Accessible high-throughput single-cell whole-genome sequencing with paired chromatin accessibility.

Single-cell whole-genome sequencing (scWGS) enables the assessment of genome-level molecular differences between individual cells with particular relevance to genetically diverse systems like solid tumors. The application of scWGS was limited due to a dearth of accessible platforms capable of producing high-throughput profiles. We present a technique that leverages nucleosome disruption methodologies with the widely adopted 10× Genomics ATAC-seq workflow to produce scWGS profiles for high-throughput copy-number analysis without new equipment or custom reagents. We further demonstrate the use of commercially available indexed transposase complexes from ScaleBio for sample multiplexing, reducing the per-sample preparation costs. Finally, we demonstrate that sequential indexed tagmentation with an intervening nucleosome disruption step allows for the generation of both ATAC and WGS data from the same cell, producing comparable data to the unimodal assays. By exclusively utilizing accessible commercial reagents, we anticipate that these scWGS and scWGS+ATAC methods can be broadly adopted by the research community.

Challenges and Approaches to Genotyping Repetitive DNA.

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

Cross-Platform DNA Encoding for Single-Cell Imaging of Gene Expression.

Integration of imaging data across different molecular target types can provide in-depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target-type-specific labeling methods. We show that cross-platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.

Wide variation in antibiotic resistance proteins identified by functional metagenomic screening of a soil DNA library.

Most genes for antibiotic resistance present in soil microbes remain unexplored because most environmental microbes cannot be cultured. Only recently has the identification of these genes become feasible through the use of culture-independent methods. We screened a soil metagenomic DNA library in an Escherichia coli host for genes that can confer resistance to kanamycin, gentamicin, rifampin, trimethoprim, chloramphenicol, or tetracycline. The screen revealed 41 genes that encode novel protein variants of eight protein families, including aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, and transporters. Several proteins of the same protein family deviate considerably from each other yet confer comparable resistance. For example, five dihydrofolate reductases sharing at most 44% amino acid sequence identity in pairwise comparisons were equivalent in conferring trimethoprim resistance. We identified variants of aminoglycoside acetyltransferases and transporters that differ in the specificity of the drugs for which they confer resistance. We also found wide variation in protein structure. Two forms of rifampin ADP-ribosyltransferases, one twice the size of the other, were similarly effective at conferring rifampin resistance, although the short form was expressed at a much lower level. Functional metagenomic screening provides insight into the large variability in antibiotic resistance protein sequences, revealing divergent variants that preserve protein function.