Omni-ATAC

A method to identify open chromatin regions, such as promotor areas, using a transposase.

Omni-ATAC, an improved ATAC-seq protocol for chromatin accessibility profiling. The Omni-ATAC protocol lowers sequencing costs by generating fewer sequencing reads that map to mitochondrial DNA, consequently a higher percentage of reads map to peaks of chromatin accessibility.

ATAC-seq primarily works by incorporating primer sequences into the chromatin using a TN5 enzyme (tagmentation). Areas in the chromatin that are densely packed, for example in heterochromatic regions, will prevent the incorporation. As such, the primer sequences will only be added to open, euchromatic areas. One typical region containing open chromatin would be the regulatory area of gene promotors.

Currently, the Omni-ATAC method at NGI is only applicable for freshly-isolated 50.000 cells, frozen nuclei pellets or tagmented DNA which is prepared using the Omni-ATAC protocol provided by NGI.

Sample requirements

• Sample type: 
  • Cells
    • freshly isolated. Viability >90%.
    • counted and resuspended in media.
  • Nuclei
    • snap frozen pellet.
    • preferably be obtained by using sucrose sedimentation.
  • Tagmented DNA
    • purified DNA fragments after the tagmentation reaction performed by the user following NGI’s protocol.
• Sample amount: 
  • Cells or nuclei: 50 000 per tube for a ~3Gbp genome organism.
  • Tagmented DNA: result from the NGI’s tagmentation protocol.
• Sample delivery:
  • Cells: should be delivered on ice at a time agreed upon with the responsible lab personnel at NGI.
  • Nuclei: snap frozen pellet, on dry ice. Can be sent by post and the package should be labeled as “Cold Delivery, –20˚C”
  • Tagmented DNA: can be sent by post and the package should be labeled as “Cold Delivery, -20˚C”
    

Although the protocol allows for some margin of error the input amount should be kept the same across samples. Too few cells may cause the library prep chemistry to fail due to insufficient DNA input, while too many will result in a higher than optimal DNA-to-tagmentase ratio generating longer DNA fragments that will perform poorly on the sequencing. Therefore it is required to count the cells or nuclei before submission and turn in 50,000 cells per tube.

For samples from sources with significantly higher or lower genome sizes (for example flies), the number of cells needs to be adjusted accordingly. Please ask your project coordinator for details.

How to evaluate the sample quality

We cannot check the correct amount of cells/nuclei or the quality of your samples. You should make sure of the following:

• count the cells/nuclei as accurately as possible.
• don’t deliver more than 50.000 cells/nuclei per sample.
• make sure that your counted cells are alive – a high proportion of dead cells in the sample will reduce data quality.

If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.

What we do with your samples

When receiving cells from cell culture, we first spin down the cells and then resuspend the cells in a mild lysis buffer to generate a crude nuclei extract. The nuclei then are spun down and the transposition reaction is added directly.

Library preparation

The Omni ATAC protocol includes the use of multiple detergents for cell lysis to improve permeabilization and remove mitochondrial DNA. For the tagmentation reaction, the TN5 enzyme is used to simultaneously fragment DNA and insert primers into the cut site. The chromatin is subsequently cleaned up using DNA purification columns. At this stage, the tagmented DNA has single-stranded overhangs due to the primers added during the tagmentation reactions. These are repaired by a single elongation step prior to the first PCR amplification with 5 cycles.

In order to amplify the libraries to the lowest extent possible, additional cycles are determined by subjecting an aliquot of the PCR reaction to a qPCR. The number of cycles is chosen where the amplification signal reaches 1/4 of the final signal level.

After the library is further amplified by the additional cycles, the final library is purified using AMPure XP beads and the concentration and fragment sizes are determined.

Library QC and sequencing

In this step, we evaluate the yield obtained and the size distribution of the libraries generated. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are queued for sequencing to be carried out according to the setup stated in the agreement.

We have access to 96 (combinatorial) dual indexes so that is the highest level of multiplexing possible at the moment. Typically we recommend 50 million paired-end reads per library for human samples. The optimal depth likely depends on genome size and level of open chromatin, so for samples where these are expected to be very different from the typical sample, the sequencing depth may need to be adjusted.

Expected results

We expect more sequencing reads from euchromatic regions than from heterochromatic regions; areas with open chromatin conformation, for example, the transcriptional start sites of active genes, are also expected to get more reads. Results will also depend on the characteristics of the sample and how they are sequenced:

• Paired-end sequencing is required when information on nucleosome positioning is of interest.
• For general information on chromatin status changes, single-end sequencing can be sufficient.
• Duplicate detection is significantly improved with paired-end sequencing.
• If too much DNA was provided, the fragment length distribution of the library will be shifted towards longer, high molecular weight fragments. We do not recommend the sequencing of such samples.
• If the nuclei were lysed before submission, we would expect higher background noise levels in the sequencing data.

Bioinformatics

The bioinformatics analysis that we provide for ATAC-seq is based on the nf-core/atacseq pipeline. It provides alignment, QC, filtering, peak calling, normalization, and differential accessibility analysis. Read about the pipeline outputs here: https://nf-co.re/atacseq/docs/output.

References and links

1. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues
2. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide
3. nf-co.re/atacseq analysis pipeline

Last Updated: 16th March 2023