Stage I. Classification of Individual Evidence Types
Description![]() |
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Single evidence is classified into weak or strong evidence (see above), depending on the confidence level of the associated methodologies. |
1. Promoters and transcription start sites (TSSs)![]() |
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Promoters are defined in bacteria by the DNA region specifically bound
by RNA polymerase to initiate transcription. A TSS is the precise first nucleotide that is transcribed, different methods identify promoters or TSSs. They are jointly classified here. |
Evidence Code | Evidence Category | |
Strong Evidence |
1.1 In vitro transcription assay |
TA | Classical experiment |
1.2 RNA-seq using two enrichment strategies for primary transcripts, consistent biological replicates, and evidence for a non-coding gene. |
RS-EPT-ENCG-CBR | High-throughput protocol | |
1.3 cross validation(GEA/GS) |
CV(GEA/GS) | independent cross-validation | |
1.4 cross validation(GEA/ROMA) |
CV(GEA/ROMA) | independent cross-validation | |
1.5 High-throughput transcription initiation mapping |
HTTIM | nd | |
1.6 RNA polymerase footprinting |
RPF | nd | |
1.7 RNA-seq using two enrichment strategies for primary transcripts and consistent biological replicates |
RS-EPT-CBR | nd | |
1.8 Transcription initiation mapping |
TIM | nd | |
Weak Evidence |
1.9 ChIP analysis |
CHIP | High-throughput protocol |
1.10 ROMA |
ROMA | High-throughput protocol | |
1.11 RNA-seq |
RS | High-throughput protocol | |
1.12 Automated inference of promoter position |
AIPP | nd | |
1.13 Human inference of promoter position |
HIPP | nd |
2. Regulatory interactions![]() |
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A regulatory interaction is defined, depending on the type of evidence, as the transcription factor (TF)-regulated gene interaction (TF-gene), or more specifically as the TF-DNA binding site interaction. | Evidence Code | Evidence Category | |
Strong Evidence |
2.1 In vitro transcription assay |
TA | Classical experiment |
2.2 ChIP analysis and statistical validation of TFBSs |
CHIP-SV | High-throughput protocol | |
2.3 cross validation(GEA/GS) |
CV(GEA/GS) | independent cross-validation | |
2.4 cross validation(GEA/ROMA) |
CV(GEA/ROMA) | independent cross-validation | |
Weak Evidence |
2.5 ChIP analysis |
CHIP | High-throughput protocol |
2.6 Mapping of signal intensities |
MSI | High-throughput protocol | |
2.7 ROMA |
ROMA | High-throughput protocol |
3. Transcription factor functional conformation ![]() |
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Most dedicated TFs have usually two conformations, one with a non-covalent bound allosteric metabolite, or a covalent phosphorylation (holo conformation), and one as a free protein or multimer (the apo conformation). There are exceptions to this statement. We call functional conformation the one that is capable of binding to its specific binding sites and perform its activation or repression activity. For the sake of functional conformation evidence the experiments below have to be considered with and without effector. | Evidence Code | Evidence Category |
4. Transcription units![]() |
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Evidence Code | Evidence Category | ||
Strong Evidence |
4.1 Mapping of signal intensities, evidence for a single gene, consistent biological replicates |
MSI-ESG-CBR | High-throughput protocol |
4.2 paired end di-tagging |
PET | High-throughput protocol | |
Weak Evidence |
4.3 Mapping of signal intensities |
MSI | High-throughput protocol |
Stage II. Analytical Cross-Validation
Description![]() |
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For each object, the types of evidence are given, which can be combined with each other to allow an upgrade to confirmed confidence. Any two methods from different rows can be combined. Types of evidence in the same row cannot be combined with each other. For instance, different protocols for transcription initiation mapping cannot be combined with each other for cross-validation, since these methods use mRNA as the starting material and therefore share a common source of false positives, which is RNA processing or degradation. Cross-validation of TF binding sites and promoters requires that the exact location of the object is specified for each individual evidence. Evidence codes: Each combination of two types of independent evidence is described as an evidence code, of the type CV(EC1/EC2). For instance, the evidence code for the combination of genomic SELEX (GSELEX) and gene expression analysis (GEA) is CV(GSELEX/GEA), that for the combination of footprinting (BPP) with site mutation (SM) is CV(BPP/SM). |
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1. Promoters and transcription start sites (TSSs)![]() |
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Confirmed Evidence |
Objects supported by two types of independent strong evidence are classified as confirmed. |
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CV(GEA/ROMA/SM) |
