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Getting started with Dovetail™ proximity-ligation assays 

Chromosome conformation capture (3C), also known as proximity-ligation is a molecular biology approach designed to analyze the spatial organization of chromatin. When the ligation events are tagged with biotinand isolated with streptavidin subjected to paired-end whole genome sequencing it is known as Hi-C. For further information, please see this2017 review paper by Davis et al.  https://doi.org/10.1038/nmeth.4146 

Dovetail Genomics offers both kits and services. For more information go to (https://dovetailgenomics.com/products-services/) 

The Dovetail proximity-ligation kits and analysis are user friendly and designed for novice Hi-C users. You will also have access to our world-class support team should you run into difficulties or have questions 

These novel Hi-C assays available only from Dovetail Genomicsdiffer from traditional Hi-C in the following important ways:  

  • Omni-C replaces restriction enzymes with DNase to generate uniform genomic coverage. 
  • Micro-C replaces restriction enzymes with MNase to provide the best resolution of any Hi-C assay, down to nucleosome-level folding. 
  • HiChIP combines the chromatin immunoprecipitation ofChIP-seq with the conformation capture of Hi-C to provide a protein-directed view of chromatin folding. 

Please view the assay selection guide for different applications (https://dovetailgenomics.com/products-services/) 

Dovetail™ Omni-C™ Assay & Kit

Dovetail™ Micro-C Assay & Kit

Dovetail™ HiChIP MNase Assay & Kit

Dovetail™ Omni-C™ Assay & Kit

General 

Omni-C leverages DNase to digest chromatin as opposed to restriction enzymes. This approach provides superior coverage across the genome enabling more applications such as genotyping and haplotype phasing. 

Sample Support 

The standard input for Omni-C is one million cells. A low input protocol is also supported down to one thousand cells.

Cells, animal and plant tissues, and blood. 

The Omni-C chemistry is robust and works across a variety of genome types.  

Workflow 

The Omni-C Kit supplies 8 reactions.

The workflow from sample to sequencingready library is split over two days.

The Omni-C kit is compatible with hybrid capture approaches. The Omni-C workflow integrates easily with Agilent SureSelect, Illumina TruSight and IDT xGEN. 

Omni-C libraries exhibit WGS-like coverage of the genome and do not use restriction enzymes; therefore, you can use off-the-shelf probe sets or design your own probes without an inclusion of a restriction enzyme site. 

Yes, frozen cells previously cross-linked with formaldehyde concentration  1% can be used as input to the assay Simply skip the formaldehyde fixation step of our recommended two step fixation process. 

Dovetail offers library preparation and index primers modules to streamline the library preparation workflowOmni-C is compatible with third party library preparation kits as wellnamely NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, Kapa® HyperPrep, and Swift Biosciences Accel-NGS® 2S Plus DNA Library Kit. 

The Omni-C workflow does not require sonication. The fragmentation is enzymatic and is carried out at Stage 1 of the protocol.

Magnetic separation rack for 0.2 mL and 1.5 mL tubescentrifuge, vortex mixer, thermal cycler, agitating thermal mixer, Qubit fluorometer, and TapeStation (Fragment Analyzer or Bioanalyzer can also be used).  

Workflow QC 

The Omni-C workflow has three built-in quality control steps, including an early QC step used to assess the digestion reaction. Optional shallow Illumina sequencing is recommended as a final QC step prior to deep sequencing. An easy-to-use sequence QC analysis pipeline is available to assess final success 

Analysis

Omni-C libraries are Illumina compatible. We recommend sequencing 2 x 150 bp. One library is typically sufficient for the generation of ~300M total read-pairs. Depending upon final sequencing depth desired, multiple libraries may need to be pooled  prior to the sequencing run. 

Dovetail has compiled a comprehensive best practices step-by-step workflow with full documentation (https://omni-c.readthedocs.io/en/latest/) that covers:

  • Alignment, Pairs generation, and filtering for high-quality proximity-ligation reads
  • Contact matrix generation for both .hic and .cool files
  • Commonly used applications

Dovetail tools for QC and Contact Matrix generation require the Omni-C raw sequence data (*.fq.gz) and a draft assembly (genome *.fa).  

