====Picard 报错==== 1、使用MarkDuplicates命令时。 Exception in thread "main" htsjdk.samtools.SAMException: /TJPROJ6/AFS_RESEQ/Proj/liangjifeng/5.mRNA/X101SC23092911-Z01-J005.20240407.RNAmapping_unmapNR/mapping/101/tmp/CSPI.4553797415123961586.tmp/12125.tmpnot found at htsjdk.samtools.util.FileAppendStreamLRUCache$Functor.makeValue(FileAppendStreamLRUCache.java:64) at htsjdk.samtools.util.FileAppendStreamLRUCache$Functor.makeValue(FileAppendStreamLRUCache.java:49) at htsjdk.samtools.util.ResourceLimitedMap.get(ResourceLimitedMap.java:76) at htsjdk.samtools.CoordinateSortedPairInfoMap.getOutputStreamForSequence(CoordinateSortedPairInfoMap.java:180) at htsjdk.samtools.CoordinateSortedPairInfoMap.put(CoordinateSortedPairInfoMap.java:164) at picard.sam.markduplicates.util.DiskBasedReadEndsForMarkDuplicatesMap.put(DiskBasedReadEndsForMarkDuplicatesMap.java:65) at picard.sam.markduplicates.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:566) at picard.sam.markduplicates.MarkDuplicates.doWork(MarkDuplicates.java:257) at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:301) at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:103) at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:113) Caused by: java.io.FileNotFoundException: /TJPROJ6/AFS_RESEQ/Proj/liangjifeng/5.mRNA/X101SC23092911-Z01-J005.20240407.RNAmapping_unmapNR/mapping/101/tmp/CSPI.4553797415123961586.tmp/12125.tmp (Too many open files) at java.io.FileOutputStream.open(Native Method) at java.io.FileOutputStream.(FileOutputStream.java:213) at htsjdk.samtools.util.FileAppendStreamLRUCache$Functor.makeValue(FileAppendStreamLRUCache.java:61) ... 10 more 由于打开文件个数超出系统限制,解决办法增加参数即可: MAX_RECORDS_IN_RAM=1000000 MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=1000