Answers.Body: <p>The ENCODE Data Coordination Center, that developed and maintains the ENCODE portal, have implemented scripts validating adherence to the standards the ENCODE Consortium agreed upon when evaluating ChIP-seq analysis results. For any ChIP-seq experiment, except for H3K9me3, the adherence to the read depth standard is assessed looking on the number of &quot;usable fragemnts&quot; (<a href="https://www.encodeproject.org/data-standards/terms/#read-depth" rel="nofollow noreferrer">https://www.encodeproject.org/data-standards/terms/#read-depth</a>) (see answer by James Hawley). For H3K9me3 the read depth is assessed not by looking on usable fragments, but by looking on the total number of mapped reads (if it is a PE sequencing run, the number of mapped read pairs). It is an exception, because the number of mapped reads, especially for H3K9me3 is substantially higher than the number of usable fragments (where the duplicated and low mapping quality reads are filtered out). The decision to apply different read depth standard in case of H3K9me3 came up due to the logic explained here (<a href="https://www.encodeproject.org/chip-seq/histone/" rel="nofollow noreferrer">https://www.encodeproject.org/chip-seq/histone/</a>), briefly: because the H3K9me3 mark is enriched in repetitive regions of the genome, the number of usable fragments (post-filtering) is not expected to be as high as other marks (45M), despite high-quality libraries and sufficient sequencing depth. Instead of marking these experiments as failing read depth standard, the script checks the total number of mapped reads (or read pairs for PE sequencing runs) and makes sure it is above the read depth threshold (45M).</p>
  
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Answers.Text: I think that "usable fragments" should also be uniquely mapped. Although the definition in Encode sates only "Fragments are considered usable if they pass the various filters in the ChIP-seq uniform processing pipelines", when I go to the filtering script at Encode's github https://github.com/ENCODE-DCC/chip-seq-pipeline/tree/master/dnanexus/filter_qc  I see "Remove unmapped reads, non-primary alignments, reads failing platform, duplicates, **multi-mapping reads**, and PCR duplicates."
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Answers.UserDisplayName: Idan Gabdank
Answers.Body: <p>In the initial description for &quot;usable fragments&quot;, ENCODE has the following (emphasis mine):</p>
  <blockquote>
  <p>Usable fragment – A fragment is defined as the sequencing output <strong>corresponding to one location in the genome</strong>. If single-ended sequencing is performed, one read is considered a fragment. If paired-ended sequencing is performed, one pair of reads is considered a fragment. <strong>Fragments are considered usable if they pass the various filters in the ChIP-seq uniform processing pipelines</strong>. Used to evaluate ChIP-seq data.</p>
  </blockquote>
  <p>Aligning reads to repetitive regions is difficult because they don't give unique alignments.
  Reads from these regions will often have poor alignment scores and may not pass alignment QC checks.
  Reads may also be counted as multi-mapped, and often methods call for uniquely mapped reads, which again, may not be true for a large number of reads from H3K9me3 ChIP-seq.</p>
  <p>So for this particular histone mark, ENCODE loosens the definition for what you should count towards a minimum sequencing depth threshold.</p>
  
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Answers.CreationDate: 2020-10-14T12:05:51.117Z
Answers.Text: Why is the more difficult alignment a reason to loosen the definition? It will result in lower-quality analysis for H3K9ME3.
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Answers.Text: Btw, if you have a reference for your explanation, or can tell me how can I find one, it'd be great.
Answers.UserDisplayName: Sam
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Answers.Text: You're right, but the tradeoff seems to be between not having any usable information from H3K9me3 experiments and having a good amount of slightly lower-quality information, compared to other histone modifications.
  
  I don't have a specific reference for that point, aside from the ENCODE documentation itself, but ENCODE has a list of [all its related publications](https://www.encodeproject.org/search/?searchTerm=chip-seq&type=Publication) that you could search to find one.
Answers.UserDisplayName: James Hawley
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Answers.Text: So to be 100% sure, a "Usable fragment" is a unique alignment, right? "Corresponding to one location in the genome" means corresponding to **only** one location in the genome?
Answers.UserDisplayName: Sam
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Answers.Text: Yes. [As per ENCODE](https://www.encodeproject.org/data-standards/terms/#read-depth): "Usable reads – A fragment is considered “usable” if it uniquely maps to the genome and remains after removing PCR duplicates (defined as two fragments that map to the same genomic position and have the same unique molecular identifier)."
Answers.UserDisplayName: James Hawley
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Answers.UserDisplayName: James Hawley
Body: <p>According to <a href="https://www.encodeproject.org/chip-seq/histone/" rel="nofollow noreferrer">ENCODE recommendations</a></p>
  <blockquote>
  <p>For broad-peak histone experiments, each replicate should have 45 million usable fragments</p>
  </blockquote>
  <p>According to my understanding, a usable fragment means a uniquely mapped read / pair of reads.</p>
  <p>However</p>
  <blockquote>
  <p>H3K9me3 is an exception as it is enriched in repetitive regions of the genome. ... Tissues and primary cells should have 45 million total mapped reads per replicate.</p>
  </blockquote>
  <p>I fail to grasp Encode's language.</p>
  <ul>
  <li>Does &quot;total mapped reads&quot; mean uniquely mapped reads?</li>
  <li>If so, why is 45 million &quot;an exception&quot; ? It is the same as the other broad peaks.</li>
  </ul>
  <p>Any ideas where else can I look for an explanation?</p>
  
Tags: <chip-seq>
Title: Recommended coverage depth for ChIP Seq (H3K9me3 )
ThreadText: # Recommended coverage depth for ChIP Seq (H3K9me3 )
  
  (Asked by: username_0 on 2020-10-09)
  
  According to [ENCODE recommendations](https://www.encodeproject.org/chip-seq/histone/)
  
  > For broad-peak histone experiments, each replicate should have 45 million usable fragments
  
  According to my understanding, a usable fragment means a uniquely mapped read / pair of reads.
  
