Answers.Body: <p>option <code>-pc</code> is for</p>
  <pre><code>-pc     Calculate coverage of pair-end fragments.
  </code></pre>
  <p>but your reads are single-end reads</p>
  
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Answers.CreationDate: 2024-02-19T14:58:28.190Z
Answers.Text: What tells you they're single ends? Is it because you know this would happen with `-pc` and single end files or was it something in the bam?
Answers.UserDisplayName: terdon
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Answers.Text: "What tells you they're single ends? Is it b" : the sam flags of each alignemt is zero. Paired would have the '1' bit set . see https://broadinstitute.github.io/picard/explain-flags.html
Answers.UserDisplayName: Pierre
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Answers.CreationDate: 2024-02-19T15:44:42.227Z
Answers.Text: Using that page, I can see that 11S139M, the first one, is "read paired", "read mapped in proper pair" and "mate unmapped". Could you explain a bit more please? Ideally, adding the detail in the answer so the next user who is confused can also find the info?
Answers.UserDisplayName: terdon
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Answers.CreationDate: 2024-02-19T17:49:47.013Z
Answers.Text: the second column is the read flag (section 1.4 of https://samtools.github.io/hts-specs/SAMv1.pdf) . it's always 0  in your example.  0 is single end read mapped on plus strand.
Answers.UserDisplayName: Pierre
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Answers.CreationDate: 2024-02-20T11:24:35.143Z
Answers.Text: Ah thanks, yes, I was looking at the cigar string, duh. It would be great if you could add some more detail to the answer to make all this clear. Especially since I don't see mention of 0 meaning "single end" either in the specs (but perhaps I missed it) or when using the explain-flags page (which just ignores 0).
Answers.UserDisplayName: terdon
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Answers.Text: "I don't see mention of 0 meaning "single end" either in the specs (but perhaps I missed it)" it's a array of bits https://en.wikipedia.org/wiki/Bit_array . The first bit is for "Read Paired". As it's not set, the read is not part of a "Read Paired", hence it's single end.
Answers.UserDisplayName: Pierre
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Answers.Text: @Pierre Some of my bam files have a mix of 0 and 16 for my read flags. So to my understanding I have a mix of single-end and paired-end reverse strand reads. Do I include or omit `-pc`?
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Body: <p>Also posted <a href="https://www.biostars.org/p/9587718/" rel="nofollow noreferrer">on biostars</a></p>
  <p>I have bam files that I've obtained through STAR alignment. Here is the first 10 lines of one of my bam files:</p>
  <pre><code>GWNJ-1013:671:GW2304233104th:2:1126:15501:17879 0       chromosome      1       255     11S139M *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:137        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1126:15130:18427 0       chromosome      1       255     11S139M *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:137        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1206:9272:29857  0       chromosome      1       255     28S108M14S      *       0       0       GGCGGTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGAGATCGGAAGAGCG  FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:106        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1266:30734:10692 0       chromosome      1       255     24S126M *       0       0       GTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF  NH:i:1  HI:i:1  AS:i:124        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1266:31412:33411 0       chromosome      1       255     24S126M *       0       0       GTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTC  FFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF::FFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFF:FFFF,FFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FFF::FFFFFFFF,FFFFFFFF:FFFFFFFFFFF:F,FFFFF  NH:i:1  HI:i:1  AS:i:124        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1330:21938:10708 0       chromosome      1       255     16S134M *       0       0       TGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACA  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFF:F  NH:i:1  HI:i:1  AS:i:132        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1367:17074:4726  0       chromosome      1       255     11S82M  *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGG   FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1   HI:i:1  AS:i:80 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1367:11939:11584 0       chromosome      1       255     11S82M  *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGG   FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:F:FFFFFFFFFFFFFFFFFFF  NH:i:1   HI:i:1  AS:i:80 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1372:24740:30843 0       chromosome      1       255     16S48M  *       0       0       TGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAA        FFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF        NH:i:1  HI:i:1  AS:i:47 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1401:11107:19319 0       chromosome      1       255     5S144M1S        *       0       0       ATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGGAATTGA  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF  NH:i:1  HI:i:1  AS:i:142        nM:i:0
  </code></pre>
  <p>This is the script I'm using that contains the <code>genomecov -d -pc -ibam</code> command:</p>
  <pre><code>BAM_DIR='./sorted_bam_files'
  
