Answers.Body: <p>STAR versions 2.6.0b and 2.6.0a are unstable,  as written <a href="https://bioinformatics.stackexchange.com/questions/4449/issues-performing-variant-calling-with-gatk">Issues performing variant calling with GATK</a> you are using version 2.6.0b. You should switch to version 2.6.0c which is stable.  Also avoid to perform multiple alignments in the same directory. </p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-06-10T13:07:15.620Z
Answers.LastActivityDate: 2018-06-10T13:07:15.620Z
Answers.UserDisplayName: bilal malik
Body: <p>Hi am trying to align RNA-seq reads using STAR through following commands,</p>
  
  <pre><code>STAR --runMode alignReads --runThreadN 26  --outSAMtype BAM  SortedByCoordinate Unsorted --sjdbOverhang 100  --genomeDir wheat --readFilesIn G3_cleaned_R1.fastq G3_cleaned_R2.fastq --sjdbGTFtagExonParentTranscript wheat.gff3 
  Jun 05 13:53:59 ..... started STAR run
  Jun 05 13:53:59 ..... loading genome
  Jun 05 14:07:12 ..... started mapping
  Jun 05 18:42:47 ..... started sorting BAM
  
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin   does not agree with the actual size on disk: 509186583   0   45                                                                                        
  
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 648593301   0   47                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 660412146   0   46                                                                                        
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 470931698   0   44                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 653155717   0   43                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  Segmentation fault (core dumped)
  </code></pre>
  
  <p>Although there is much free space on my system as shown below:</p>
  
  <pre><code>&gt; df -Th
    Filesystem     Type      Size  Used Avail Use% Mounted on
    devtmpfs       devtmpfs   63G     0   63G   0% /dev
    tmpfs          tmpfs      63G  136K   63G   1% /dev/shm
    tmpfs          tmpfs      63G  1.9M   63G   1% /run
    tmpfs          tmpfs      63G     0   63G   0% /sys/fs/cgroup
    /dev/sda3      ext4      3.6T  1.4T  2.2T  39% /
    /dev/sda2      ext4      9.8G   57M  9.2G   1% /boot
    tmpfs          tmpfs      13G     0   13G   0% /run/user/480
    tmpfs          tmpfs      13G   16K   13G   1% /run/user/1000
  </code></pre>
  
  <p>How this problem can be solved?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T08:07:07.177Z
Comments.Text: Are you an older version of the STAR aligner? A similar error has been discussed here: https://groups.google.com/forum/#!topic/rna-star/OTUG26gKKmo
Comments.UserDisplayName: Kasper Thystrup Karstensen
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T08:15:29.050Z
Comments.Text: You are using 26 threads, while you have 125GB of RAM, this means per thread you have 4.8 GB RAM. Your genome size is 13 GB (according to your previous [post](https://bioinformatics.stackexchange.com/questions/4340/error-creating-indices-using-star)). An answer to your previous post suggested to reduce the number of threads. Did you try to run with lower number of threads?
Comments.UserDisplayName: benn
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T08:30:46.223Z
Comments.Text: @b.nota I ran the job discussed in previous post with same number of threads.
Comments.UserDisplayName: Ammar Sabir Cheema
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T08:33:01.977Z
Comments.Text: @Kasper I am using latest version of STAR
Comments.UserDisplayName: Ammar Sabir Cheema
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T09:04:33.213Z
Comments.Text: Report the bug on github and sort with samtools.
Comments.UserDisplayName: Devon Ryan
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T18:54:10.667Z
Comments.Text: @DevonRyan ok I will post it there. Can I use --limitBAMsortRAM to solve this problem?
Comments.UserDisplayName: Ammar Sabir Cheema
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-06-06T21:16:36.303Z
Comments.Text: @AmmarSabirCheema I don't think so, since any time you need to increase that there's an error message saying so. I think this is just a bug somewhere. Just sort the unsorted output with samtools or sambamba.
Comments.UserDisplayName: Devon Ryan
Tags: <sam><rna-alignment><star>
Title: Disk space error while aligning reads using STAR
ThreadText: # Disk space error while aligning reads using STAR
  
  (Asked by: username_0 on 2018-06-06)
  
  Hi am trying to align RNA-seq reads using STAR through following commands,
  
  ```
  STAR --runMode alignReads --runThreadN 26  --outSAMtype BAM  SortedByCoordinate Unsorted --sjdbOverhang 100  --genomeDir wheat --readFilesIn G3_cleaned_R1.fastq G3_cleaned_R2.fastq --sjdbGTFtagExonParentTranscript wheat.gff3 
  Jun 05 13:53:59 ..... started STAR run
  Jun 05 13:53:59 ..... loading genome
  Jun 05 14:07:12 ..... started mapping
  Jun 05 18:42:47 ..... started sorting BAM
  
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin   does not agree with the actual size on disk: 509186583   0   45                                                                                        
  
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 648593301   0   47                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 660412146   0   46                                                                                        
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 470931698   0   44                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  EXITING because of FATAL ERROR: number of bytes expected from the BAM bin does not agree with the actual size on disk: 653155717   0   43                                                                                        
  
  Jun 05 18:42:47 ...... FATAL ERROR, exiting                                                                                                                                                                                      
  Segmentation fault (core dumped)
  
  ```
  
  Although there is much free space on my system as shown below:
  
  ```
  > df -Th
    Filesystem     Type      Size  Used Avail Use% Mounted on
    devtmpfs       devtmpfs   63G     0   63G   0% /dev
    tmpfs          tmpfs      63G  136K   63G   1% /dev/shm
    tmpfs          tmpfs      63G  1.9M   63G   1% /run
    tmpfs          tmpfs      63G     0   63G   0% /sys/fs/cgroup
    /dev/sda3      ext4      3.6T  1.4T  2.2T  39% /
    /dev/sda2      ext4      9.8G   57M  9.2G   1% /boot
    tmpfs          tmpfs      13G     0   13G   0% /run/user/480
    tmpfs          tmpfs      13G   16K   13G   1% /run/user/1000
  
  ```
  
  How this problem can be solved?
  
  **Tags:** sam, rna-alignment, star
  
  > **Comments:**
  > **username_1**: Are you an older version of the STAR aligner? A similar error has been discussed here: https://groups.google.com/forum/#!topic/rna-star/OTUG26gKKmo  
  > **username_2**: You are using 26 threads, while you have 125GB of RAM, this means per thread you have 4.8 GB RAM. Your genome size is 13 GB (according to your previous [post](https://bioinformatics.stackexchange.com/questions/4340/error-creating-indices-using-star)). An answer to your previous post suggested to reduce the number of threads. Did you try to run with lower number of threads?  
  > **username_0**: @b.nota I ran the job discussed in previous post with same number of threads.  
  > **username_0**: @username_1 I am using latest version of STAR  
  > **username_3**: Report the bug on github and sort with samtools.  
  > **username_0**: @username_3 ok I will post it there. Can I use --limitBAMsortRAM to solve this problem?  
  > **username_3**: @username_0 I don't think so, since any time you need to increase that there's an error message saying so. I think this is just a bug somewhere. Just sort the unsorted output with samtools or sambamba.  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_4 on 2018-06-10. Score: 4 | Accepted answer
  
  STAR versions 2.6.0b and 2.6.0a are unstable, as written [Issues performing variant calling with GATK](https://bioinformatics.stackexchange.com/questions/4449/issues-performing-variant-calling-with-gatk) you are using version 2.6.0b. You should switch to version 2.6.0c which is stable. Also avoid to perform multiple alignments in the same directory.
  
  
  ---
  
  