Single-cell RNA-sequencing: The future of genome biology is now

Abstract

Genome-wide single-cell analysis represents the ultimate frontier of genomics research. In particular, single-cell RNA-sequencing (scRNA-seq) studies have been boosted in the last few years by an explosion of new technologies enabling the study of the transcriptomic landscape of thousands of single cells in complex multicellular organisms. More sensitive and automated methods are being continuously developed and promise to deliver better data quality and higher throughput with less hands-on time. The outstanding amount of knowledge that is going to be gained from present and future studies will have a profound impact in many aspects of our society, from the introduction of truly tailored cancer treatments, to a better understanding of antibiotic resistance and host-pathogen interactions; from the discovery of the mechanisms regulating stem cell differentiation to the characterization of the early event of human embryogenesis.

Keywords: CEL-seq; CytoSeq; Drop-seq; MARS-seq; Nanopore; RNA-sequencing; SMART-seq2; STRT/C1; Tn5 transposase; single-cell.

Figure 1.

(see previous page) (A)Tang method. Polyadenylated mRNA is reverse transcribed from an oligo-dT primer carrying the Universal Primer 1 (UP1) sequence at its 5´-end. After RT a poly(A) tail is added to the 3´-end of the first strand cDNA by using a terminal deoxynucleotidyl transferase. The second cDNA strand is synthesized by using a complementary poly(T) primer carrying the Universal Primer 2 (UP2) sequence at its 5´-end. Double-stranded cDNA is then amplified via PCR using complementary primers to the UP1 and UP2 sequences. After shearing and adaptor ligation the fragments undergo a second PCR that allows the introduction of SOLiD-compatible sequences (SOLiD P1 and SOLiD P2). (B) Smart-seq2. Polyadenylated mRNA is reverse transcribed from an oligo-dT primer (SMARTdT₃₀VN). When the reverse transcriptase reaches the end of the RNA template, 2–5 cytosines are added to the newly synthesized cDNA (3 in the figure). The reaction mix also contains a TS oligonucleotide (TSO-LNA) carrying 2 riboguanosines (rG) and a LNA-modified guanosine (+G) at its 3´-end. After annealing of the 3 terminal nucleotides of the LNA-TSO with the 3 cytosines, the reverse transcriptase synthesizes a cDNA strand using the LNA-TSO as template (red arrow). The cDNA is then amplified via PCR using only one primer (ISPCR), since both the SMARTdT₃₀VN and LNA-TSO oligonucleotides share the same sequence at their 5´-end (here in orange). The amplified cDNA is then fragmented by tagmentation using the Tn5 transposase. Simultaneously, the Tn5 ligates different 5′ and 3′ primers to the fragments (red and blue sequences). Another round of PCR introduces Illumina-compatible sequences (Illumina P5 and P7) as well as index sequences (i5 and i7 indices) to allow sample multiplexing. (C) CEL-seq. Polyadenylated mRNA is reverse transcribed from an oligo-dT primer containing the T7 promoter, the Illumina P1 adaptor and a cell barcode. In MARS-seq and CEL-seq2 the sequencing primer also accommodates a UMI downstream of the cell barcode. After RT and second-strand synthesis the cDNA from all the cells is pooled and amplified by IVT from the T7 promoter to generate aRNA. The Illumina P2 adaptor is ligated after heat fragmentation, followed by generation of double-stranded DNA and sequencing of the 3´-terminal fragments. (D) STRT/C1. Polyadenylated mRNA is reverse transcribed from a biotinylated oligo-dT primer (C1-P1-T₃₁) containing the Illumina P1 adaptor (here in yellow) and a PvuI restriction site. The TS reaction occurs in a similar way as for Smart-seq2 but the TSO used in STRT/C1 is different. The TSO is biotinylated and contains the Illumina P1 adaptor, a UMI and 3 riboguanosines at the 3´-end. After TS the cDNA is amplified via PCR using a single primer (C1-P1-PCR) as in the Smart-seq2 method. Fragmentation and ligation of the Illumina P2 adaptor and the cell barcode are performed simultaneously utilizing in-house Tn5 transposase. After pooling all the samples, streptavidin-coated magnetic beads are used to collect only the biotinylated fragments (5´- and 3´-ends of the transcripts). The 3′-ends are then digested by the PvuI restriction enzyme and only the 5´-ends are used for sequencing.

Figure 2.

(A) Structure of the transposon Tn5. In the transposon Tn5 2 near-identical Insertion Sequences (IS50L and IS50R, where “L” and “R” stand for “Left” and “Right,” respectively) bracket 3 antibiotic resistance genes [kan (kanamycin resistance), ble (bleomycin resistance) and str (streptomycin resistance)]. Each IS50 sequence contains 2 inverted 19-bp End Sequences (ESs), an Outside End (OE) and an Inside End (IE). IS50R encodes the functional transposase protein (Tnp) as well as an inhibitor of transposition (Inh). Wild-type ESs have a limited utility due their relatively low activity and were therefore replaced in vitro by hyperactive Mosaic End (ME) sequences which is, as the name indicates, contains elements from both the ES. Adapted and modified from ref. 45. (B) Tn5 transposase-mediated library preparation. Each monomer of Tn5 transposase contains one of 2 partly double-stranded oligonucleotides (here indicated in cyan and red). The double-stranded portion of each oligonucleotide is the hyperactive ME sequence necessary for transposition and is always 19 bp long. The gray-color bar is a Connecting Sequence (CS), which in the Nextera applications is 14 bp long. In the presence of magnesium chloride 2 Tn5 transposase monomers dimerize and become capable of cutting double-stranded DNA in a near-to-random fashion. The ME sequences are then appended to the DNA in a 5-minutes reaction, creating a 9 bp gap in the non-transferred strand. The gap is later filled by a DNA Polymerase. All the fragments carrying different adaptors are amplified by an “enrichment PCR” that also introduces a 8 bp index sequence (for multiplexing purposes) as well as the Illumina P5 and P7 adaptors (necessary for sequencing).