text: user2594: I am running Cufflinks for transcriptome assembly using the .bam file generated by Hisat2. I tried both bam and sorted bam files
  cufflinks --no-update-check -o ./ -p 15 accepted_hits.bam
  
  it gives the following error:
  Error: sort order of reads in BAMs must be the same
  
  UPDATE: It is working fine now. It seems there were some issues with the reference file. The BAM files generated from Tophat2 also gave the same error. I downloaded the updated reference file and it worked great.
  
  user294: Just add --dta-cufflinks to your Hisat2 command, so that the output alignment file provides the attributes needed by Cufflinks (XS flags). This should do the job.  
  From manual:
  
  Report alignments tailored specifically for Cufflinks. In addition to
    what HISAT2 does with the above option (--dta), With this option,
    HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG,
    AT/AC), but all user-provided splice sites are used irrespective of
    their signals. HISAT2 produces an optional field, XS:A:[+-], for every
    spliced alignment.
  
  
original_text: 2594: <p>I am running <a href="http://cole-trapnell-lab.github.io/cufflinks/" rel="nofollow noreferrer">Cufflinks</a> for transcriptome assembly using the .bam file generated by <a href="https://ccb.jhu.edu/software/hisat2/index.shtml" rel="nofollow noreferrer">Hisat2</a>. I tried both bam and sorted bam files</p>
  
  <pre><code>cufflinks --no-update-check -o ./ -p 15 accepted_hits.bam
  </code></pre>
  
  <p>it gives the following error:</p>
  
  <pre><code>Error: sort order of reads in BAMs must be the same
  </code></pre>
  
  <p>UPDATE: It is working fine now. It seems there were some issues with the reference file. The BAM files generated from <a href="https://ccb.jhu.edu/software/tophat/index.shtml" rel="nofollow noreferrer">Tophat2</a> also gave the same error. I downloaded the updated reference file and it worked great.</p>
   294: <p>Just add <code>--dta-cufflinks</code> to your Hisat2 command, so that the output alignment file provides the attributes needed by Cufflinks (XS flags). This should do the job.  </p>
  
  <p>From <a href="https://ccb.jhu.edu/software/hisat2/manual.shtml" rel="noreferrer">manual</a>:</p>
  
  <blockquote>
    <p>Report alignments tailored specifically for Cufflinks. In addition to
    what HISAT2 does with the above option (--dta), With this option,
    HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG,
    AT/AC), but all user-provided splice sites are used irrespective of
    their signals. HISAT2 produces an optional field, XS:A:[+-], for every
    spliced alignment.</p>
  </blockquote>
  