Transcriptomics technologies

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of
all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome
and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the
information network, whilst non-coding RNAs perform additional diverse functions. A transcriptome
captures a snapshot in time of the total transcripts present in a cell. Transcriptomics technologies provide
a broad account of which cellular processes are active and which are dormant. A major challenge in
molecular biology is to understand how a single genome gives rise to a variety of cells. Another is how
gene expression is regulated.

The first attempts to study whole transcriptomes began in the early 1990s. Subsequent technological
advances since the late 1990s have repeatedly transformed the field and made transcriptomics a
widespread discipline in biological sciences. There are two key contemporary techniques in the field:
microarrays, which quantify a set of predetermined sequences, and RNA-Seq, which uses high-
throughput sequencing to record all transcripts. As the technology improved, the volume of data produced
by each transcriptome experiment increased. As a result, data analysis methods have steadily been
adapted to more accurately and efficiently analyse increasingly large volumes of data. Transcriptome
databases have consequently been growing bigger and more useful as transcriptomes continue to be
collected and shared by researchers. It would be almost impossible to interpret the information contained
in a transcriptome without the knowledge of previous experiments.

Measuring the expression of an organism's genes in different tissues or conditions, or at different times,
gives information on how genes are regulated and reveals details of an organism's biology. It can also be
used to infer the functions of previously unannotated genes. Transcriptome analysis has enabled the study
of how gene expression changes in different organisms and has been instrumental in the understanding of
human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends
which cannot be discerned by more targeted assays.

History
Transcriptomics has been characterised by the development of new techniques which have redefined what
is possible every decade or so and rendered previous technologies obsolete. The first attempt at capturing
a partial human transcriptome was published in 1991 and reported 609 mRNA sequences from the human
brain.[2] In 2008, two human transcriptomes, composed of millions of transcript-derived sequences
covering 16,000 genes, were published,[3][4] and by 2015 transcriptomes had been published for hundreds
of individuals.[5][6] Transcriptomes of different disease states, tissues, or even single cells are now
routinely generated.[6][7][8] This explosion in transcriptomics has been driven by the rapid development
of new technologies with improved sensitivity and economy.[9][10][11][12]
Before transcriptomics
Studies of individual transcripts were being
performed several decades before any
transcriptomics approaches were available. Libraries
of silkmoth mRNA transcripts were collected and
converted to complementary DNA (cDNA) for
storage using reverse transcriptase in the late
1970s.[13] In the 1980s, low-throughput sequencing
using the Sanger method was used to sequence
random transcripts, producing expressed sequence
tags (ESTs).[2][14][15][16] The Sanger method of
Transcriptomics method use over time. Published
sequencing was predominant until the advent of
papers referring to RNA-Seq (black), RNA
high-throughput methods such as sequencing by microarray (red), expressed sequence tag (blue),
synthesis (Solexa/Illumina). ESTs came to digital differential display (green), and serial/cap
prominence during the 1990s as an efficient method analysis of gene expression (yellow) since 1990.[1]
to determine the gene content of an organism
without sequencing the entire genome.[16] Amounts
of individual transcripts were quantified using Northern blotting, nylon membrane arrays, and later
reverse transcriptase quantitative PCR (RT-qPCR) methods,[17][18] but these methods are laborious and
can only capture a tiny subsection of a transcriptome.[12] Consequently, the manner in which a
transcriptome as a whole is expressed and regulated remained unknown until higher-throughput
techniques were developed.

Early attempts
The word "transcriptome" was first used in the 1990s.[19][20] In 1995, one of the earliest sequencing-
based transcriptomic methods was developed, serial analysis of gene expression (SAGE), which worked
by Sanger sequencing of concatenated random transcript fragments.[21] Transcripts were quantified by
matching the fragments to known genes. A variant of SAGE using high-throughput sequencing
techniques, called digital gene expression analysis, was also briefly used.[9][22] However, these methods
were largely overtaken by high throughput sequencing of entire transcripts, which provided additional
information on transcript structure such as splice variants.[9]

