RNA-Seq
RNA-Seq is a particular technology-based sequencing technique which uses next-generation sequencing to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing cellular transcriptome.
Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing and in situ sequencing of fixed tissue.
Prior to RNA-Seq, gene expression studies were done with hybridization-based microarrays. Issues with microarrays include cross-hybridization artifacts, poor quantification of lowly and highly expressed genes, and needing to know the sequence a priori. Because of these technical issues, transcriptomics transitioned to sequencing-based methods. These progressed from Sanger sequencing of Expressed Sequence Tag libraries, to chemical tag-based methods, and finally to the current technology, next-gen sequencing of cDNA.
Methods
Library preparation
The general steps to prepare a complementary DNA library for sequencing are described below, but often vary between platforms.- RNA Isolation: RNA is isolated from tissue and mixed with deoxyribonuclease. DNase reduces the amount of genomic DNA. The amount of RNA degradation is checked with gel and capillary electrophoresis and is used to assign an RNA integrity number to the sample. This RNA quality and the total amount of starting RNA are taken into consideration during the subsequent library preparation, sequencing, and analysis steps.
- RNA selection/depletion: To analyze signals of interest, the isolated RNA can either be kept as is, filtered for RNA with 3' polyadenylated tails to include only mRNA, depleted of ribosomal RNA, and/or filtered for RNA that binds specific sequences. The RNA with 3' poly tails are mature, processed, coding sequences. Poly selection is performed by mixing RNA with poly oligomers covalently attached to a substrate, typically magnetic beads. Poly selection ignores noncoding RNA and introduces 3' bias, which is avoided with the ribosomal depletion strategy. The rRNA is removed because it represents over 90% of the RNA in a cell, which if kept would drown out other data in the transcriptome.
- cDNA synthesis: RNA is reverse transcribed to cDNA because DNA is more stable and to allow for amplification and leverage more mature DNA sequencing technology. Amplification subsequent to reverse transcription results in loss of strandedness, which can be avoided with chemical labeling or single molecule sequencing. Fragmentation and size selection are performed to purify sequences that are the appropriate length for the sequencing machine. The RNA, cDNA, or both are fragmented with enzymes, sonication, or nebulizers. Fragmentation of the RNA reduces 5' bias of randomly primed-reverse transcription and the influence of primer binding sites, with the downside that the 5' and 3' ends are converted to DNA less efficiently. Fragmentation is followed by size selection, where either small sequences are removed or a tight range of sequence lengths are selected. Because small RNAs like miRNAs are lost, these are analyzed independently. The cDNA for each experiment can be indexed with a hexamer or octamer barcode, so that these experiments can be pooled into a single lane for multiplexed sequencing.
| Strategy | Type of RNA | Ribosomal RNA content | Unprocessed RNA content | Genomic DNA content | Isolation method |
| Total RNA | All | High | High | High | - |
| PolyA selection | Coding | Low | Low | Low | Hybridization with poly oligomers |
| rRNA depletion | Coding, noncoding | Low | High | High | Removal of oligomers complementary to rRNA |
| RNA capture | Targeted | Low | Moderate | Low | Hybridization with probes complementary to desired transcripts |
Small RNA/non-coding RNA sequencing
When sequencing RNA other than mRNA, the library preparation is modified. The cellular RNA is selected based on the desired size range. For small RNA targets, such as miRNA, the RNA is isolated through size selection. This can be performed with a size exclusion gel, through size selection magnetic beads, or with a commercially developed kit. Once isolated, linkers are added to the 3' and 5' end then purified. The final step is cDNA generation through reverse transcription.Direct RNA sequencing
Because converting RNA into cDNA, ligation, amplification, and other sample manipulations have been shown to introduce biases and artifacts that may interfere with both the proper characterization and quantification of transcripts, single molecule direct RNA sequencing has been explored by companies including Helicos, Oxford Nanopore Technologies, and others. This technology sequences RNA molecules directly in a massively-parallel manner.Single-cell RNA sequencing (scRNA-Seq)
Standard methods such as microarrays and standard bulk RNA-Seq analysis analyze the expression of RNAs from large populations of cells. In mixed cell populations, these measurements may obscure critical differences between individual cells within these populations.Single-cell RNA sequencing provides the expression profiles of individual cells. Although it is not possible to obtain complete information on every RNA expressed by each cell, due to the small amount of material available, patterns of gene expression can be identified through gene clustering analyses. This can uncover the existence of rare cell types within a cell population that may never have been seen before. For example, rare specialized cells in the lung called pulmonary ionocytes that express the Cystic Fibrosis Transmembrane Conductance Regulator were identified in 2018 by two groups performing scRNA-Seq on lung airway epithelia.