GEA: Gene expression analysis ROMA: ROMA SM: Site mutation |
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CV(GEA/SM/GS) |
GEA: Gene expression analysis SM: Site mutation GS: genomic SELEX |
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CV(RPF/GEA/GS) |
RPF: RNA polymerase footprinting GEA: Gene expression analysis GS: genomic SELEX |
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CV(RPF/GEA/ROMA) |
RPF: RNA polymerase footprinting GEA: Gene expression analysis ROMA: ROMA |
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CV(RPF/RS-EPT-CBR) |
RPF: RNA polymerase footprinting RS-EPT-CBR: RNA-seq using two enrichment strategies for primary transcripts and consistent biological replicates |
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CV(RPF/RS-EPT-ENCG-CBR) |
RPF: RNA polymerase footprinting RS-EPT-ENCG-CBR: RNA-seq using two enrichment strategies for primary transcripts, consistent biological replicates, and evidence for a non-coding gene. |
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CV(RPF/SM) |
RPF: RNA polymerase footprinting SM: Site mutation |
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CV(RPF/TA) |
RPF: RNA polymerase footprinting TA: In vitro transcription assay |
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CV(RPF/TIM) |
RPF: RNA polymerase footprinting TIM: Transcription initiation mapping |
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CV(RS-EPT-CBR/SM) |
RS-EPT-CBR: RNA-seq using two enrichment strategies for primary transcripts and consistent biological replicates SM: Site mutation |
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CV(RS-EPT-CBR/TA) |
RS-EPT-CBR: RNA-seq using two enrichment strategies for primary transcripts and consistent biological replicates TA: In vitro transcription assay |
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CV(RS-EPT-ENCG-CBR/SM) |
RS-EPT-ENCG-CBR: RNA-seq using two enrichment strategies for primary transcripts, consistent biological replicates, and evidence for a non-coding gene. SM: Site mutation |
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CV(RS-EPT-ENCG-CBR/TA) |
RS-EPT-ENCG-CBR: RNA-seq using two enrichment strategies for primary transcripts, consistent biological replicates, and evidence for a non-coding gene. TA: In vitro transcription assay |
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CV(SM/TA) |
SM: Site mutation TA: In vitro transcription assay |
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CV(SM/TIM) |
SM: Site mutation TIM: Transcription initiation mapping |
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CV(TA/TIM) |
TA: In vitro transcription assay TIM: Transcription initiation mapping |
2. Regulatory interactions ![]() |
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Strong Evidence |
Objects supported by two types of independent weak evidence are classified as strong. |
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CV(GEA/GS) |
GEA: Gene expression analysis GS: genomic SELEX |
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CV(GEA/ROMA) |
GEA: Gene expression analysis ROMA: ROMA |
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Confirmed Evidence |
Objects supported by two types of independent strong evidence are classified as confirmed. |
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CV(CHIP-SV/GEA/GS) |
CHIP-SV: ChIP analysis and statistical validation of TFBSs GEA: Gene expression analysis GS: genomic SELEX |
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CV(CHIP-SV/GEA/ROMA) |
CHIP-SV: ChIP analysis and statistical validation of TFBSs GEA: Gene expression analysis ROMA: ROMA |
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CV(CHIP-SV/RPF) |
CHIP-SV: ChIP analysis and statistical validation of TFBSs RPF: RNA polymerase footprinting |
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CV(CHIP-SV/SM) |
CHIP-SV: ChIP analysis and statistical validation of TFBSs SM: Site mutation |
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CV(GEA/ROMA/SM) |
GEA: Gene expression analysis ROMA: ROMA SM: Site mutation |
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CV(GEA/SM/GS) |
GEA: Gene expression analysis SM: Site mutation GS: genomic SELEX |
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CV(RPF/GEA/GS) |
RPF: RNA polymerase footprinting GEA: Gene expression analysis GS: genomic SELEX |
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CV(RPF/GEA/ROMA) |
RPF: RNA polymerase footprinting GEA: Gene expression analysis ROMA: ROMA |
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CV(RPF/SM) |
RPF: RNA polymerase footprinting SM: Site mutation |
3. Transcription units ![]() |
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Confirmed Evidence |
Objects supported by two types of independent strong evidence are classified as confirmed. |
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CV(LTED/PM) |
LTED: Length of transcript experimentally determined PM: Polar mutation |
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CV(PET/PM) |
PET: paired end di-tagging PM: Polar mutation |