special feature | PRIMER

Single-cell sequencing A brief overview of how to derive a genome or transcriptome from a single cell.

Genome and transcriptome sequencing require orders of magnitude PCR’, and pooling barcoded cDNA from different samples to provide more starting material than what is found in an individual cell, pushing enough starting material for linear amplification by in vitro transcrip- the limits of amplification technology. Handling such small quantities tion. Unique molecular identifier sequences can also be used to label means that degradation, sample loss and contamination can have a individual RNA molecules, allowing the absolute number of origi- pronounced effect on sequence quality and robustness. Heavy ampli- nal molecules to be counted directly even after subsequent (uneven) fication also propagates errors and biases, which can lead to uneven amplification. coverage, noise and inaccurate quantification. Recent technical advances have helped mitigate these challenges, Single genomes making single-cell sequencing an appealing way to address an expand- Whole-genome amplification starts with a tiny amount of material: ing set of problems. Rare cell types, heterogeneous samples, pheno- just a single molecule of DNA. This makes uneven coverage and pref- types associated with mosaicism or erential amplification of one allele— variability, and microbes that cannot Dissociate or loss, known as allelic dropout—a be cultured are good candidates for widespread problem. The most com- single-cell approaches. Single-cell mon approach is multiple displace- sequencing can enable the discovery Solid tissue Cell suspension ment amplification, which uses ran- of clonal mutations, cryptic cell types dom primers that bind throughout Cell sort or enrich or transcriptional features that would Micromanipulate the genome and a polymerase that be diluted or averaged out in bulk tis- Tube- or - can displace other fragments on the sue. based steps same template that it copies, forming Captured single cell iterative branching structures that Picking the right cell massively amplify DNA. Early cycles Micromanipulation is a precise but have a strong effect on the uniformity laborious way to target a single cell, of amplification. One variation uses and microcapillaries can be used to DNA RNA special primers that cause amplicons RT and extract a cell’s contents directly. Many Preamplify second-strand to form closed loops and prevent fur- tissues can be dissociated to produce PCR synthesis ther copying, allowing a few cycles cell suspensions, which are easier to or of linear amplification prior to PCR. Looped MDA handle and allow cells expressing amplicons IVT Scaling up and monitoring reactions specific markers to be enriched with a in real time can help to overcome cDNA PCR

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature cell sorter. Instruments that trap very low success rates in genome ampli- Amplified RNA rare cells on the basis of their surface PCR fication, and lower-input sequencing Amplified DNA markers are also being used to isolate library preparations that rely on less tumor cells that circulate in blood. Amplified cDNA amplification are another promising npg Prepare sequencing library direction. Single transcriptomes Many single-cell RNA sequencing Sequence One cell for all protocols are now available, though Genomic sequence, Gene or transcript Scaling up is important to ensure that every variation begins with the con- allele copy number abundance biological variability is well sampled Marina Corral Spence version of RNA to the first strand of in single-cell studies. Microfluidics or complementary DNA (cDNA) by Workflows for amplifying and sequencing the RNA or genomic microwell technologies provide high- . Some methods DNA of a cell. MDA, multiple displacement amplification; RT, er throughput and standardized han- reverse ; IVT, in vitro transcription. sequence full transcripts and others dling, and are often efficient because sequence tags only at the 5ʹ or 3ʹ end. reactions are concentrated in small The common goal of these methods is to capture the original RNA volumes; however, microfluidics can be restricted to certain cell size population and amplify it evenly and accurately. Capture efficiency ranges. Barcoding and pooling will also help to increase throughput. is influenced by how comprehensively reverse transcriptase samples Technologies for single-cell amplification and sequencing RNA from the cell—a stochastic process that can be improved using are maturing. As the cost and ease of examining individual cells small reaction volumes and, potentially, better enzymes. In addition, improves, the approach will enter the hands of more researchers as a a technique known as template switching may ensure that a greater standard tool for understanding biology at high resolution. proportion of captured transcripts are full length. Tal Nawy Amplification can also be improved by minimizing the number ACKNOWLEDGMENTS of cycles, inhibiting primer byproduct amplification by ‘suppression We thank J. Levin and K. Zhang for help preparing this Primer.

18 | VOL.11 NO.1 | JANUARY 2014 | nature methods