An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Study the forest, not only the trees: Environmental exposures, not genomes, generate most health disparities.

As sequencing and analysis techniques provide increasingly detailed data at a plummeting cost, it is increasingly popular to seek the answers to medical and public health challenges in the DNA sequences of affected populations. This is methodologically attractive in its simplicity, but a genomics-only approach ignores environmentally mediated health disparities, which are well-documented at multiple national and global scales. While genetic differences exist among populations, it is unlikely that these differences overcome social and environmental factors in driving the gap in health outcomes between privileged and oppressed communities. We advocate for following the lead of communities in addressing their self-identified interests, rather than treating widespread suffering as a convenient natural experiment.

Master Transcription Regulators and Transcription Factors Regulate Immune-Associated Differences Between Patients of African and European Ancestry With Colorectal Cancer.

Background and Aims: Individuals of African (AFR) ancestry have a higher incidence of colorectal cancer (CRC) than those of European (EUR) ancestry and exhibit significant health disparities. Previous studies have noted differences in the tumor microenvironment between AFR and EUR patients with CRC. However, the molecular regulatory processes that underpin these immune differences remain largely unknown. Methods: Multiomics analysis was carried out for 55 AFR and 456 EUR patients with microsatellite-stable CRC using The Cancer Genome Atlas. We evaluated the tumor microenvironment by using gene expression and methylation data, transcription factor, and master transcriptional regulator analysis to identify the cell signaling pathways mediating the observed phenotypic differences. Results: We demonstrate that downregulated genes in AFR patients with CRC showed enrichment for canonical pathways, including chemokine signaling. Moreover, evaluation of the tumor microenvironment showed that cytotoxic lymphocytes and neutrophil cell populations are significantly decreased in AFR compared with EUR patients, suggesting AFR patients have an attenuated immune response. We further demonstrate that molecules called "master transcriptional regulators" (MTRs) play a critical role in regulating the expression of genes impacting key immune processes through an intricate signal transduction network mediated by disease-associated transcription factors (TFs). Furthermore, a core set of these MTRs and TFs showed a positive correlation with levels of cytotoxic lymphocytes and neutrophils across both AFR and EUR patients with CRC, thus suggesting their role in driving the immune infiltrate differences between the two ancestral groups. Conclusion: Our study provides an insight into the intricate regulatory landscape of MTRs and TFs that orchestrate the differences in the tumor microenvironment between patients with CRC of AFR and EUR ancestry.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding.

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.