Due to the use of restriction enzymes and sonication, traditional Hi-C valid reads require the insert size (or physical coverage) of a read-pair to be greater than the library fragment and to be adjacent to a restriction site. Omni-C libraries do not use restriction enzymes or require sonication (a key source of noise in Hi-C libraries). Therefore, a valid Omni-C read only requires that the insert size (or physical coverage) of a read-pair be greater than the library fragment as defined by HiC-Pro. A more in-depth analysis can be read here: https://dovetailgenomics.com/hi-c_pro_valid_reads/ 

HiC-Pro is also compatible with Omni-C libraries. Note that there are custom configuration files that are required. Please reach out to us at Dovetail for guidance on how to run HiC-Pro. 

This largely depends on application. The following recommendations are good starting points: 

  1. Genome Assembly – 30X coverage; 1 library for genomes < 3 Gbp
  2. Genotyping and Haplotype Phasing – 40X coverage; 2 libraries
  3. Topology Analysis – A/B compartments (3X coverage; 1 library), TADs (30X; 1-2 libraries), Loops (> 90X coverage; 3-4 libraries) 
  4. For QC we recommend 1 – 2 million paired-end reads (2 x 150 bp) 

Did we miss you question? Please submit your question to our support team: support@dovetail-genomics.com 

Dovetail™ Micro-C Assay & Kit

General 

Micro-C leverages micrococcal nuclease (MNase) to digest chromatin as opposed to restriction enzymes. This approach provides superior signaltonoise enabling nucleosome resolution/nucleosome phasing and a more granular view of chromatin folding including enhancer-promoter/promoter-promoter interactions and loop extrusion features. 

While coverage is reduced in nucleosome-free regions, it rarely drops to zero because the digestion profile contains di– and tri-nucleosomes as well as mononucleosomes. This ensures that some fragments contain linker-DNA and thereby provides some coverage at nucleosome-free regions. 

Sample Support 

The standard input for Micro-C assay is one million cells  Low input is also supported down to 10,000 cells. 

Currently Micro-C is validated for mammalian cells. If you are interested in another sample type, please contact our Technical Support team at support@dovetail-genomics.com. 

The quantity of the reagents supplied in the Dovetail™ Micro-C kit supports the optimization of the digestion reaction. 

Workflow 

The Micro-C Kit supplies 8 reactions.

The workflow from sample to sequencingready library is split over two days. 

Yes, frozen cells previously cross-linked with formaldehyde concentration  1% can be used as input to the assay. Simply skip the formaldehyde fixation step of our recommended two step fixation process. 

Dovetail offers library preparation and index primers modules to streamline the library preparation workflow. Omni-C is compatible with third party library preparation kits as well, namely NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, Kapa® HyperPrep, and Swift Biosciences Accel-NGS® 2S Plus DNA Library Kit. 

The Micro-C workflow does not require sonication. The fragmentation is enzymatic and is carried out at Stage 1 of the protocol. 

Magnetic separation rack for 0.2 mL and 1.5 mL tubes, centrifuge, vortex mixer, thermal cycler, agitating thermal mixer, Qubit fluorometer, and TapeStation (Fragment Analyzer or Bioanalyzer can also be used). 

Workflow QC 

The HiChIP workflow has built-in quality control steps, including an early QC step used to assess the digestion reaction. Optional shallow Illumina sequencing can also be carried out as a final QC step prior to deep sequencing. We offer our kit users access to an easy-to-use QC analysis pipeline. 

Analysis

Micro-C libraries are Illumina compatible. We recommend sequencing 2 x 150 bp.

Dovetail has compiled a comprehensive best practices step-by-step workflow with full documentation (https://micro-c.readthedocs.io/en/latest/) that covers:

  • Alignment, Pairs generation, and filtering for high-quality proximity-ligation reads
  • Contact matrix generation for both .hic and .cool files
  • Commonly used applications

Dovetail tools for QC and Contact Matrix generation require the Micro-C raw sequence data (*.fq.gz) and a reference assembly  (genome *.fa).

Due to the use of restriction enzymes and sonication, traditional Hi-C valid reads require the insert size (or physical coverage) of a read-pair to be greater than the library fragment and to be adjacent to a restriction site. Micro-C libraries do not use restriction enzymes or require sonication (a key source of noise in Hi-C libraries). Therefore, a valid Micro-C read only requires that the insert size (or physical coverage) of a read-pair be greater than the library fragment as defined by HiC-Pro. A more in-depth analysis can be read here: https://dovetailgenomics.com/hi-c_pro_valid_reads/ 

HiC-Pro is also compatible with Micro-C libraries. Note that there are custom configuration files that are required. Please reach out to us at Dovetail for guidance on how to run HiC-Pro. 

