Answers.Body: <p>This is essentially a duplicate of <a href="https://bioinformatics.stackexchange.com/questions/13997/samtools-index-chromosome-blocks-not-continuous">Samtools Index: Chromosome Blocks not Continuous</a> but with different error messages, and a duplicate of <a href="https://bioinformatics.stackexchange.com/questions/5352/error-given-while-trying-to-index-a-bam-file-with-samtools-index-no-coor">Error given while trying to index a BAM file with Samtools Index - NO COOR?</a>.</p>
  <pre><code>samtools sort -n ${file} -o ${file}_sorted.bam
  </code></pre>
  <p>These error messages also indicate that the reads are not sorted by coordinate — in particular, that the unplaced unmapped reads with RNAME <code>*</code> are not all together at the end of the file.</p>
  <p>This is because <code>samtools sort -n</code> has been used to sort the reads by name instead. Remove <code>-n</code> to sort by position, which is what is needed to prepare a BAM file for indexing with <code>samtools index</code>.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-08-20T08:12:39.520Z
Answers.Text: Thank you! problem solved :)
Answers.UserDisplayName: user9393
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-08-19T07:29:20.887Z
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Answers.UserDisplayName: John Marshall
Body: <p>I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse  &quot;GCA_000001635.9_GRCm39_genomic.fna&quot;.</p>
  <p>Apparently everything works well up to here. Now I'm using samtools 1.9 to convert sam to bam, to sort those bam and create the index, just like this:</p>
  <pre><code>samtools view -bS <span class="math-container">${file} &gt; $</span>{file}.bam
  
  samtools sort -n <span class="math-container">${file} -o $</span>{file}_sorted.bam
  
  samtools index  ${file}_sorted.bam
  </code></pre>
  <p>But when indexing I get this error:</p>
  <blockquote>
  <p>[bam_sort_core] merging from 11 files and 1 in-memory blocks...
  [E::hts_idx_push] NO_COOR reads not in a single block at the end 0 -1
  samtools index: failed to create index for
  &quot;CD4_1A_concat.bam_sorted.bam&quot;: No such file or directory</p>
  <p>[bam_sort_core] merging from 11 files and 1 in-memory blocks...
  [E::hts_idx_push] NO_COOR reads not in a single block at the end 23 -1
  samtools index: failed to create index for
  &quot;CD4_1B_concat.bam_sorted.bam&quot;: No such file or directory
  [bam_sort_core] merging from 11 files and 1 in-memory blocks...</p>
  </blockquote>
  <p>And more...</p>
  <p>Any help would be appreciated!</p>
  
Tags: <rna-seq><samtools>
Title: NO_COOR reads not in a single block at the end 0 -1
ThreadText: # NO_COOR reads not in a single block at the end 0 -1
  
  (Asked by: username_0 on 2020-08-19)
  
  I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse "GCA\_000001635.9\_GRCm39\_genomic.fna".
  
  Apparently everything works well up to here. Now I'm using samtools 1.9 to convert sam to bam, to sort those bam and create the index, just like this:
  
  ```
  samtools view -bS ${file} > ${file}.bam
  
  samtools sort -n ${file} -o ${file}_sorted.bam
  
  samtools index  ${file}_sorted.bam
  
  ```
  
  But when indexing I get this error:
  
  > [bam\_sort\_core] merging from 11 files and 1 in-memory blocks...
  > [E::hts\_idx\_push] NO\_COOR reads not in a single block at the end 0 -1
  > samtools index: failed to create index for
  > "CD4\_1A\_concat.bam\_sorted.bam": No such file or directory
  >
  > [bam\_sort\_core] merging from 11 files and 1 in-memory blocks...
  > [E::hts\_idx\_push] NO\_COOR reads not in a single block at the end 23 -1
  > samtools index: failed to create index for
  > "CD4\_1B\_concat.bam\_sorted.bam": No such file or directory
  > [bam\_sort\_core] merging from 11 files and 1 in-memory blocks...
  
  And more...
  
  Any help would be appreciated!
  
  **Tags:** rna-seq, samtools
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2020-08-19. Score: 2
  
  This is essentially a duplicate of [Samtools Index: Chromosome Blocks not Continuous](https://bioinformatics.stackexchange.com/questions/13997/samtools-index-chromosome-blocks-not-continuous) but with different error messages, and a duplicate of [Error given while trying to index a BAM file with Samtools Index - NO COOR?](https://bioinformatics.stackexchange.com/questions/5352/error-given-while-trying-to-index-a-bam-file-with-samtools-index-no-coor).
  
  ```
  samtools sort -n ${file} -o ${file}_sorted.bam
  
  ```
  
  These error messages also indicate that the reads are not sorted by coordinate — in particular, that the unplaced unmapped reads with RNAME `*` are not all together at the end of the file.
  
  This is because `samtools sort -n` has been used to sort the reads by name instead. Remove `-n` to sort by position, which is what is needed to prepare a BAM file for indexing with `samtools index`.
  
  > **Comments:**
  > **username_0**: Thank you! problem solved :)  
  
  ---
  
  