Getting started with Dovetail®proximity-ligation assays

General

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 biotin and isolated with streptavidin subjected to paired-end whole genome sequencing it is known as Hi-C.

For further information, check out this review paper by Davis et al. (2017).

We offer both! Check out our kits and services to learn more about each.

The Dovetail proximity-ligation kits are user friendly and designed for novice Hi-C users. Plus, our world-class support team is also available to help out if you run into difficulties or have questions.

They are novel Hi-C assays available only from Dovetail Genomics® and differ from traditional Hi-C in the following important ways:

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

Check out the assay selection guide to find the best assay for you!

Don’t see your question? Submit it to our support team: support@dovetail-genomics.com

Dovetail® HiChIP MNase Assay & Kit

General

The Dovetail HiChIP MNase assay leverages the Micro-C (MNase-based Hi-C) workflow to conduct chromatin digestions. With the HiChIP MNase assay:

• No sonication is required in the protocol, improving assay robustness
• Nucleosome position is preserved
• 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.

Inclusion of ChIP-seq data simplifies data QC and interpretation but is not an absolute requirement. You can use publicly available data from sources such as ENCODE if you have not generated ChIP-seq data. However, it will not be truly reflective of your experiment or sample 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.

The Dovetail HiChIP MNase assay is currently validated for mammalian cells. If you are interested in analyzing another sample source, please contact our Technical Support team.

Workflow

It only takes three days to go from sample to sequencing-ready library!

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.

Workflow QC

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

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:
• Low-occurrence – 100 M paired-end reads (2 x 150 bp)
• High-occurrence – 400 M paired-end reads (2 x 150 bp)
• QC – 1 M paired-end reads (2 x 150 bp) to assess the proximity-ligation qualities of the libraries. 40 M 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 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 that assesses 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. Download our white paper to learn more.


Don’t see your question? Submit it to our support team:
support@dovetail-genomics.com

Dovetail® Micro-C Assay & Kit

General

Micro-C uses micrococcal nuclease (MNase) to digest chromatin instead of the restriction enzymes other Hi-C methods use. This approach provides superior signal–to–noise, enabling nucleosome resolution/nucleosome phasing and a more granular view of chromatin folding that includes 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 mono–nucleosomes. 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.

Yes, the quantity of the reagents supplied in the Dovetail Micro-C kit will allow you to optimize the digestion reaction.

Workflow

You will get 8 reactions with each Micro-C kit.

It only takes two days to go from sample to sequencing-ready library!

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 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

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 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. Check out this white paper to learn more.

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)

Don’t see your question? Submit it to our support team: support@dovetail-genomics.com

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

You will get 8 reactions with each Omni-C kit.

The workflow from sample to sequencing–ready 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 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 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 tubes, centrifuge, 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 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. 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. Check out this white paper to learn more.

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

Don’t see your question? Submit it to our support team: support@dovetail-genomics.com