  • Topology Analysis – A/B compartments (3X coverage; 1 library), TADs (30X; 1-2 libraries), Loops (80X coverage; 2-3 libraries) 
  • For QC we recommend 1– 2 million paired-end reads (2 x 150 bp) 

Did we miss you question? Please submit your question to our support team: support@dovetail-genomics.com 

Dovetail™ HiChIP MNase Assay & Kit

General 

Inclusion of ChIP-seq data while simplify data QC and interpretation, however, is not an absolute requirement. If you have not generated ChIP-seq data, you may use publicly available data from sources such as ENCODE but will not be truly reflective of your experiment or sample of interest. 

Dovetail HiChIP leverages the Micro-C (MNase-based Hi-C) workflow to conduct chromatin digestions which provides the following benefits to the user: 

  1. No sonication is required in the protocol 
  2. Nucleosome position is preserved 
  3. Peak adjustment based on restriction enzyme-proximity to DNA-protein interaction of interest is not required. The use of restriction enzymes enriches libraries at the restriction size closest to the protein-DNA interaction, not on the interaction itself. As a consequence, in the data analyses HiChIP peaks must be adjusted to reflect the real protein-DNA site, not the restriction site closest to the interaction of interest. 

While coverage is reduced at nucleosome-free regions, it rarely drops to zero because the digestion profile contains di- and tri-nucleosomes as well as mono-nucleosomes. This ensures that some fragments contain linker-DNA and there by provide some coverage at nucleosome-free regions. 

Sample Support 

The recommended cell input amount for each of the Dovetail validated antibody can be found here. If you are using a non-validated antibody, we recommend starting with 10 million cells. 

Currently Dovetail HiChIP MNase assay is validated for mammalian cells. If you are interested in another sample source, please contact our Technical Support team at support@dovetail-genomics.com . 

Workflow 

Yes, frozen cells previously crosslinked with formaldehyde concentration  1% can be used as input to the assay.  Simply skip the formaldehyde fixation step of our recommended two step fixation process. 

The Dovetail HiChIP assay is a three-day workflow from sample to sequencing-ready library. 

Workflow QC 

The Micro-C workflow has three built-in quality control steps, including an early QC step used to assess the digestion reaction. Optional shallow Illumina sequencing is recommended as a final QC step prior to deep sequencing. An easy-to-use sequence QC analysis pipeline is available to assess final success.

Analysis

This will vary depending on the occurrence rate of Protein-DNA interactions of the protein of interest and the genome in question. The following are recommended starting guidelines: 

  1. Low-occurrence – 100M paired-end reads (2 x 150 bp) 
  2. High-occurrence – 400M paired-end reads (2 x 150 bp) 
  3. QC – 1M paired-end reads (2 x 150 bp) to assess the proximity-ligation qualities of the libraries. 40M paired-end reads (2×150) are need to assess the success of the ChIP enrichment 

Dovetail has compiled a comprehensive best practices step-by-step workflow with full documentation (https://hichip.readthedocs.io/en/latest/) that covers:

  • Alignment, Pairs generation, and filtering for high-quality proximity-ligation reads
  • IP-enrichment assessment
  • Contact matrix generation for both .hic and .cool files
  • Commonly used applications

The Dovetail HiChIP QC tool designed to assess the success of a Dovetail HiChIP library requires the raw HiChIP sequence data (*.fq.gz), a reference (genome *.fa) and a file of 1D ChIP-seq peak locations (*.bed).  

Due to the use of restriction enzymes and sonication, traditional Hi-C valid reads require the insert size (or physical coverage) of a read-pair to be greater than the library fragment and to be adjacent to a restriction site. Dovetail HiChIP libraries do not use restriction enzymes or require sonication (a key source of noise in Hi-C libraries). Therefore, a valid HiChIP read only requires that the insert size (or physical coverage) of a read-pair be greater than the library fragment as defined by HiC-Pro. A more in-depth analysis can be read here: https://dovetailgenomics.com/hi-c_pro_valid_reads/ 

Did we miss you question? Please submit your question to our support team: support@dovetail-genomics.com