45-1 Ramsey Road

Shirley, NY 11967
USA
Email: contact@cd-genomics.com

Bioinformatics Workflow of RNA-Seq

RNA-sequencing (RNA-seq) has a wide range of applications, and there is no optimal pipeline for all
cases. We review all of the major steps in RNA-seq data analysis, including quality control, read
alignment, quantification of gene and transcript levels, differential gene expression, functional profiling,
and advanced analysis. They will be discussed later.

Figure 1. The general workflow of RNA-seq analysis.

Quality control of raw reads

Quality control of RNA-seq raw reads consists of analysis of sequence quality, GC content, adaptor
content, overrepresented k-mers, and duplicated reads, dedicated to detecting sequencing errors,
contaminations, and PCR artifacts. Read quality decreases towards the 3’ end of reads, bases with low
quality, therefore, they should be removed to improve mappability. In addition to the quality of raw data,
quality control of raw reads also includes the analysis of read alignment (read uniformity and GC
content), quantification (3’ bias, biotypes, and low-counts), and reproducibility (correlation, principal
component analysis, and batch effects).

Table 1. The tools for quality control of RNA-seq raw reads.

Tools Applications

NGSQC Quality control of raw reads generated by Illumina platforms.

 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

FastQC Quality control of raw reads generated by any platforms.

FASTX-Toolkit Discard of low-quality reads, trim adaptor sequences, and elimination

Trimmonmatic of poor quality bases.

Picard
Quality control in read alignment, including the determination of read
RSeQC
uniformity and GC content.
Qualimap

NOISeq Provide useful plots for quality control of count data.

EDASeq

Read alignment

There are generally three strategies for read alignment, genome mapping, transcriptome mapping, and
de novo assembly. Regardless of whether a genome or transcriptome reference is available, reads may
map uniquely or be assigned to multiple position in the reference, which are referred to as
multi-mapped reads or multireads. Genomic multireads are generally due to repetitive sequences or
shared domains of paralogous genes. Transcriptome multi-mapping arises more often due to gene
isoforms. Therefore, transcript identification and quantification are important challenges for alternatively
expressed genes. When a reference is not available, RNA-seq reads are assembled de novo using
packages such as SOAPdenovo-Trans, Oases, Trans-ABySS, or Trinity. PE strand-specific and
long-length reads are preferred since they are more informative. Emerging long-read technologies,
such as PacBio SMRT technology, can generate full-length transcripts for most genes.

Figure 2. Three basic strategies for RNA-seq read mapping (Conesa et al. 2016). Abbreviations: GFF, General
Feature Format; GTF, gene transfer format; RSEM, RNA-seq by Expectation Maximization.
 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

Table 2. The comparison of genome-based and de novo assembly strategies for RNA-seq analysis.

Genome-based De novo assembly

Method Alignment to a reference genome Not using a reference genome

Advantages ⚫ Efficient computing ⚫ Reference genome is not required

⚫ Eliminates contaminating reads ⚫ Correct alignment of reads to known

splice site is not required
⚫ Very sensitive and can assemble
transcripts of low abundance ⚫ Trans-spliced transcripts can be
assembled
⚫ Can discover novel transcripts
without annotation

Disadvantages Requires high-quality reference genome ⚫ More computational intense

⚫ Sensitive to sequencing error

Recommended depth Approximately 10x Beyond 30x

Table 3. The public sources of RNA-seq data.

Transcriptomic Database Data Type Website

Gene Expression Omnibus Both microarray and sequencing https://www.ncbi.nlm.nih.gov/ge

(GEO) data o/

ArrayExpress Both microarray and sequencing https://www.ebi.ac.uk/arrayexpre

data ss/

ENCODE: Encyclopedia of DNA Public ENCODE Consortium https://www.encodeproject.org/

Elements data

Sequence Read Archive (SRA) Sequencing data https://www.ncbi.nlm.nih.gov/sra

European Nucleotide Archive Sequencing data https://www.ebi.ac.uk/ena

(ENA)

DDBJ Sequence Read Archive Sequencing data https://www.ddbj.nig.ac.jp/dra

(DRA)

Transcript quantification

Transcript quantification can be used to estimate gene and transcript expression levels.

Table 4. The common tools for transcript quantification.

 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

Tools Principles and Applications

TopHat Using an expectation-maximization approach that estimates transcript abundances.

Designed to take advantage of PE reads, and may use GTF information to identify expressed
Cufflinks
transcripts, or can infer transcripts de novo from the mapping data alone.

RSEM
Quantify expression from transcriptome mapping.
eXpress
Allocate multi-mapping reads among transcript and output within-sample normalized values
Sailfish
corrected for sequencing biases.
kallisto

Provides an efficient way of estimating transcript expression from SE reads with a low
NURD
memory and computing cost.

Figure 3. The tools for isoform expression quantification.

Differential expression testing

Differential expression testing is used to evaluate if one gene is differentially expressed in one condition
compared to the other(s). Normalizing methods need to be adopted before comparing different samples.
RPKM and TPM normalize away the most important factor, sequencing depth. TMM, DESeq, and
UpperQuartile can ignore highly variable and/or highly expressed features. Other factors that interfere
with intra-sample comparisons involve transcript length, positional biases in coverage, average
fragment size, and GC content, which can be normalized by tools, such as DESeq, edgeR, baySeq,
and NOISeq. Batch effects may still be present after normalization, which can be minimized by
 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

appropriate experimental design, or removed by methods such as COMBAT or ARSyN.

