Mostly natural sequencing-by-synthesis for scRNA-seq using Ultima sequencing

Abstract

Here we introduce a mostly natural sequencing-by-synthesis (mnSBS) method for single-cell RNA sequencing (scRNA-seq), adapted to the Ultima genomics platform, and systematically benchmark it against current scRNA-seq technology. mnSBS uses mostly natural, unmodified nucleotides and only a low fraction of fluorescently labeled nucleotides, which allows for high polymerase processivity and lower costs. We demonstrate successful application in four scRNA-seq case studies of different technical and biological types, including 5′ and 3′ scRNA-seq, human peripheral blood mononuclear cells from a single individual and in multiplex, as well as Perturb-Seq. Benchmarking shows that results from mnSBS-based scRNA-seq are very similar to those using Illumina sequencing, with minor differences in results related to the position of reads relative to annotated gene boundaries, owing to single-end reads of Ultima being closer to gene ends than reads from Illumina. The method is thus compatible with state-of-the-art scRNA-seq libraries independent of the sequencing technology. We expect mnSBS to be of particular utility for cost-effective large-scale scRNA-seq projects.

Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring

“…Traditional sequencing by synthesis technologies add all 4 bases (A,T,C,G) per sequencing cycle and determine the template nucleotide based on the fluorescence of the complemented base (one base pair is measured per sequencing cycle). However, Ultima’s flow-based technology uses a mix of fluorescently-labelled and unlabeled nucleotides (all nucleotides added in one flow are either A, T, C or G) and relies on the intensity of fluorescence to assign the number of consecutive identical bases that complement the template 32 . Therefore, Ultima technologies may be less susceptible to single-nucleotide substitution errors, given that only a single base type is added per cycle, at the cost of increased difficulty in gauging the size of homopolymers (typically, homopolymers greater than 11 base pairs 31 ).…”

“…Ultima Genomics has recently debuted a mostly natural sequencing-by-synthesis platform that generates low-cost (1USD/Gb) whole genome sequencing 31,32 . To date, Ultima sequencing has been benchmarked in germline variant calling 31 and single-cell RNA sequencing 32 .…”

“…Ultima Genomics has recently debuted a mostly natural sequencing-by-synthesis platform that generates low-cost (1USD/Gb) whole genome sequencing 31,32 . To date, Ultima sequencing has been benchmarked in germline variant calling 31 and single-cell RNA sequencing 32 . While these data are informative for these specific experimental contexts, several important technical performance parameters in the context of deep ccfDNA WGS remained unknown.…”

“…The Ultima platform produces single-end reads at ∼10 billion reads per run for 1USD/Gb, thus substantially lowering sequencing costs compared with current platforms. This cost efficiency holds great promise for many genomics applications, and this approach has now been applied to Genome-In-A-Bottle and 1000 Genomes reference samples 31 and adapted for single-cell RNA-seq studies 32 . However, to date mnSBS/Ultima sequencing has not been harnessed for application to clinical ccfDNA samples for ctDNA sequencing.…”

Impact of the Human Cell Atlas on medicine

“…Much of this progress has been driven by the rise of experimental technologies (Table 1) and computational algorithms that are applicable in studies at all stages of biomedicine, from understanding mechanisms to diagnosing and treating disease. As technological advances in sequencing, cell manipulation and spatial profiling are rapidly growing in scale and resolution (and dropping in cost) 136,137 , they enable the collection of diverse reference atlases across genders, age, ancestry and demographics that are needed for clinical work. They also enable the sort of large-scale sampling within and across human patients that is required to understand and monitor disease, as well as screening experiments that are crucial to drug discovery.…”