Answers.Body: <pre><code>samtools sort blasted_SRR6649368.bam -o sorted_SRR6649368.bam -n
  </code></pre>
  <p>These error messages indicate that the reads are not sorted by coordinate — in particular, that the reads mapped to <em>ScdB1pO_646;HRSCAF=880</em> are not all together, and that the reads at positions 11159020 &lt; 11158717 are not sorted by position on their chromosome.</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: 2020-07-27T16:17:27.050Z
Answers.Text: Thank you, it worked! As for why I used the -n flag, I am trying replicate the results of a journal article. The journal article did not provide explicit instructions for sorting/indexing the files, and this is my first time sorting/indexing files. I think "sort by reads" simply _sounded_ like the correct thing to do, but it looks like I was incorrect
Answers.UserDisplayName: annabelperry
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-07-25T10:06:24.230Z
Answers.LastActivityDate: 2020-07-25T10:06:24.230Z
Answers.UserDisplayName: John Marshall
Body: <p>I am working with short-read whole-genome sequences from the NCBI's SRA. I have aligned and sorted all of my short-read sequences and am attempting to index each sequence into .bai format using samtools index, but am running into a couple of errors.</p>
  <p>I unpacked the original .sra files in the following manner:</p>
  <pre><code>fastq-dump --defline-seq '@<span class="math-container">$sn[_$</span>rn]/$ri' --split-files SRR6509138.sra
  </code></pre>
  <p>I then aligned each fastq paired-end read, removed duplicate reads, and converted each to bam format like so:</p>
  <pre><code>bwa mem xiphophorus_birchmanni_10x_12Sep2018_yDAA6.fasta SRR6509136_1.fastq SRR6509136_2.fastq | samblaster -e -r| samtools view -Sb - &gt; blasted_SRR6509136.bam
  </code></pre>
  <p>Some of my files did not come as paired-end reads. For those files, I used the --ignoreUnmated flag on samblaster.</p>
  <p>I then sorted each bam file like so:</p>
  <pre><code>samtools sort blasted_SRR6649368.bam -o sorted_SRR6649368.bam -n
  </code></pre>
  <p>I am attempting to obtain a bai file for each bam using the following command:</p>
  <pre><code>samtools index sorted_SRR6649368.bam sorted_SRR6649368.bam.bai
  </code></pre>
  <p>This is the error I run into for the unpaired reads:</p>
  <pre><code>[E::hts_idx_push] Chromosome blocks not continuous
  [E::sam_index] Read 'HWI-ST387:164:D0CJWACXX:4:1101:1129:13264' with ref_name='ScdB1pO_646;HRSCAF=880', ref_length=33092979, flags=16, pos=23260108 cannot be indexed
  samtools index: failed to create index for &quot;sorted_SRR791885.bam&quot;: No such file or directory
  </code></pre>
  <p>This is the error I run into for the paired reads:</p>
  <pre><code>[E::hts_idx_push] Unsorted positions on sequence #79: 11159020 followed by 11158717
  samtools index: failed to create index for &quot;sorted_SRR6649368.bam&quot;
  </code></pre>
  <p>Can anyone help me figure out why this is happening?</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-07-25T10:07:55.987Z
Comments.Text: Are there some instructions you are following that tell you to use these commands? Where has the idea that you need to use `-n` on the `samtools sort` command here come from?
Comments.UserDisplayName: John Marshall
Tags: <genome><samtools><bwa><indexing>
Title: Samtools Index: Chromosome Blocks not Continuous
ThreadText: # Samtools Index: Chromosome Blocks not Continuous
  
  (Asked by: username_0 on 2020-07-25)
  
  I am working with short-read whole-genome sequences from the NCBI's SRA. I have aligned and sorted all of my short-read sequences and am attempting to index each sequence into .bai format using samtools index, but am running into a couple of errors.
  
  I unpacked the original .sra files in the following manner:
  
  ```
  fastq-dump --defline-seq '@$sn[_$rn]/$ri' --split-files SRR6509138.sra
  
  ```
  
  I then aligned each fastq paired-end read, removed duplicate reads, and converted each to bam format like so:
  
  ```
  bwa mem xiphophorus_birchmanni_10x_12Sep2018_yDAA6.fasta SRR6509136_1.fastq SRR6509136_2.fastq | samblaster -e -r| samtools view -Sb - > blasted_SRR6509136.bam
  
  ```
  
  Some of my files did not come as paired-end reads. For those files, I used the --ignoreUnmated flag on samblaster.
  
  I then sorted each bam file like so:
  
  ```
  samtools sort blasted_SRR6649368.bam -o sorted_SRR6649368.bam -n
  
  ```
  
  I am attempting to obtain a bai file for each bam using the following command:
  
  ```
  samtools index sorted_SRR6649368.bam sorted_SRR6649368.bam.bai
  
  ```
  
  This is the error I run into for the unpaired reads:
  
  ```
  [E::hts_idx_push] Chromosome blocks not continuous
  [E::sam_index] Read 'HWI-ST387:164:D0CJWACXX:4:1101:1129:13264' with ref_name='ScdB1pO_646;HRSCAF=880', ref_length=33092979, flags=16, pos=23260108 cannot be indexed
  samtools index: failed to create index for "sorted_SRR791885.bam": No such file or directory
  
  ```
  
  This is the error I run into for the paired reads:
  
  ```
  [E::hts_idx_push] Unsorted positions on sequence #79: 11159020 followed by 11158717
  samtools index: failed to create index for "sorted_SRR6649368.bam"
  
  ```
  
  Can anyone help me figure out why this is happening?
  
  **Tags:** genome, samtools, bwa, indexing
  
  > **Comments:**
  > **username_1**: Are there some instructions you are following that tell you to use these commands? Where has the idea that you need to use `-n` on the `samtools sort` command here come from?  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2020-07-25. Score: 5 | Accepted answer
  
  ```
  samtools sort blasted_SRR6649368.bam -o sorted_SRR6649368.bam -n
  
  ```
  
  These error messages indicate that the reads are not sorted by coordinate — in particular, that the reads mapped to *ScdB1pO\_646;HRSCAF=880* are not all together, and that the reads at positions 11159020 < 11158717 are not sorted by position on their chromosome.
  
  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, it worked! As for why I used the -n flag, I am trying replicate the results of a journal article. The journal article did not provide explicit instructions for sorting/indexing the files, and this is my first time sorting/indexing files. I think "sort by reads" simply _sounded_ like the correct thing to do, but it looks like I was incorrect  
  
  ---
  
  