Experimental procedures
Current scRNA-Seq protocols involve the following steps: isolation of single cell and RNA, reverse transcription, amplification, library generation and sequencing. Early methods separated individual cells into separate wells; more recent methods encapsulate individual cells in droplets in a microfluidic device, where the reverse transcription reaction takes place, converting RNAs to cDNAs. Each droplet carries a DNA "barcode" that uniquely labels the cDNAs derived from a single cell. Once reverse transcription is complete, the cDNAs from many cells can be mixed together for sequencing; transcripts from a particular cell are identified by the unique barcode.Challenges for scRNA-Seq include preserving the initial relative abundance of mRNA in a cell and identifying rare transcripts. The reverse transcription step is critical as the efficiency of the RT reaction determines how much of the cell's RNA population will be eventually analyzed by the sequencer. The processivity of reverse transcriptases and the priming strategies used may affect full-length cDNA production and the generation of libraries biased toward 3’ or 5' end of genes.
In the amplification step, either PCR or in vitro transcription is currently used to amplify cDNA. One of the advantages of PCR-based methods is the ability to generate full-length cDNA. However, different PCR efficiency on particular sequences may also be exponentially amplified, producing libraries with uneven coverage. On the other hand, while libraries generated by IVT can avoid PCR-induced sequence bias, specific sequences may be transcribed inefficiently, thus causing sequence drop-out or generating incomplete sequences.
Several scRNA-Seq protocols have been published:
Tang et al.,
STRT,
SMART-seq,
CEL-seq,
RAGE-seq,
, Quartz-seq.
and C1-CAGE. These protocols differ in terms of strategies for reverse transcription, cDNA synthesis and amplification, and the possibility to accommodate sequence-specific barcodes or the ability to process pooled samples.
In 2017, two approaches were introduced to simultaneously measure single-cell mRNA and protein expression through oligonucleotide-labeled antibodies known as REAP-seq, and CITE-seq.
Applications
scRNA-Seq is becoming widely used across biological disciplines including Development, Neurology, Oncology, Autoimmune disease, and Infectious disease.scRNA-Seq has provided considerable insight into the development of embryos and organisms, including the worm Caenorhabditis elegans, and the regenerative planarian Schmidtea mediterranea. The first vertebrate animals to be mapped in this way were Zebrafish and Xenopus laevis. In each case multiple stages of the embryo were studied, allowing the entire process of development to be mapped on a cell-by-cell basis. Science recognized these advances as the 2018 Breakthrough of the Year.
Experimental considerations
A variety of parameters are considered when designing and conducting RNA-Seq experiments:- Tissue specificity: Gene expression varies within and between tissues, and RNA-Seq measures this mix of cell types. This may make it difficult to isolate the biological mechanism of interest. Single cell sequencing can be used to study each cell individually, mitigating this issue.
- Time dependence: Gene expression changes over time, and RNA-Seq only takes a snapshot. Time course experiments can be performed to observe changes in the transcriptome.
- Coverage : RNA harbors the same mutations observed in DNA, and detection requires deeper coverage. With high enough coverage, RNA-Seq can be used to estimate the expression of each allele. This may provide insight into phenomena such as imprinting or cis-regulatory effects. The depth of sequencing required for specific applications can be extrapolated from a pilot experiment.
- Data generation artifacts : The reagents, personnel involved, and type of sequencer can result in technical artifacts that might be mis-interpreted as meaningful results. As with any scientific experiment, it is prudent to conduct RNA-Seq in a well controlled setting. If this is not possible or the study is a meta-analysis, another solution is to detect technical artifacts by inferring latent variables and subsequently correcting for these variables.
- Data management: A single RNA-Seq experiment in humans is usually on the order of 1 Gb. This large volume of data can pose storage issues. One solution is compressing the data using multi-purpose computational schemas or genomics-specific schemas. The latter can be based on reference sequences or de novo. Another solution is to perform microarray experiments, which may be sufficient for hypothesis-driven work or replication studies.