  # Loop over each BAM file in the directory
  for file in &quot;<span class="math-container">$BAM_DIR"/*_high_mapq_reads.sorted.bam
  do
      echo "Calculating coverage for $</span>file...&quot;
      # Get the basename of the file
      base=<span class="math-container">$(basename "$</span>file&quot;)
      # Calculate coverage
      bedtools genomecov -d -pc -ibam <span class="math-container">$file &gt; "$</span>{base%.sorted.bam}.coverage&quot;
  done
  </code></pre>
  <p>This is the output I keep getting (first 10 lines only shown). I've tried this with at least 30 other bam files but the output is always the same.</p>
  <pre><code>chromosome      1       0
  chromosome      2       0
  chromosome      3       0
  chromosome      4       0
  chromosome      5       0
  chromosome      6       0
  chromosome      7       0
  chromosome      8       0
  chromosome      9       0
  chromosome      10      0
  </code></pre>
  <p>Any ideas on why this keeps happening? I don't think there's something wrong with my bam files because I was able to use them for DGE analyses and also checked to see if most reads were getting unmapped but they weren't. Any help is appreciated.</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-02-15T21:47:25.430Z
Comments.Text: Why is your first chromosome called "chromosome", that's odd.
Comments.UserDisplayName: ATpoint
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-02-16T14:48:40.400Z
Comments.Text: @ATpoint also second column `FLAG == 0` and the fifth `MAPQ ==255` meaning: "A value 255 indicates that the mapping quality is not available.". Then the CIGARs looking like the read was indeed mapped `11S82M` etc.
Comments.UserDisplayName: darked89
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2024-02-19T15:53:56.530Z
Comments.Text: @ATpoint The data I have is from bacteria, so I'm only working with 1 chromosome
Comments.UserDisplayName: gcabebe
Tags: <rna-seq><bedtools>
Title: Bedtools genomecov gives me coverage files showing zeros at all nucleotide positions
ThreadText: # Bedtools genomecov gives me coverage files showing zeros at all nucleotide positions
  
  (Asked by: username_0 on 2024-02-15)
  
  Also posted [on biostars](https://www.biostars.org/p/9587718/)
  
  I have bam files that I've obtained through STAR alignment. Here is the first 10 lines of one of my bam files:
  
  ```
  GWNJ-1013:671:GW2304233104th:2:1126:15501:17879 0       chromosome      1       255     11S139M *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:137        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1126:15130:18427 0       chromosome      1       255     11S139M *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGG  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:137        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1206:9272:29857  0       chromosome      1       255     28S108M14S      *       0       0       GGCGGTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGAGATCGGAAGAGCG  FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1  HI:i:1  AS:i:106        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1266:30734:10692 0       chromosome      1       255     24S126M *       0       0       GTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTC  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF  NH:i:1  HI:i:1  AS:i:124        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1266:31412:33411 0       chromosome      1       255     24S126M *       0       0       GTTTGGCGTGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTC  FFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF::FFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFF:FFFF,FFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FFF::FFFFFFFF,FFFFFFFF:FFFFFFFFFFF:F,FFFFF  NH:i:1  HI:i:1  AS:i:124        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1330:21938:10708 0       chromosome      1       255     16S134M *       0       0       TGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACA  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFF:F  NH:i:1  HI:i:1  AS:i:132        nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1367:17074:4726  0       chromosome      1       255     11S82M  *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGG   FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF  NH:i:1   HI:i:1  AS:i:80 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1367:11939:11584 0       chromosome      1       255     11S82M  *       0       0       ATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGG   FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:F:FFFFFFFFFFFFFFFFFFF  NH:i:1   HI:i:1  AS:i:80 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1372:24740:30843 0       chromosome      1       255     16S48M  *       0       0       TGCGTATTTCCATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAA        FFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF        NH:i:1  HI:i:1  AS:i:47 nM:i:0
  GWNJ-1013:671:GW2304233104th:2:1401:11107:19319 0       chromosome      1       255     5S144M1S        *       0       0       ATCACAACTGCTCCTCGGAAGTCGACCAACAAGTCAGCTATGACTTGGCATAATTTGTGCCGACAAAATGCGCGCAGAGTATAGGGGTGGATTAACCCCTATTCAACTCTTCGGTAGTGATTTCCGACTTCACGCTACAACAGGAATTGA  FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF  NH:i:1  HI:i:1  AS:i:142        nM:i:0
  
  ```
  
