Single-cell chromatin accessibility sequencing technology based on single-molecule sequencing platform

On October 11, 2022, the Tang Fuyi research group of the Center for Biomedical Frontier Innovation (BIOPIC) of Peking University published a paper entitled scNanoATAC-seq: a long-read single-cell ATAC sequencing method to detect chromatin accessibility and genetic variants at Cell Research The paper simultaneously within an individual cell first reports a single-cell chromatin accessibility sequencing technology
based on a third-generation sequencing platform (single-molecule sequencing platform) called scNanoATAC-seq 。 This technology combines the advantages of a long-read single-molecule sequencing platform and single-cell chromatin accessibility sequencing (scATAC-seq: Single cell assay for Transposase accessible chromatin with high-throughput sequencing) to simultaneously detect chromatin open state and genomic structural variation
in a single cell.

scNanoATAC-seq expands the understanding
of chromatin accessibility sequencing analysis.
The scATAC-seq technique based on next-generation sequencing platforms (NGS) enriches only short fragments of genomic DNA (typically 80-300 bp) on locally open regions of chromatin, which are treated as signals
for open chromatin.
However, the use of excess Tn5 transposase to cut and label locally open regions of chromatin also produces some long pieces of genomic DNA, and it is unclear
whether these long pieces of DNA contain chromatin status information.

This study developed a single-cell ATAC-seq sequencing technology based on a single-molecule sequencing platform (shown in Figure 1) and explored its application to
biological problems.
First, using five human cell lines and in vivo human peripheral blood mononuclear cells, this study confirmed that scNanoATAC-seq technology can accurately group different cell types according to chromatin open state and reveal key chromatin open state regulatory information like scATAC-seq based on next-generation sequencing platforms (as shown in Figure 2).

Figure 1.
scNanoATAC-seq experimental flowchart (left) and analytical schematic (right)

Figure 2.
The effect of scNanoATAC-seq on clustering different cell types

Next, the study took advantage of the scNanoATAC-seq long-read segment to accurately identify
allele-specific chromatin open regions.
The vast majority of cell types in the human body are diploid cells
.
For diploid cells, ATAC-seq technology based on next-generation sequencing platforms to distinguish between two alleles in an open region of chromatin requires heterozygous single nucleotide polymorphisms (SNP) sites within that open region (typically between 80-300 bp in length
).
The scNanoATAC-seq technology based on the third-generation sequencing platform needs to distinguish two alleles in a chromatin open region, which does not require heterozygous SNP sites in the open region, but only requires heterozygous SNP sites or heterozygous structural variants within 4000bp on both sides of the open region
.
Compared with ATAC-seq technology based on next-generation sequencing platforms, scNanoATAC-seq technology based on third-generation sequencing platforms can detect more than ten times more allele-specific chromatin open regions
in the same cell line.
scNanoATAC-seq technology accurately genotypes chromatin accessibility signals (99.
1% accuracy for maternal alleles and 88.
1% accuracy for paternal alleles, as shown in Figure 3).

。 For example, using scNanoATAC-seq technology, the study detected allele-specific chromatin open regions in the promoter differential methylation region of the imprinted gene TRIM61 of the human B lymphoid cell line GM12878 (as shown in Figure 4, the chromatin in the promoter region of the maternal allele is open and the chromatin of the promoter region of the paternal allele is off), and there are no heterozygous SNP sites in the GM12878 cell line in this chromatin open region.
It cannot be detected by the ATAC-seq method for short read
segments.

In addition, the study found that allele-specific chromatin open regions detected in the GM12878 cell line using scNanoATAC-seq technology are mainly enriched on the
X chromosome.
This is consistent with
previous findings using DNase I highly sensitive site detection methods.
The reason why allele-specific chromatin openings tend to occur on the X chromosome is that far more cells are silenced on the paternal X chromosome than on the maternal X chromosome in the GM12878 cell line
.

Figure 3.
Schematic diagram of identification of allele-specific chromatin open regions using scNanoATAC-seq technique

Figure 4.
Allele-specific chromatin open regions of the promoter differential methylation region of the imprinted gene TRIM61 identified by scNanoATAC-seq technique

Later, the study used scNanoATAC-seq technology to detect chromatin open state in a single cell, while also detecting various structural variation events (insertions, deletions, duplicates, inversions, translocations, etc.
)
in the genome 。 Taking a large number of cell genome three-generation sequencing data from the human chronic myeloid leukemia (CML) cell line K562 as a benchmark, the study found that scNanoATAC-seq detected 7688 insertion events (64.
6% of the benchmark) and 6120 deletion events (67.
7% of the benchmark) in the K562 cell line (at least 5 single-cell supports), with an accuracy of 93.
8% and 75.
5%,
respectively 。 In addition to the classic BCR-ABL1 translocation event, the study can also detect deletion events up to 89 kb that simultaneously truncate the ZRANB1 and CTBP2 genes (as shown in Figure 5).