Analysis
Transcriptome assembly
Two methods are used to assign raw sequence reads to genomic features :- De novo: This approach does not require a reference genome to reconstruct the transcriptome, and is typically used if the genome is unknown, incomplete, or substantially altered compared to the reference. Challenges when using short reads for de novo assembly include 1) determining which reads should be joined together into contiguous sequences robustness to sequencing errors and other artifacts, and 3) computational efficiency. The primary algorithm used for de novo assembly transitioned from overlap graphs, which identify all pair-wise overlaps between reads, to de Bruijn graphs, which break reads into sequences of length k and collapse all k-mers into a hash table. Overlap graphs were used with Sanger sequencing, but do not scale well to the millions of reads generated with RNA-Seq. Examples of assemblers that use de Bruijn graphs are Velvet, Trinity, Oases, and Bridger. Paired end and long read sequencing of the same sample can mitigate the deficits in short read sequencing by serving as a template or skeleton. Metrics to assess the quality of a de novo assembly include median contig length, number of contigs and N50.
- Genome guided: This approach relies on the same methods used for DNA alignment, with the additional complexity of aligning reads that cover non-continuous portions of the reference genome. These non-continuous reads are the result of sequencing spliced transcripts align short portions of the read use dynamic programming to find an optimal alignment, sometimes in combination with known annotations. Software tools that use genome-guided alignment include Bowtie, TopHat de novo assembly metrics comparisons to known transcript, splice junction, genome, and protein sequences using precision, recall, or their combination. In addition, in silico assessment could be performed using simulated reads.
Gene expression quantification
Expression is quantified to study cellular changes in response to external stimuli, differences between healthy and diseased states, and other research questions. Gene expression is often used as a proxy for protein abundance, but these are often not equivalent due to post transcriptional events such as RNA interference and nonsense-mediated decay.Expression is quantified by counting the number of reads that mapped to each locus in the transcriptome assembly step. Expression can be quantified for exons or genes using contigs or reference transcript annotations. These observed RNA-Seq read counts have been robustly validated against older technologies, including expression microarrays and qPCR. Examples of tools that quantify counts are HTSeq, FeatureCounts, Rcount, maxcounts, FIXSEQ, and Cuffquant. The read counts are then converted into appropriate metrics for hypothesis testing, regressions, and other analyses. Parameters for this conversion are:
- Sequencing depth/coverage: Although depth is pre-specified when conducting multiple RNA-Seq experiments, it will still vary widely between experiments. Therefore, the total number of reads generated in a single experiment is typically normalized by converting counts to fragments, reads, or counts per million mapped reads. Sequencing depth is sometimes referred to as library size, the number of intermediary cDNA molecules in the experiment.
- Gene length: Longer genes will have more fragments/reads/counts than shorter genes if transcript expression is the same. This is adjusted by dividing the FPM by the length of a gene, resulting in the metric fragments per kilobase of transcript per million mapped reads. When looking at groups of genes across samples, FPKM is converted to transcripts per million by dividing each FPKM by the sum of FPKMs within a sample.
- Total sample RNA output: Because the same amount of RNA is extracted from each sample, samples with more total RNA will have less RNA per gene. These genes appear to have decreased expression, resulting in false positives in downstream analyses.
- Variance for each gene's expression: is modeled to account for sampling error, increase power, and decrease false positives. Variance can be estimated as a normal, Poisson, or negative binomial distribution and is frequently decomposed into technical and biological variance.
Absolute quantification
Differential expression
The simplest but often most powerful use of RNA-Seq is finding differences in gene expression between two or more conditions ; this process is called differential expression. The outputs are frequently referred to as differentially expressed genes and these genes can either be up- or down-regulated. There are many tools that perform differential expression. Most are run in R, Python, or the Unix command line. Commonly used tools include DESeq, edgeR, and voom+limma, all of which are available through R/Bioconductor. These are the common considerations when performing differential expression:- Inputs: Differential expression inputs include an RNA-Seq expression matrix and a design matrix containing experimental conditions for N samples. The simplest design matrix contains one column, corresponding to labels for the condition being tested. Other covariates can include batch effects, known artifacts, and any metadata that might confound or mediate gene expression. In addition to known covariates, unknown covariates can also be estimated through unsupervised machine learning approaches including principal component, surrogate variable, and PEER analyses. Hidden variable analyses are often employed for human tissue RNA-Seq data, which typically have additional artifacts not captured in the metadata.
- Methods: Most tools use regression or non-parametric statistics to identify differentially expressed genes, and are either count-based or assembly-based. Following regression, most tools employ either familywise error rate or false discovery rate p-value adjustments to account for multiple hypotheses.
