Search

BackgroundSingle-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 are available and have been used in many scRNA-seq studies. On the other hand, while several methods have been published, the 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.ResultsWe performed multiplexing snATAC-seq analyses on the mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The demultiplexing accuracy of NuHash was high, and only ten out of 9,144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. We compared results between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples and found that most of the peaks detected in snATAC-seq were also detected in deeply sequenced bulk ATAC-seq. The bulk ATAC-seq signal intensity was positively correlated with the number of cell subtype clusters detected in snATAC-seq, but not the subset of peaks detected in all clusters. These subsets of snATAC-seq peaks showed different distributions over different genomic features, suggesting that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci.ConclusionsOur multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high accuracy demultiplexing of samples. The NuHash protocol is straightforward, it works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Background: African Americans with colorectal cancer (CRC) have significantly worse outcomes than Caucasians with higher incidence, later stage at presentation, and worse overall survival. Even when controlling for socioeconomic status and stage at presentation, African Americans compared to Caucasians have a poorer response to medical therapy. We hypothesized that African Americans with colorectal cancer have a distinct genomic mutational profile that explains the observed differences in the natural history and response to treatment. Furthermore, self-reported data on race are imprecise, especially in individuals of mixed race as often found in under-represented communities. Methods: Using unpublished data from The Cancer Genome Atlas (TCGA), we utilized genomic data to accurately capture a person's ancestry (i.e., European, African, Asian). For each patient with CRC, we used a microarray of single-nucleotide polymorphisms (SNPs) and matched their genome with the publicly available 1000 Genomes project, which is a well-characterized database of the major world populations. Each individual's self-reported race was reclassified using a microarray of their SNPs using Ancestry Informative Markers with Principal Component Analysis. After genotypically defining each individual's ancestry, we studied differences in their somatic mutational profiles. To determine somatic mutational variations, we utilized MuTect2 software to study whole-exome sequencing data of the tumor and normal tissue. The functional impact of each mutation was then classified as high or low. Somatic mutation results were separated based on mutation type (e.g., single-nucleotide variants or insertions/deletions). Mutation-specific tables were separated by genotypically defined race. Permutation analysis was performed using 1000 random samplings to determine whether the observed gene frequency for each race was statistically significant (p Results: 633 patients with colorectal cancer were analyzed and their baseline characteristics were: females (62%), self-reported African Americans (10%) and Caucasians (47%), stage III/IV (35%) and proximal CRC (50%). Preliminary data reveal unique somatic mutations that were distinct in individuals from African ancestry with specific genes playing key roles in oncogenesis. Additional data, including genotypically defined ancestry and somatic mutational differences, will be reported at the time of the conference. Conclusions: To our knowledge, this is the first application of ancestry informative markers using whole-exome sequencing to study health disparities in colorectal cancer. Differences in tumor mutational profiles may help explain disparities in outcomes between individuals of European and African ancestry. Citation Format: Taylor Thompson, John Greally, Parvathi Myer. Health disparities and the molecular genomics of colorectal cancer by ancestry: The translational impact on colorectal cancer screening, surveillance, and treatment [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B060.

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., Functional variants have been proposed to alter transcription factor binding. Here, the authors provide direct evidence that functional variants within the TBC1D4 gene, encoding an NFκB binding site, can alter transcription factor binding, and use CRISPR-Cas9 to reveal localization of the transcription factor to be the regulator of chromatin accessibility and p65 binding and ultimately TBC1D4 expression.