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.<init>(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