Tn5 transposase-based epigenomic profiling methods are prone to open chromatin bias

Abstract

Epigenetic studies of rare biological samples like mammalian oocytes and preimplantation embryos require low input or even single cell epigenomic profiling methods. To reduce sample loss and avoid inefficient immunoprecipitation, several chromatin immuno-cleavage-based methods using Tn5 transposase fused with Protein A/G have been developed to profile histone modifications and transcription factor bindings using small number of cells. The Tn5 transposase-based epigenomic profiling methods are featured with simple library construction steps in the same tube, by taking advantage of Tn5 transposase’s capability of simultaneous DNA fragmentation and adaptor ligation. However, the Tn5 transposase prefers to cut open chromatin regions. Our comparative analysis shows that Tn5 transposase-based profiling methods are prone to open chromatin bias. The high false positive signals due to biased cleavage in open chromatin could cause misinterpretation of signal distributions and dynamics. Rigorous validation is needed when employing and interpreting results from Tn5 transposase-based epigenomic profiling methods.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag)

“…CUT&Tag is a Tn5 transposase-based method 9,10 and there are concerns of possible bias in epigenomic profiling caused by the preference of Tn5 transposase to cut open chromatin regions 12 . We validated this point in the following analyses.…”

“…2b). This may be due to the bias of Tn5 transposase to preferentially cut open chromatin regions 12 . To estimate the magnitude of this bias, we evaluated the generation of false positive peaks by the comparison of NTU-CAT, ChIP-seq 11 , and ATAC-seq peaks 13 .…”

“…In order to identify the open chromatin regions, we used publicly available ATAC-seq data of bovine inner cell mass (GSM4516360) deposited by Halstead et al, 13 and mapping and peak calling generated 137,149 ATAC-seq peaks. We then calculated the false positive rate (FPR) possibly caused by the bias of Tn5 transposase toward open chromatin, where the FPR was the number of peaks that did not overlap with ChIP-seq, but did overlap with the ATAC-seq peaks, divided by the total number of peaks, as proposed by Wang et al 12 . As a result, the FPR rate was 10%-15% (Fig.…”

“…(a) A girasol plot showing the number of peaks and false positive rate (FPR). TheFPR was calculated by the number of NTU-CAT peaks that did not overlap with ChIP-seq (Blastocyst 1 of our previous report11 ) but did overlap with the ATAC-seq peaks13 , divided by the total number of peaks12 . (b) Average profile plot of the H3K4me3 signals in total NTU-CAT peaks and false positive peaks in Rep1 (Embryo 1 in Fig.1a).…”

Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag

“…38 However, compared to CUT&RUN, current protocols may have more limited effectiveness at detecting transient protein-DNA interactions involving transcription factors, and in some cases, the use of Tn5 transposase could potentially bias data toward open chromatin. 38,39 Thus far, CUT&Tag has been applied to assay enrichment of modified histones in plant and mammalian tissues. 38,[40][41][42][43][44][45] Here we describe successful implementation of CUT&-RUN and CUT&Tag methods for profiling protein-DNA interactions in zebrafish embryos.…”