  However
  
  > H3K9me3 is an exception as it is enriched in repetitive regions of the genome. ... Tissues and primary cells should have 45 million total mapped reads per replicate.
  
  I fail to grasp Encode's language.
  
  * Does "total mapped reads" mean uniquely mapped reads?
  * If so, why is 45 million "an exception" ? It is the same as the other broad peaks.
  
  Any ideas where else can I look for an explanation?
  
  **Tags:** chip-seq
  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_1 on 2020-11-25. Score: 1 | Accepted answer
  
  The ENCODE Data Coordination Center, that developed and maintains the ENCODE portal, have implemented scripts validating adherence to the standards the ENCODE Consortium agreed upon when evaluating ChIP-seq analysis results. For any ChIP-seq experiment, except for H3K9me3, the adherence to the read depth standard is assessed looking on the number of "usable fragemnts" (<https://www.encodeproject.org/data-standards/terms/#read-depth>) (see answer by username_2 username_2). For H3K9me3 the read depth is assessed not by looking on usable fragments, but by looking on the total number of mapped reads (if it is a PE sequencing run, the number of mapped read pairs). It is an exception, because the number of mapped reads, especially for H3K9me3 is substantially higher than the number of usable fragments (where the duplicated and low mapping quality reads are filtered out). The decision to apply different read depth standard in case of H3K9me3 came up due to the logic explained here (<https://www.encodeproject.org/chip-seq/histone/>), briefly: because the H3K9me3 mark is enriched in repetitive regions of the genome, the number of usable fragments (post-filtering) is not expected to be as high as other marks (45M), despite high-quality libraries and sufficient sequencing depth. Instead of marking these experiments as failing read depth standard, the script checks the total number of mapped reads (or read pairs for PE sequencing runs) and makes sure it is above the read depth threshold (45M).
  
  > **Comments:**
  > **username_0**: I think that "usable fragments" should also be uniquely mapped. Although the definition in Encode sates only "Fragments are considered usable if they pass the various filters in the ChIP-seq uniform processing pipelines", when I go to the filtering script at Encode's github https://github.com/ENCODE-DCC/chip-seq-pipeline/tree/master/dnanexus/filter_qc  I see "Remove unmapped reads, non-primary alignments, reads failing platform, duplicates, **multi-mapping reads**, and PCR duplicates."  
  
  ---
  
  ### Answer by username_2 on 2020-10-13. Score: 1
  
  In the initial description for "usable fragments", ENCODE has the following (emphasis mine):
  
  > Usable fragment – A fragment is defined as the sequencing output **corresponding to one location in the genome**. If single-ended sequencing is performed, one read is considered a fragment. If paired-ended sequencing is performed, one pair of reads is considered a fragment. **Fragments are considered usable if they pass the various filters in the ChIP-seq uniform processing pipelines**. Used to evaluate ChIP-seq data.
  
  Aligning reads to repetitive regions is difficult because they don't give unique alignments.
  Reads from these regions will often have poor alignment scores and may not pass alignment QC checks.
  Reads may also be counted as multi-mapped, and often methods call for uniquely mapped reads, which again, may not be true for a large number of reads from H3K9me3 ChIP-seq.
  
  So for this particular histone mark, ENCODE loosens the definition for what you should count towards a minimum sequencing depth threshold.
  
  > **Comments:**
  > **username_0**: Why is the more difficult alignment a reason to loosen the definition? It will result in lower-quality analysis for H3K9ME3.  
  > **username_0**: Btw, if you have a reference for your explanation, or can tell me how can I find one, it'd be great.  
  > **username_2**: You're right, but the tradeoff seems to be between not having any usable information from H3K9me3 experiments and having a good amount of slightly lower-quality information, compared to other histone modifications.  I don't have a specific reference for that point, aside from the ENCODE documentation itself, but ENCODE has a list of [all its related publications](https://www.encodeproject.org/search/?searchTerm=chip-seq&type=Publication) that you could search to find one.  
  > **username_0**: So to be 100% sure, a "Usable fragment" is a unique alignment, right? "Corresponding to one location in the genome" means corresponding to **only** one location in the genome?  
  > **username_2**: Yes. [As per ENCODE](https://www.encodeproject.org/data-standards/terms/#read-depth): "Usable reads – A fragment is considered “usable” if it uniquely maps to the genome and remains after removing PCR duplicates (defined as two fragments that map to the same genomic position and have the same unique molecular identifier)."  
  
  ---
  
  