Targeting individual cells by barcode in pooled sequence libraries.

Navpreet Ranu,Nir Hacohen,Paul C Blainey,Alexandra-Chloé Villani

doi:10.1093/nar/gky856

Abstract

Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.

Highlights

Intensive interest exists in applying single-cell genomic analyses including gene expression, chromatin accessibility, and DNA copy number variation to resolve differences between cells in a population
In instances where rare cells are of interest, investigators must cope with applying extremely high sequencing effort or the sample loss and perturbation associated with enrichment by fluorescence-activated cell sorting (FACS), which limits the types of samples that can be processed [15]
Cells that initially lack sequence coverage can be targeted for deeper follow-up sequencing and rare cell populations too small in quantity or too sensitive to perturbation for pre-selection by FACS can be enriched from the original pooled sequence library

Summary

Introduction

Intensive interest exists in applying single-cell genomic analyses including gene expression, chromatin accessibility, and DNA copy number variation to resolve differences between cells in a population. Pooled analysis of thousands of single cells is routinely practiced by introducing cell-specific DNA barcodes early in cell processing protocols to produce a pooled library that is sequenced as a single sample and deconvoluted in silico. While such pooled experimental workflows are a mainstream approach in life science research including cell atlasing efforts [1,2,3,4,5,6,7,8], these workflows do not currently enable cell targeting, even in cases when only a few rare cells are of interest [9,10,11]. The PCR enrichment approach can be combined with complementary enrichment approaches like FACS to target ultra-rare cell types

Methods

Results

Conclusion