A modular; customizable sequencing system for simultaneous genotyping and transcript analysis in single cells


Year of Award:
2019
Status:
Active
Award Type:
R21
Project Number:
CA236594
RFA Number:
RFA-CA-18-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
GARBER, MANUEL
Other PI or Project Leader:
Not Applicable
Institution:
UNIV OF MASSACHUSETTS MED SCH WORCESTER
Project SummarySingle cell sequencing has revolutionized the way in which we define cell types and understand tissues, and hastremendous potential for analyzing the heterogeneity of complex tumors or large perturbation screens. Atpresent, however, this powerful technology has serious limitations, particularly in what cellular informationcan be detected and analyzed. Current high-throughput microfluidics single cell RNA sequencing (scRNA-seq)methods can conveniently process tens of thousands of cells but only capture the extreme 3? ends of the mostabundant transcripts from each cell. The low sensitivity and partial transcript coverage hampers thesemethods? abilities to detect allele-specific or subtle perturbation effects, while also dramatically increasing theper-cell sequencing cost. Moreover, no existing single-cell sequencing method can read DNA genotype andRNA expression from the same cell, which is crucial to studying non-coding regulatory elements and somaticrearrangements -- where the most genetic variation associated with cancer and human disease resides.Here we propose to overcome these limitations by developing a flexible high throughput single-cell sequencingsystem that (1) Can target many different regulatory DNA elements and ?transcripts simultaneously in thesame cell?, (2) Is sensitive enough to measure subtle, allele-specific effects, (3) measures the effects of differentmutations across tens-of-thousands of cells in a single assay, and (4) is easily and rapidly adaptable forapplication to any biological system with a heterogeneous cell population. We iteratively develop thistechnology, ensuring that each step independently creates new capabilities that address current scRNA-Seqlimitations and enables allele specific expression analysis and perturbation screens of non-coding elements.First?, we will modify the the inDrop bead manufacturing process to make it flexible and rapidly customizableso that one large batch of universal barcoded beads can be conveniently adapted to target many specifictranscript pools, SNP-containing portions of transcripts, and even genomic DNA, in just 8 hours.Second?, we test the sensitivity and allele-specificity of our new method in a predictably heterogeneous system:random X inactivation in hybrid female (XX) cells. Using single-molecule RNA-FISH as a gold standard, wewill measure the sensitivity, specificity, and efficiency of our targeted scRNA-Seq approach.Third?, we enable simultaneous DNA genotyping and transcript quantification by adapting our custom beadsand reaction conditions for isothermal amplification of genomic DNA loci, simultaneous with RT of targetedtranscripts in the same cells.Fourth?, we will combine our approaches above in a proof-of-principle application to characterize enhancerfunction using a CRISPR mutagenesis screen. CRISPR mutagenesis randomly creates different alleles in eachcell. We then use targeted sequencing of neighboring genes and DNA genotyping to evaluate the effect of eachallele on its target(s) in ?cis?.