  This is the script I'm using that contains the `genomecov -d -pc -ibam` command:
  
  ```
  BAM_DIR='./sorted_bam_files'
  
  # Loop over each BAM file in the directory
  for file in "$BAM_DIR"/*_high_mapq_reads.sorted.bam
  do
      echo "Calculating coverage for $file..."
      # Get the basename of the file
      base=$(basename "$file")
      # Calculate coverage
      bedtools genomecov -d -pc -ibam $file > "${base%.sorted.bam}.coverage"
  done
  
  ```
  
  This is the output I keep getting (first 10 lines only shown). I've tried this with at least 30 other bam files but the output is always the same.
  
  ```
  chromosome      1       0
  chromosome      2       0
  chromosome      3       0
  chromosome      4       0
  chromosome      5       0
  chromosome      6       0
  chromosome      7       0
  chromosome      8       0
  chromosome      9       0
  chromosome      10      0
  
  ```
  
  Any ideas on why this keeps happening? I don't think there's something wrong with my bam files because I was able to use them for DGE analyses and also checked to see if most reads were getting unmapped but they weren't. Any help is appreciated.
  
  **Tags:** rna-seq, bedtools
  
  > **Comments:**
  > **username_1**: Why is your first chromosome called "chromosome", that's odd.  
  > **username_2**: @username_1 also second column `FLAG == 0` and the fifth `MAPQ ==255` meaning: "A value 255 indicates that the mapping quality is not available.". Then the CIGARs looking like the read was indeed mapped `11S82M` etc.  
  > **username_0**: @username_1 The data I have is from bacteria, so I'm only working with 1 chromosome  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_3 on 2024-02-16. Score: 3 | Accepted answer
  
  option `-pc` is for
  
  ```
  -pc     Calculate coverage of pair-end fragments.
  
  ```
  
  but your reads are single-end reads
  
  > **Comments:**
  > **username_4**: What tells you they're single ends? Is it because you know this would happen with `-pc` and single end files or was it something in the bam?  
  > **username_3**: "What tells you they're single ends? Is it b" : the sam flags of each alignemt is zero. Paired would have the '1' bit set . see https://broadinstitute.github.io/picard/explain-flags.html  
  > **username_4**: Using that page, I can see that 11S139M, the first one, is "read paired", "read mapped in proper pair" and "mate unmapped". Could you explain a bit more please? Ideally, adding the detail in the answer so the next user who is confused can also find the info?  
  > **username_3**: the second column is the read flag (section 1.4 of https://samtools.github.io/hts-specs/SAMv1.pdf) . it's always 0  in your example.  0 is single end read mapped on plus strand.  
  > **username_4**: Ah thanks, yes, I was looking at the cigar string, duh. It would be great if you could add some more detail to the answer to make all this clear. Especially since I don't see mention of 0 meaning "single end" either in the specs (but perhaps I missed it) or when using the explain-flags page (which just ignores 0).  
  > **username_3**: "I don't see mention of 0 meaning "single end" either in the specs (but perhaps I missed it)" it's a array of bits https://en.wikipedia.org/wiki/Bit_array . The first bit is for "Read Paired". As it's not set, the read is not part of a "Read Paired", hence it's single end.  
  > **username_0**: @username_3 Some of my bam files have a mix of 0 and 16 for my read flags. So to my understanding I have a mix of single-end and paired-end reverse strand reads. Do I include or omit `-pc`?  
  
  ---
  
  