CTBP2 is known to inhibit leukemia cell proliferation, which means that scNanoATAC-seq technology detected potential structural variation events
in the K562 cell line that lead to loss of tumor suppressor function.
In addition, scNanoATAC-seq technology can accurately detect genomic copy number variation in individual cells, accurately distinguishing between euploidy cells and aneuploidy cells
.

Figure 5.
Structural variation (deletion of large fragments) on two genes, ZRANB1 and CTBP2 identified by scNanoATAC-seq (top) and PCR verification results (bottom)

Finally, the study took advantage of the long read segment of scNanoATAC-seq technology to detect 3868 chromatin co-opening events
in the GM12878 cell line.
Taking the co-open events found on the SOX4 gene as an example, these adjacent co-open genomic functional elements are supported by long read segments that directly connect two open regions, and their read length distribution is significantly different from that of non-common open regions (as shown in Figure 6 and Figure 7).

However, the analysis of chromatin co-opening events by scATAC-seq technology based on next-generation sequencing platform is highly dependent on the detection coverage (or detection sensitivity) of each single cell, and if the detection coverage of scATAC-seq is relatively low, it is difficult to detect chromatin co-opening events
。 More importantly, even in the case of high detection coverage of scATAC-seq, half of the chromatin co-opening events are signaled by the co-opening of two different alleles in a single cell (e.
g.
, the enhancer of allele #1 is "co-open" with the promoter of allele #2), which is a technical artifact.
However, the signals of chromatin co-opening events detected by scNanoATAC-seq technology based on the third-generation sequencing platform are all direct linkage information from a single DNA molecule, and are all real co-opening events from the same allele in a single cell (e.
g.
, the enhancer of allele #1 is co-open with the promoter of allele #1, or the enhancer of allele #2 is co-open with the promoter of allele #2), and there is no such technical artifact.

Figure 6.
The principle of identifying co-open events of adjacent regulatory elements using scNanoATAC-seq is used

Figure 7.
Co-opening events occur near regulatory elements near the SOX4 gene

In summary, scNanoATAC-seq technology has a wide range of biological application prospects
.
This method can simultaneously detect chromatin accessibility and genomic structural variation
in a single cell.
This method takes advantage of the long read segment to discover in a single cell an open region of chromatin that is not labeled with a heterozygous SNP site, which is not possible with the scATAC-seq technique of the short read segment
.
The method can also detect co-open events
that occur on different regulatory elements of the same allele with direct evidence support.
The advent of scNanoATAC-seq heralds the era of three-generation sequencing of
single-cell epigenomes.

It is worth discussing that in the exposed regions of genomic DNA with a high degree of chromatin openness, the higher the concentration of Tn5 transposase, the shorter
the resulting DNA library fragment 。 It can be inferred from this that in the case of excessive Tn5 transposase, in addition to the usual open chromatin, there may be less open chromatin regions, such as permissive chromatin, which cannot be detected
by short-read sequencing 。 In fact, scNanoATC-seq based on next-generation sequencing platforms detected more nucleosome mass information than scATAC-seq technology based on next-generation sequencing platforms (as shown in Figure 8).

In addition, regarding the study of chromatin open status of difficult genomic regions such as repeating sequence regions, scNanoATAC-seq based on third-generation sequencing platforms also has obvious advantages
over scATAC-seq based on next-generation sequencing platforms.
The above problems are worthy of in-depth exploration by applying scNanoATAC-seq technology based on the third-generation sequencing platform, which also reflects the necessity of
developing scNanoATAC-seq technology.

Figure 8.
Signal enrichment of scNanoATAC-seq and 10x scATAC-seq sequenced fragments near transcription start sites

Yuqiong Hu, a postdoctoral fellow at the School of Life Sciences, Peking University, and doctoral students Zhenhuan Jiang and Kuxuan Chen are the joint first authors
of the paper.
Professor Tang Fufu of the Center for Biomedical Frontier Innovation of Peking University is the corresponding author
of the paper.
The research project was supported
by the Peking University-Tsinghua Joint Center for Life Sciences, the National Natural Science Foundation of China, the Beijing Municipal Science and Technology Commission, and the Beijing Advanced Innovation Center for Future Genetic Diagnosis.

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