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Applications of RNA-Seq
What is RNA-Seq?

Regulation of gene expression is fundamental to link genotypes with phenotypes. RNAs shape
complex gene expression networks which drive biological processes. An in-depth understanding of the
underlying mechanisms about how to govern these complex gene expression networks is vital for the
treatment of complex disease such as cancer. Hybridization-based microarrays are used to allow the
simultaneous monitoring of expression levels of annotated genes in cell populations. However,
genome-wide approaches are proved to provide more valuable insights into transcriptomes. These
next/third sequencing platforms allow the rapid and cost-effective generation of massive amounts of
sequence data. The RNA profiling by utilizing high-throughput sequencing technologies are known as
RNA-seq.

What are the applications of RNA-Seq?

Since RNA-seq is quantitative, it is useful to determine RNA expression levels. In addition to this basic
function, RNA-seq can be used for differential gene expression, variants detection and allele-specific
expression, small RNA profiling, characterization of alternative splicing patterns, system biology, and
single-cell RNA-seq.
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Figure 1. Overview of the typical RNA-seq analysis pipeline (Han et al. 2015).

⚫ Differential gene expression

An important application of RNA-seq is the comparison of transcriptomes across different


developmental stages, treatments, or disease conditions. This analysis, also known as differential
gene expression analysis, requires identification of genes along their isoforms and precise assessment
of their expression levels. It is important to illustrate functional elements of the genome and uncover
the biological mechanisms of development and disease.

The common tools for differential gene expression include Cuffdiff, DESeq, DESeq2, EdgeR,
PoissonSeq, Limma voom, and MISO.
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⚫ Variants detection and allele-specific expression

RNA-seq allows identification of variants and allele-specific expression. Single-nucleotide


polymorphisms (SNPs) refer to the variation in a single nucleotide that occurs at a specific position in
the genome, which may lead to allele-specific expression (ASE). ASE means that one of two alleles is
highly transcribed into mRNA and the other is lowly transcribed or even not transcribed at all. Recent
studies have also associated ASE to the susceptibility of a number of human diseases. RNA-seq and
whole-genome DNA sequencing (WGS) allow identification of common disease variants, including
SNPs and ASE.

The common tools used for variants detection are GATK, ANNOVAR, SNPiR, SNiPlay3.

⚫ Small RNA profiling

Small RNA species generally involve microRNA (miRNA), small interfering RNA (siRNA), and piwi-
interacting RNA (piRNA), as well as other types of small RNA, such as small nucleolar RNA (snoRNA)
and small nuclear RNA (snRNA). Small RNAs play a role in gene silencing and post-transcriptional
regulation of gene expression. Small RNAs have been demonstrated to be involved in biological
processes, including development, cell proliferation and differentiation, and apoptosis. Most initial
small RNA discovery studies used pyrosequencing, and subsequently, other NGS platforms with
higher throughput, which resulted in genome-wide surveys and the discovery of an increasing number
of small RNA species. Common bioinformatic tools for small RNA sequencing data are shown in Table
1.

Table 1. sRNA-seq web application comparison (Rahman et al. 2018).

Features Oasis 2 omiRas mirTools 2.0 MAGI Chimira sRNAtoolbox

FASTQ compression √ √ √

miRNA modifications and √ √ √ √ √


edits

Novel miRNA database √ √

Infection and cross- √


species analysis

Non-model organism √ √

Differential expression √ √ √ √ √ √

Multivariate differential √ √
expression

Classification √

Novel miRNA target √ √ √


prediction

Pathway/GO analysis √ √ √ √ √
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Batch job submission √


(API)

Genome browser √

⚫ Characterization of alternative splicing patterns

Alternative splicing patterns are important to understand development and human diseases since
altered splicing patterns contribute to development, cell differentiation, and human disease. RNA-seq
is a powerful tool for characterization of alternative splicing patterns. Paired-end sequencing enables
sequence information from both ends, thereby detecting splicing patterns without a requirement for
previous knowledge of transcript annotations. PacBio SMRT sequencing allows examination of splicing
patterns and transcript connectivity in an unbiased and genome-scale manner by generating full-length
transcript sequences.

The common tools for characterization of alternative splicing patterns include TopHat, MapSplice,
SpliceMap, SplitSeek, GEM mapper, SpliceR, SplicingCompass, GIMMPS, MATS, and rMATS.
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Figure 2. RNA-seq for detection of alternative splicing events (Ozsolak and Milos 2011).

⚫ System biology

Creating lists of differential expression (DE) genes is not the final step of RNA-seq analysis. Further
biological insight into an experimental system can be acquired by looking at the expression changes of
sets of genes. This process, known as system biology, is based on the understanding that the whole is
greater than the sum of the parts. Pathway analysis and co-expression network analysis are two
important included parts.
Table 2. The tools for pathway analysis and co-expression network analysis using RNA-seq data.

GSEA A knowledge-based approach for genome-wide expression profiling.


Pathway
analysis GSVA A non-parametric, unsupervised method for estimating variation of
gene set enrichment through the samples of an expression data set.
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SeqGSEA Provides methods for gene set enrichment analysis by integrating


differential expression and splicing.

GAGE An evaluation of the very latest large-scale genome assembly


algorithms.

SPIA Identifies the pathways most relevant to the condition

TAPPA A java-based tool for identification of phenotype-associated genetic


pathways.

DEAP Identifies important regulatory patterns from differential expression


data.

GSAASeqSP Can identify pathways or gene sets significantly associated with a


disease or phenotype.

GSCA help researchers make discoveries by using massive amounts of


publicly available gene expression data.

Co-expression DICER Detects differentially co-expressed gene sets by using a novel


network probabilistic score for differential correlation.

WGCNA A powerful method to isolate co-expressed groups of genes from


microarray or RNA-seq data.

⚫ Single-cell RNA-seq

The single-cell RNA-seq offers opportunities to dissect of the interplay between intrinsic cellular
processes and extrinsic stimuli in cell fate determination. It also contributes to a better understanding
of how an ‘outlier cell’ may determine the outcome of an infection. In addition, a majority of living cells
cannot be cultivated in vitro, single-cell RNA-seq may discover novel species or regulatory processes
of biotechnological or medical relevance. The workflow of single-cell RNA-seq generally involves the
following steps: single-cell isolation, cDNA library construction, RNA-seq, and bioinformatics (Figure 2).
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Figure 3. The general workflow of single-cell RNA-seq.

Applications of single-cell RNA-seq

➢ Stem cell differentiation

➢ Embryogenesis

➢ Whole-tissue analysis

➢ Single-cell RNA-seq for whole-organism studies

➢ Disease biology and treatment

References:

1. Ozsolak F, Milos P M. RNA sequencing: advances, challenges and opportunities. Nature reviews
genetics, 2011, 12(2): 87.

2. Rahman R U, Gautam A, Bethune J, et al. Oasis 2: improved online analysis of small RNA-seq data.
BMC bioinformatics, 2018, 19(1): 54.

3. Han Y, Gao S, Muegge K, et al. Advanced applications of RNA sequencing and challenges.
Bioinformatics and biology insights, 2015, 9: BBI. S28991.

4. Saliba A E, Westermann A J, Gorski S A, et al. Single-cell RNA-seq: advances and future


challenges. Nucleic acids research, 2014, 42(14): 8845-8860.