RNA sequencing: advances, challenges and opportunities

Abstract

In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.

Figure 1. RNA-seq for detection of alternative splicing events

a | Sequence reads are mapped to genomic DNA or to a transcriptome reference to detect alternative isoforms of an RNA transcript. Mapping is based simply on read counts to each exon and reads that span the exonic boundaries. One infers the absence of the genomic exon in the transcript by virtue of no reads mapping to the genomic location. b | Paired sequence reads provide additional information about exonic splicing events, as demonstrated by matching the first read in one exon and placing the second read in the downstream exon, creating a map of the transcript structure.

Figure 2. Use of RNA-seq for BCR–ABL fusion gene detection

a | Breakpoint cluster region (BCR) and ABL1 gene transcripts. b | BCR–ABL fusion gene transcript. c | Sequence reads mapping across the BCR–ABL fusion gene site demonstrating the ability to accurately identify the site of gene fusion. The data were derived from RNA-seq analysis of the K562 transcriptome using the HeliScope (the raw data files are available at the University of California Santa Cruz Genome Browser and the Helicos Technology Center).

Figure 3. Alternative methods for targeted RNA-seq

a | Using poly(A)⁺ RNA converted to double-stranded cDNA the Agilent SureSelect method uses RNA probes to enrich selected cDNA. b | A custom NimbleGen array may allow selection of cDNAs of interest. c | The generation of DNA molecules with sequence-specific complementary targeting sites allows the targeting of cDNAs,. d | Helicos sequencing surfaces containing target-specific oligonucleotides can be used to select desired RNA, DNA and cDNA species and sequence regions of interest in a single step. nt, nucleotide.

Figure 4. Direct RNA sequencing using the Helicos approach

a | RNA that is polyadenylated and 3′ deoxy-blocked with poly(A) polymerase is captured on poly(dT)-coated surfaces. A ‘fill-and-lock’ step is performed, in which the ‘fill’ step is performed with natural thymidine and polymerase, and the ‘lock’ step is performed with fluorescently labelled A, C and G Virtual Terminator (VT) nucleotides and polymerase. This step corrects for any misalignments that may be present in poly(A) and poly(T) duplexes, and ensures that the sequencing starts in the RNA template rather than the polyadenylated tail. b | Imaging is performed to locate the positions of the templates. Then, chemical cleavage of the dye–nucleotide linker is performed to release the dye and prepare the templates for nucleotide incorporation. c | Incubation of this surface with one labelled nucleotide (C-VT is shown as an example) and a polymerase mixture is carried out. After this step, imaging is performed to locate the templates that have incorporated the nucleotide. Chemical cleavage of the dye allows the surface and DNA templates to be ready for the next nucleotide-addition cycle. Nucleotides are added in the C, T, A, G order for 120 total cycles (30 additions of each nucleotide).

Figure 5. Emerging technologies for single-cell or low-quantity-cell gene expression profiling

a | Single-molecule DNA and RNA sequencing technologies could be modified for single-cell applications. Cells can be delivered to flow cells using fluidics systems, followed by cell lysis and capture of mRNA species on the poly(dT)-coated sequencing surfaces by hybridization. Standard sequencing runs could take place on channels with a 127.5 mm² surface area, requiring 2,750 images to be taken per cycle to image the entire channel area. The surface area needed to accommodate ~350,000 mRNA molecules contained in a single cell is ~0.4 mm²; thus, only eight images per cycle would be needed. Sequence analysis can be done with direct RNA sequencing (DRS) or on-surface cDNA synthesis followed by single-molecule DNA sequencing. b | Counter system workflow. Two probes are used for each target site: the capture probe (shown in red) contains a target-specific sequence and a modification that allows the immobilization of the molecules on a surface; the reporter probe contains a different target-specific sequence (shown in blue) and a fluorescent barcode (shown by a green circle) that is unique to each target being examined. After hybridization of the capture and reporter probe mixture to RNA samples in solution, excess probes are removed. The hybridized RNA duplexes are then immobilized on a surface and imaged to identify and count each transcript with the unique fluorescent signals on the capture and reporter probes.