System and Methods for Massively Parallel Analysis of Nucleic Acids in Single Cells

Abstract

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 459,600, filed Dec. 16, 2010, which is hereby incorporated in its entirety by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention relates to the fields of molecular biology and molecular diagnostics, and more specifically to methods for massively parallel genetic analysis of nucleic acids in single cells.[0004]2. Description of the Related Art[0005]Certain quantitative genetic analyses of biological tissues and organisms are best performed at the single cell level. However, single cells only contain picograms of genetic material. Conventional methods, such as polymerase chain reaction (PCR), RNA sequencing (Mortazavi et al., 2008 Nature Methods 5:621-8), chromatin immunoprecipitation sequencing (Johnson et al., 2007 Science 316:1497-502), or whole genome sequencing (Lander et al., 2001 Nature 409:860-921), require more genetic ma...

AI Technical Summary

Benefits of technology

The patent describes a method for analyzing nucleic acid sequences in single cells. The method involves using multiple probes to amplify specific sequences and create fused complexes that can be detected in a population of cells. The fused complexes can be generated by co-localizing two target sequences with two probes and a third probe, or by amplifying the first and second target sequences separately and then combining them using a fourth probe. The method can also involve performing a bulk sequencing reaction to generate sequence information for at least 100,000 fused complexes from at least 10,000 cells. The nucleic acid sequences can include rare gene sequences, which can be present in fewer than 5% of cells or even less than 1% of cells. The method can be used to analyze cells in emulsion microdroplets or in reaction containers.

Problems solved by technology

These techniques provide useful genetic information at the cell population level, but have serious limitations for understanding biology at the single cell level.

Current biological tools also lack the capacity to assay genetic measurements in many single cells in parallel.

Conventional single cell techniques are slow, tedious, and limited in the quantity of cells that can be analyzed at once.

Applications such as PGD require time-consuming, hand-guided biopsy technology, and the largest studies include hundreds of single cells.

In another example, genetic recombination between loci of interest can be measured in single sperm cells (Jiang et al., 2005 Nucleic Acids Research 33:e91), but a manual analysis of thousands of single sperm would be time-consuming and impractical.

However, due to the physical limits of parallelization using reaction wells, FACS is only useful for analyzing hundreds of single cells, rather than hundreds of thousands of single cells.

These single cell assays are limited to single cell PCR in emulsions, or in situ PCR in single fixed and permeabilized cells.

Moreover, when analyzing large populations of cells, it is difficult to trace back each gene product to a single cell or subpopulations of cells.

Images