A simple ATAC-seq protocol for population epigenetics

Abstract

We describe here a protocol for the generation of sequence-ready libraries for population epigenomics studies, and the analysis of alignment results. We show that the protocol can be used to monitor chromatin structure changes in populations when exposed to environmental cues. The protocol is a streamlined version of the Assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) that provides a positive display of accessible, presumably euchromatic regions. The protocol is straightforward and can be used with small individuals such as daphnia and schistosome worms, and probably many other biological samples of comparable size (~10,000 cells), and it requires little molecular biology handling expertise.

Keywords: ATAC-seq; Daphnia pulex; Schistosoma mansoni; epigenetics; epigenomics.

Figure 1.. Schematic representation of experimental design.

( A) Daphnia were put into a water tank and allowed to acclimate (start population). Then, two experimental tanks were set up following strictly the same design. The only difference was the presence of a predator (a guppy trained to eat daphnia) in the floating plastic fish breeding isolation box in the stress treatment. (B) S.mansoni infected snails were used to produce cercariae that were separated into two populations, one treated with Latex in well water, the other without treatment. After one hour cercaria were used to infect mice. Adult worms were recovered at 65 days post-infection by perfusion and used for ATAC-seq.

Figure 2.. Example qPCR amplification profile.

(X-axis) Number of PCR cycles. (Y-axis) Fluorescence intensity. One-third of the maximum fluorescent intensity is shown by the orange line and the optimal number of additional cycles to perform are indicated for three example ATAC-seq libraries.

Figure 3.

Examples of fragmentation profiles of ATAC-seq libraries before ( A) and after size selection ( B). X-axis: Base pairs. Y-axis: Fluorescence intensity. ( A) Peaks correspond to nucleosome-free region, mono- to tetra-nucleosome fractions. Bottom lane: too strong fragmentation, thus Tn5 quantity needs to be decreased. ( B) Examples of BioAnalyser profiles of ATAC-seq libraries after size selection. Fragment size should be between 150 and 800 bp. Peaks at 35 bp and 10380 bp are spiked-in marker peaks for the BioAnalyser.

Figure 4.. Superposed metagene ATAC profiles of four individuals after sequencing and analysis.

X-axis in base-pairs. TSS = Transcription start site, TES = transcription end site. Y-axis average enrichment of ATAC-seq reads over genes and upstream and downstream regions. Enrichment of accessible chromatin occurs along the entire length of the genes. The metagene profiles are almost identical for Daphnia ( A), while there is more heterogeneity of the profiles in schistosomes ( B). Nevertheless, the profiles are in both cases very reproducible indicating the robustness of the ATAC-seq procedure.

Figure 5.

( A) Schematic representation of the measures taken on daphnia. SL = short length, LL = Long length. ( B) Boxplot of morphometric ratios of (LL-SL)/SL in control and stressed daphnia populations (control: n = 12; stress: n = 14).

Figure 6.. Combined metagene ATAC profiles of stressed and control daphnia populations.

X-axis in base-pairs. TSS = Transcription start site, TES = transcription end site. Y-axis average enrichment of ATAC-seq reads over genes and upstream and downstream regions.

Figure 7.. Clustering of individual daphnia based on their ATAC-seq profiles.

Heat map indicating similarity in the HMM ChromstaR results. Generally, samples from the stressed and the control populations each cluster together.

Figure 8.

( A) MA-Plot and ( B) Principal component analysis of individual schistosoma based on their ATAC-seq profiles. MA-plots are symmetric. Red dots indicate significantly different ATAC regions in control vs “Latex” population. On the PCA every point represents an individual male adult schistosome. Populations are colour coded. Samples from the control (blue) and “Latex” population (red) are well separated.