售后

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

2.Picard 内存不够

Exception in thread "main" java.lang.OutOfMemoryError: Java heap space
      at htsjdk.samtools.util.BlockCompressedInputStream.inflateBlock(BlockCompressedInputStream.java:548)
      at htsjdk.samtools.util.BlockCompressedInputStream.processNextBlock(BlockCompressedInputStream.java:532)
      at htsjdk.samtools.util.BlockCompressedInputStream.nextBlock(BlockCompressedInputStream.java:468)
      at htsjdk.samtools.util.BlockCompressedInputStream.readBlock(BlockCompressedInputStream.java:458)
      at htsjdk.samtools.util.BlockCompressedInputStream.available(BlockCompressedInputStream.java:196)
      at htsjdk.samtools.util.BlockCompressedInputStream.read(BlockCompressedInputStream.java:331)
      at java.base/java.io.DataInputStream.read(DataInputStream.java:149)
      at htsjdk.samtools.util.BinaryCodec.readBytesOrFewer(BinaryCodec.java:421)
      at htsjdk.samtools.util.BinaryCodec.readBytes(BinaryCodec.java:394)
      at htsjdk.samtools.util.BinaryCodec.readByteBuffer(BinaryCodec.java:507)
      at htsjdk.samtools.util.BinaryCodec.readInt(BinaryCodec.java:518)
      at htsjdk.samtools.BAMRecordCodec.decode(BAMRecordCodec.java:261)
      at htsjdk.samtools.BAMFileReader$BAMFileIterator.getNextRecord(BAMFileReader.java:866)
      at htsjdk.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:840)
      at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:834)
      at htsjdk.samtools.BAMFileReader$BAMFileIterator.next(BAMFileReader.java:802)
      at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:574)
      at htsjdk.samtools.SamReader$AssertingIterator.next(SamReader.java:553)
      at picard.sam.markduplicates.MarkDuplicates.buildSortedReadEndLists(MarkDuplicates.java:524)
      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)
      

由于内存不够引起，可以增加参数

picard -Xmx20G -XX:ParallelGCThreads=4 CollectRnaSeqMetrics