- Outputs: A typical output consists of rows corresponding to the number of genes and at least three columns, each gene's log fold change, p-value, and p-value adjusted for multiple comparisons. Genes are defined as biologically meaningful if they pass cut-offs for effect size and statistical significance. These cut-offs should ideally be specified a priori, but the nature of RNA-Seq experiments is often exploratory so it is difficult to predict effect sizes and pertinent cut-offs ahead of time.
- Pitfalls: The raison d'etre for these complex methods is to avoid the myriad of pitfalls that can lead to statistical errors and misleading interpretations. Pitfalls include increased false positive rates, sample preparation artifacts, sample heterogeneity, highly correlated samples, unaccounted for multi-level experimental designs, and poor experimental design. One notable pitfall is viewing results in Microsoft Excel. Although convenient, Excel automatically converts some gene names into dates or floating point numbers.
- Choice of tools and benchmarking: There are numerous efforts that compare the results of these tools, with DESeq2 tending to moderately outperform other methods. As with other methods, benchmarking consists of comparing tool outputs to each other and known gold standards.
Alternative splicing
is integral to eukaryotes and contributes significantly to protein regulation and diversity, occurring in >90% of human genes. There are multiple alternative splicing modes: exon skipping, mutually exclusive exons, alternative donor or acceptor sites, intron retention, alternative transcription start site, and alternative polyadenylation. One goal of RNA-Seq is to identify alternative splicing events and test if they differ between conditions. Long-read sequencing captures the full transcript and thus minimizes many of issues in estimating isoform abundance, like ambiguous read mapping. For short-read RNA-Seq, there are multiple methods to detect alternative splicing that can be classified into three main groups:- Count-based : estimate exon retention. Examples are DEXSeq, MATS, and SeqGSEA.
- Isoform-based : estimate isoform abundance first, and then relative abundance between conditions. Examples are Cufflinks 2 and DiffSplice.
- Intron excision based: calculate alternative splicing using split reads. Examples are MAJIQ and Leafcutter.
Coexpression networks
Coexpression networks are data-derived representations of genes behaving in a similar way across tissues and experimental conditions. Their main purpose lies in hypothesis generation and guilt-by-association approaches for inferring functions of previously unknown genes. RNA-Seq data has been used to infer genes involved in specific pathways based on Pearson correlation, both in plants and mammals. The main advantage of RNA-Seq data in this kind of analysis over the microarray platforms is the capability to cover the entire transcriptome, therefore allowing the possibility to unravel more complete representations of the gene regulatory networks. Differential regulation of the splice isoforms of the same gene can be detected and used to predict and their biological functions.Weighted gene co-expression network analysis has been successfully used to identify co-expression modules and intramodular hub genes based on RNA seq data. Co-expression modules may correspond to cell types or pathways. Highly connected intramodular hubs can be interpreted as representatives of their respective module. An eigengene is a weighted sum of expression of all genes in a module. Eigengenes are useful biomarkers for diagnosis and prognosis. Variance-Stabilizing Transformation approaches for estimating correlation coefficients based on RNA seq data have been proposed.
Variant discovery
RNA-Seq captures DNA variation, including single nucleotide variants, small insertions/deletions. and structural variation. Variant calling in RNA-Seq is similar to DNA variant calling and often employs the same tools with adjustments to account for splicing. One unique dimension for RNA variants is allele-specific expression : the variants from only one haplotype might be preferentially expressed due to regulatory effects including imprinting and expression quantitative trait loci, and noncoding rare variants. Limitations of RNA variant identification include that it only reflects expressed regions and has lower quality when compared to direct DNA sequencing.RNA editing (post-transcriptional alterations)
Having the matching genomic and transcriptomic sequences of an individual can help detect post-transcriptional edits. A post-transcriptional modification event is identified if the gene's transcript has an allele/variant not observed in the genomic data.Fusion gene detection
Caused by different structural modifications in the genome, fusion genes have gained attention because of their relationship with cancer. The ability of RNA-Seq to analyze a sample's whole transcriptome in an unbiased fashion makes it an attractive tool to find these kinds of common events in cancer.The idea follows from the process of aligning the short transcriptomic reads to a reference genome. Most of the short reads will fall within one complete exon, and a smaller but still large set would be expected to map to known exon-exon junctions. The remaining unmapped short reads would then be further analyzed to determine whether they match an exon-exon junction where the exons come from different genes. This would be evidence of a possible fusion event, however, because of the length of the reads, this could prove to be very noisy. An alternative approach is to use pair-end reads, when a potentially large number of paired reads would map each end to a different exon, giving better coverage of these events. Nonetheless, the end result consists of multiple and potentially novel combinations of genes providing an ideal starting point for further validation.