genome...single reads Mapping onto complete human genome...pair reads Reading Results... Filtering reads... Outfile saved on /home/User/MTDNA-prospective/pc_GXL/out/OUT_pc-mtGXL/OUT.sam. Done. >SAM files post-processing... >##### SORTING OUT.sam FILES WITH PICARDTOOLS... >[Thu May 16 12:23:08 JST 2024] net.sf.picard.sam.SortSam INPUT=OUT.sam OUTPUT=OUT.sam.bam SORT_ORDER=coordinate TMP_DIR=[/home/User/MTDNA-prospective/pc_GXL/out/OUT_pc-mtGXL/tmp] VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false [Thu May 16 12:23:08 JST 2024] Executing as User@at138 on Linux 5.15.0-87-generic amd64; OpenJDK 64-Bit Server VM 1.8.0_392-b08; Picard version: 1.98(1547) [Thu May 16 12:23:08 JST 2024] net.sf.picard.sam.SortSam done. Elapsed time: 0.01 minutes. Runtime.totalMemory()=2058354688 To get help, see http://picard.sourceforge.net/index.shtml#GettingHelp Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Read name M02699:57:000000000-LG9CK:1:1119:16584:12259, CIGAR M operator maps off end of reference at net.sf.samtools.SAMUtils.processValidationErrors(SAMUtils.java:448) at net.sf.samtools.SAMRecord.getCigar(SAMRecord.java:606) at net.sf.samtools.SAMRecord.getCigarLength(SAMRecord.java:617) at net.sf.samtools.SAMRecord.isValid(SAMRecord.java:1599) at net.sf.samtools.SAMLineParser.parseLine(SAMLineParser.java:328) at net.sf.samtools.SAMTextReader$RecordIterator.parseLine(SAMTextReader.java:237) at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:225) at net.sf.samtools.SAMTextReader$RecordIterator.next(SAMTextReader.java:201) at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:672) at net.sf.samtools.SAMFileReader$AssertableIterator.next(SAMFileReader.java:650) at net.sf.picard.sam.SortSam.doWork(SortSam.java:68) at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177) at net.sf.picard.cmdline.CommandLineProgram.instanceMainWithExit(CommandLineProgram.java:119) at net.sf.picard.sam.SortSam.main(SortSam.java:57) Success. >samtools index: "OUT.sam.bam" is in a format that cannot be usefully indexed >##### REALIGNING KNOWN INDELS WITH GATK INDELREALIGNER... >Realigning known indels for file OUT_pc-mtGXL/OUT.sam.bam using /home/User/MToolBox-1.2.1/MToolBox/data/MITOMAP_HMTDB_known_indels.chrM as reference... INFO 12:23:11,978 HelpFormatter - ------------------------------------------------------------------------------------ INFO 12:23:11,984 HelpFormatter - The Genome Analysis Toolkit (GATK) v3.8-1-0-gf15c1c3ef, Compiled 2018/02/19 05:43:50 INFO 12:23:11,984 HelpFormatter - Copyright (c) 2010-2016 The Broad Institute INFO 12:23:11,984 HelpFormatter - For support and documentation go to https://software.broadinstitute.org/gatk INFO 12:23:11,984 HelpFormatter - [Thu May 16 12:23:11 JST 2024] Executing on Linux 5.15.0-87-generic amd64 INFO 12:23:11,984 HelpFormatter - OpenJDK 64-Bit Server VM 1.8.0_392-b08 INFO 12:23:11,986 HelpFormatter - Program Args: -U ALLOW_N_CIGAR_READS -T IndelRealigner -R /home/User/MToolBox-1.2.1/MToolBox//data/chrM.fa -I OUT.sam.bam -o OUT.realigned.bam -targetIntervals /home/User/MToolBox-1.2.1/MToolBox//data/intervals_file_chrM.list -known /home/User/MToolBox-1.2.1/MToolBox//data/MITOMAP_HMTDB_known_indels_chrM.vcf -compress 0 INFO 12:23:11,993 HelpFormatter - Executing as User@at138 on Linux 5.15.0-87-generic amd64; OpenJDK 64-Bit Server VM 1.8.0_392-b08. INFO 12:23:11,994 HelpFormatter - Date/Time: 2024/05/16 12:23:11 INFO 12:23:11,994 HelpFormatter - ------------------------------------------------------------------------------------ INFO 12:23:11,994 HelpFormatter - ------------------------------------------------------------------------------------ INFO 12:23:12,139 NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/lustre7/home/User/MToolBox-1.2.1/MToolBox/ext_tools/GenomeAnalysisTK.jar!/com/intel/gkl/native/libgkl_compression.so INFO 12:23:12,711 GenomeAnalysisEngine - Deflater: IntelDeflater INFO 12:23:12,711 GenomeAnalysisEngine - Inflater: IntelInflater INFO 12:23:12,711 GenomeAnalysisEngine - Strictness is SILENT INFO 12:23:12,962 GenomeAnalysisEngine - Downsampling Settings: No downsampling INFO 12:23:12,967 SAMDataSource$SAMReaders - Initializing SAMRecords in serial INFO 12:23:12,985 SAMDataSource$SAMReaders - Done initializing BAM readers: total time 0.02 >##### ERROR ------------------------------------------------------------------------------------------ >##### ERROR A USER ERROR has occurred (version 3.8-1-0-gf15c1c3ef): >##### ERROR >##### ERROR This means that one or more arguments or inputs in your command are incorrect. >##### ERROR The error message below tells you what is the problem. >##### ERROR >##### ERROR If the problem is an invalid argument, please check the online documentation guide >##### ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool. >##### ERROR >##### ERROR Visit our website and forum for extensive documentation and answers to >##### ERROR commonly asked questions >https://software.broadinstitute.org/gatk >##### ERROR >##### ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself. >##### ERROR >##### ERROR MESSAGE: SAM/BAM/CRAM file OUT.sam.bam is malformed. >Please see https://software.broadinstitute.org/gatk/documentation/article?id=1317for more information. Error details: SAM file doesn't have any read groups defined in the header. The GATK no longer supports SAM files without read groups >##### ERROR ------------------------------------------------------------------------------------------ >The last process reported an error. Exit.