Table 5. The normalization tools for differential expression testing.

Package Read count distribution Input Replicates Normalization

assumptions

DESeq Negative binomial distribution Raw counts No Library size

edgeR Bayesian methods for negative Raw counts Yes Library size
binomial distribution
TMM

RLE

Upperquartile

baySeq Bayesian methods for negative Raw counts Yes Library size
binomial distribution
Quantile

TMM

NOISeq Non-parametric Raw or No Library size

normalized counts
RPKM

TMM

Upperquartile

Alternative splicing analysis

Alternative splicing (AS) is a posttranscriptional process which generates different transcripts from the
same gene and is vital in response to environmental stimuli by producing diverse protein products.
Multiple bioinformatics tools have been developed to detect AS from experimental data. The
comparison of these detection tools using RNA-seq data was conducted by Ding in 2017, and the
results are shown in Table 7. They have demonstrated that TopHat and its downstream tool, FineSplice,
are the fastest tools, whereas PASTA is the slowest program. Furthermore, AltEventFinder can detect
the highest number of junctions, and RSR detects the lowest number of junctions. Other tools, such as
TopHat, are likely to detect false positive ones. Of the two tools that detect differentially spliced isoforms,
rMATS is faster than rSeqDiff but detects less differentially spliced isoforms than rSeqDiff.
Table 7. Detected AS types or differentially spliced isoforms of these tools (Ding et al. 2017).

Number of
Running Maximum
Data Maximum Number Differentially
Tool Time Memory
Source CPU (%) of SJs Spliced
(Minutes) (GB)
Isoforms
Alt Event Finder ENCODE 12 1.364 100 30569 N/A
 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

Number of
Running Maximum
Data Maximum Number Differentially
Tool Time Memory
Source CPU (%) of SJs Spliced
(Minutes) (GB)
Isoforms
SpliceMap ENCODE 42 3.1 99.9 11882 N/A
FineSplice ENCODE 2 1.364 100 8577 N/A
RSW N/A N/A N/A N/A N/A N/A
RSR ENCODE 24 3.968 100 3143 N/A
PASTA ENCODE 350 2.17 101 14675 N/A
mouse used
rMATS in RSW 44 26.536 274 N/A 17
study
SOAPsplice ENCODE 123 5.332 99.7 10381 N/A
SplicePie N/A N/A N/A N/A N/A N/A
SplicingCompass N/A N/A N/A N/A N/A N/A
TopHat ENCODE 1.75 1.364 100 9619 N/A
TrueSight ENCODE 229 2.914 571 12360 N/A
NSMAP N/A N/A N/A N/A N/A N/A
mouse used
rSeqDiff in RSW 115 0.186 119 N/A 203
study
rSeqNP N/A N/A N/A N/A N/A N/A

Visualization

There are many bioinformatics tools for the visualization of RNA-seq data, including genome browsers,
such as ReadXplorer, UCSC browser, Integrative Gnomics Viewer (IGV), Genome Maps, Savant, tools
specifically designed for RNA-seq data, such as RNAseqViewer, as well as some packages for
differential gene expression analysis that enable the visualization, such as DESeq2 and DEXseq in
Bioconductor. Packages, such as CummeRbund and Sashimi plots, have also been developed for
visualization-exclusive purposes.

Functional Profiling

The latest step in a standard transcriptomics study is generally the characterization of the molecular
functions or pathways in which differentially expressed genes are involved. Gene Ontology,
Bioconductor, DAVID, or Babelomics contain annotation data for most model species, which can be
used for functional annotation. As for novel transcripts, protein-coding transcripts can be functionally
annotated using orthology with the help of databases such as SwissProt, Pfam, and InterPro. Gene
 45-1 Ramsey Road
Shirley, NY 11967
USA
Email: contact@cd-genomics.com

Ontology (GO) allows for some exchangeability of functional information across orthologs. Blast2GO is
a popular tool that allows massive annotation of complete transcriptome against a variety of databases
and controlled vocabularies. The Rfam database contains most well-characterized RNA families that
can be used for functional annotation of long non-coding RNAs.

Advanced analysis

The advanced analysis of RNA-seq usually includes other RNA-seq and integration with other
technologies, which is outlined in Figure 4. More information on applications of RNA-seq, please view
this article Applications of RNA-Seq.
Figure 3. The advanced analysis of RNA-seq data.

References:

1. Conesa A, Madrigal P, Tarazona S, et al. A survey of best practices for RNA-seq data analysis.
Genome biology, 2016, 17(1): 13.

2. Ding L, Rath E, Bai Y. Comparison of Alternative Splicing Junction Detection Tools Using RNASeq
Data. Current genomics, 2017, 18(3): 268-277.

3. Grabherr M G, Haas B J, Yassour M, et al. Full-length transcriptome assembly from RNA-Seq data
without a reference genome. Nature biotechnology, 2011, 29(7): 644.