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--- 4dn:phase1:working_groups:omics_data_standards:minutes-03-25-2019 [2019/03/25 13:00]
xiaoyi_cao created
+++ 4dn:phase1:working_groups:omics_data_standards:minutes-03-25-2019 [2025/04/22 16:21] (current)
@@ Line 1: / Line 1: @@
-======   Update on PLAC-seq/HiChIP protocol   ======
+====== Update on PLAC-seq/HiChIP protocol ======
-  *
+  * QC metrics
-**QC metrics**
+  * Recommended antibodies
-  *
-**Recommended antibodies**
+  * Crosslinking conditions
-  *
-**Crosslinking conditions**
+===== QC Metrics =====
-=====   QC Metrics   =====
-  *
+  * Hi-C - digestion/ligation efficiency, noise level
-**Hi-C - digestion/ligation efficiency, noise level**
+  * IP - enrichment performance
-  *
-**IP - enrichment performance**
+  * Library preparation - complexity All three aspects have both a qualitative detection methods and quantitative detection method (with shallow sequencing)
-  *
-**Library preparation - complexity**
+==== Qualitative ====
-**All three aspects have both a qualitative detection methods and quantitative detection method (with shallow sequencing)**
-====   Qualitative   ====
+  * Hi-C - DNA fragment size before digestion (should not have smear), after digestion (should be a smear at lower size range, note that the expected size should be larger than the theoretical value because of potential chromatin structures and accessibility issue) and after ligation (should be a similarly-shaped smear at a larger size range)
-  *
+  * IP - DNA fragments after sonication should be around 100~600bp, incomplete sonication will result in larger fragments and affect IP performance. IP yield (IPed DNA / input DNA) is also a good metric. In general for H3K4me3 / H3K27ac will have <1~3% IP yield, and CTCF / PolII will have < 0.1%. However, IP yield is necessary but not sufficient for a good IP.
-**Hi-C - DNA fragment size before digestion (should not have smear), after digestion (should be a smear at lower size range, note that the expected size should be larger than the theoretical value because of potential chromatin structures and accessibility issue) and after ligation (should be a similarly-shaped smear at a larger size range)**
+  * Library preparation - Libraries with good complexity require at least 10~20ng of IPed DNA, with 11~13 PCR cycles and 20~40% duplication rate at ~250M reads. Libraries with worse complexity will need more input.
-  *
-**IP - DNA fragments after sonication should be around 100~600bp, incomplete sonication will result in larger fragments and affect IP performance. IP yield (IPed DNA / input DNA) is also a good metric. In general for H3K4me3 / H3K27ac will have <1~3% IP yield, and CTCF / PolII will have < 0.1%. However, IP yield is necessary but not sufficient for a good IP.**
+==== Quantitative ====
-  *
-**Library preparation - Libraries with good complexity require at least 10~20ng of IPed DNA, with 11~13 PCR cycles and 20~40% duplication rate at ~250M reads. Libraries with worse complexity will need more input.**
-====   Quantitative   ====
 **Glossary:**
-**A - sequenced read pairs**
+A - sequenced read pairs\\ B - valid read pairs\\ C - valid read pairs after PCR duplicates removal\\ D - inter-chromosomal read pairs\\ E - intra-chromosomal read pairs\\ F - short-range (<=1kb) of E\\ G - long-range (>1kb) of E\\ H - F that overlap with ChIP peaks
-**B - valid read pairs**
-**C - valid read pairs after PCR duplicates removal**
-**D - inter-chromosomal read pairs**
-**E - intra-chromosomal read pairs**
-**F - short-range (<=1kb) of E**
-**G - long-range (>1kb) of E**
-**H - F that overlap with ChIP peaks**
-  *
+  * Hi-C - trans ratio (D/C) reflects noise level (reference < 20~40%), long-range cis ratio (G/E): (reference > 50~70%)
-**Hi-C - trans ratio (D/C) reflects noise level (reference < 20~40%), long-range cis ratio (G/E): (reference > 50~70%)**
+  * IP - on-target rate (H/F): (reference for histone marks > 20%, for TFs > 5~10%)
-  *
-**IP - on-target rate (H/F): (reference for histone marks > 20%, for TFs > 5~10%)**
+  * Library preparation - PCR duplication rate (C/B): (reference < 3%)
-  *
-**Library preparation - PCR duplication rate (C/B): (reference < 3%)**
+===== How to choose the best antibody =====
-=====   How to choose the best antibody   =====
-  *
+  * High specificity - high on-target rate
-**High specificity - high on-target rate**
+  * High affinity - large IP yield
-  *
-**High affinity - large IP yield**
+  * Highly robust - less batch effects (monoclonal Ab is better than polyclonal)
-  *
-**Highly robust - less batch effects (monoclonal Ab is better than polyclonal)**
+Currently recommended tested antibodies (all monoclonal):
-**Currently recommended tested antibodies (all monoclonal):**
-  *
+  * CTCF: Cell Signaling, 3418T
-**CTCF: Cell Signaling, 3418T**
+  * H3K4me3: Millipore, 04-745
-  *
-**H3K4me3: Millipore, 04-745**
+  * H3K27ac: Diagenode, C15200184-50; Active motif, 91193
-  *
-**H3K27ac: Diagenode, C15200184-50; Active motif, 91193**
+Bill Noble: Will ENCODE develop QC metrics on Hi-C data? Shall we establish a data quality measurement procedure? There are several software that can evaluate Hi-C datasets, like HiCRep or other tools as described in [[https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1658-7|https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1658-7]].
-**Bill Noble: Will ENCODE develop QC metrics on Hi-C data? Shall we establish a data quality measurement procedure? There are several software that can evaluate Hi-C datasets, like HiCRep or other tools as described in [[https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1658-7|https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1658-7]].**
-**Miao Yu: The current QC metrics is before deep sequencing and the evaluation can be done after data generation**
+Miao Yu: The current QC metrics is before deep sequencing and the evaluation can be done after data generation
-**PLAC-seq / HiChIP has lower IP efficiency than ChIP-seq**
+PLAC-seq / HiChIP has lower IP efficiency than ChIP-seq
-  *
+  * Hi-C may disrupt protein complexes
-**Hi-C may disrupt protein complexes**
+  * Biotin enrichment after IP may enrich DNA fragments without protein binding
-  *
-**Biotin enrichment after IP may enrich DNA fragments without protein binding**
+===== Test of crosslinking conditions =====
-=====   Test of crosslinking conditions   =====
-  *
+  * Different crosslinking conditions may affect on-target rate. Results are preliminary and higher temperature does not improve on-target rates. One DSG + HCHO test had a high on-target rate but needs further verification.
-**Different crosslinking conditions may affect on-target rate. Results are preliminary and higher temperature does not improve on-target rates. One DSG + HCHO test had a high on-target rate but needs further verification.**
+===== Discussion =====
-=====   Discussion   =====
-**Burak: What is the intended disseminate method for all this results?**
+Burak: What is the intended disseminate method for all this results?
-**Bing: We are currently preparing a protocol that will be circulated within 4DN and be submitted to Nature Protocol but the manuscript is still under work.**
+Bing: We are currently preparing a protocol that will be circulated within 4DN and be submitted to Nature Protocol but the manuscript is still under work.

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