Development of contemporary techniques

The dominant contemporary techniques, microarrays and RNA-Seq, were developed in the mid-1990s
and 2000s.[9][33] Microarrays that measure the abundances of a defined set of transcripts via their
hybridisation to an array of complementary probes were first published in 1995.[34][35] Microarray
technology allowed the assay of thousands of transcripts simultaneously and at a greatly reduced cost per
gene and labour saving.[36] Both spotted oligonucleotide arrays and Affymetrix high-density arrays were
the method of choice for transcriptional profiling until the late 2000s.[12][33] Over this period, a range of
microarrays were produced to cover known genes in model or economically important organisms.
Advances in design and manufacture of arrays improved the specificity of probes and allowed more
genes to be tested on a single array. Advances in fluorescence detection increased the sensitivity and
measurement accuracy for low abundance transcripts.[35][37]
RNA-Seq is accomplished Comparison of contemporary methods[23][24][10]
by reverse transcribing
RNA-Seq Microarray
RNA in vitro and
1 day to 1 week per
sequencing the resulting Throughput 1–2 days per experiment[10]
[10] experiment[10]
cDNAs. Transcript
abundance is derived from Input RNA Low ~ 1 ng total RNA[25] High ~ 1 μg mRNA[26]
amount
the number of counts from
High (sample preparation and
each transcript. The Labour Low[10][23]
intensity data analysis)[10][23]
technique has therefore
been heavily influenced None required, although a Reference
Prior reference genome/transcriptome is
by the development of knowledge genome/transcriptome required for design of
[23]
high-throughput sequence is useful probes[23]
sequencing Quantitation ~90% (limited by sequence >90% (limited by fluorescence
technologies. [9][11] accuracy coverage) [27]
detection accuracy)[27]
Massively parallel RNA-Seq can detect SNPs Specialised arrays can detect
signature sequencing Sequence and splice variants (limited by mRNA splice variants (limited
resolution sequencing accuracy of by probe design and cross-
(MPSS) was an early
~99%)[27] hybridisation)[27]
example based on
generating 16–20 bp 1 transcript per million 1 transcript per thousand
Sensitivity (approximate, limited by (approximate, limited by
sequences via a complex sequence coverage) [27]
fluorescence detection)[27]
series of
[38][note 1] Dynamic 100,000:1 (limited by 1,000:1 (limited by
hybridisations, range sequence coverage)[28] fluorescence saturation)[28]
and was used in 2004 to
Technical
validate the expression of reproducibility >99%[29][30] >99%[31][32]
ten thousand genes in
Arabidopsis thaliana.[39] The earliest RNA-Seq work was published in 2006 with one hundred thousand
transcripts sequenced using 454 technology.[40] This was sufficient coverage to quantify relative
transcript abundance. RNA-Seq began to increase in popularity after 2008 when new Solexa/Illumina
technologies allowed one billion transcript sequences to be recorded.[4][10][41][42] This yield now allows
for the quantification and comparison of human transcriptomes.[43]

Data gathering
Generating data on RNA transcripts can be achieved via either of two main principles: sequencing of
individual transcripts (ESTs, or RNA-Seq) or hybridisation of transcripts to an ordered array of
nucleotide probes (microarrays).[23]

Isolation of RNA
All transcriptomic methods require RNA to first be isolated from the experimental organism before
transcripts can be recorded. Although biological systems are incredibly diverse, RNA extraction
techniques are broadly similar and involve mechanical disruption of cells or tissues, disruption of RNase
with chaotropic salts,[44] disruption of macromolecules and nucleotide complexes, separation of RNA
from undesired biomolecules including DNA, and concentration of the RNA via precipitation from
solution or elution from a solid matrix.[44][45] Isolated RNA may additionally be treated with DNase to
digest any traces of DNA.[46] It is necessary to enrich messenger RNA as total RNA extracts are typically
98% ribosomal RNA.[47] Enrichment for transcripts can be performed by poly-A affinity methods or by
depletion of ribosomal RNA using sequence-specific probes.[48] Degraded RNA may affect downstream
results; for example, mRNA enrichment from degraded samples will result in the depletion of 5’ mRNA
ends and an uneven signal across the length of a transcript. Snap-freezing of tissue prior to RNA isolation
is typical, and care is taken to reduce exposure to RNase enzymes once isolation is complete.[45]

Expressed sequence tags

An expressed sequence tag (EST) is a short nucleotide sequence generated from a single RNA transcript.
RNA is first copied as complementary DNA (cDNA) by a reverse transcriptase enzyme before the
resultant cDNA is sequenced.[16] Because ESTs can be collected without prior knowledge of the organism
from which they come, they can be made from mixtures of organisms or environmental samples.[49][16]
Although higher-throughput methods are now used, EST libraries commonly provided sequence
information for early microarray designs; for example, a barley microarray was designed from 350,000
previously sequenced ESTs.[50]

Serial and cap analysis of gene expression (SAGE/CAGE)

Serial analysis of gene
expression (SAGE) was a
development of EST
methodology to increase the
throughput of the tags generated
and allow some quantitation of
transcript abundance.[21] cDNA
is generated from the RNA but
is then digested into 11 bp "tag"
fragments using restriction
enzymes that cut DNA at a
specific sequence, and 11 base
pairs along from that sequence.
These cDNA tags are then
joined head-to-tail into long
strands (>500 bp) and
sequenced using low-
throughput, but long read-length Summary of SAGE. Within the organisms, genes are transcribed and
methods such as Sanger spliced (in eukaryotes) to produce mature mRNA transcripts (red). The
sequencing. The sequences are mRNA is extracted from the organism, and reverse transcriptase is used
to copy the mRNA into stable double-stranded–cDNA (ds-cDNA; blue). In
then divided back into their
SAGE, the ds-cDNA is digested by restriction enzymes (at location ‘X’ and
original 11 bp tags using
‘X’+11) to produce 11-nucleotide "tag" fragments. These tags are
computer software in a process concatenated and sequenced using long-read Sanger sequencing
called deconvolution.[21] If a (different shades of blue indicate tags from different genes). The
high-quality reference genome sequences are deconvoluted to find the frequency of each tag. The tag
is available, these tags may be frequency can be used to report on transcription of the gene that the tag
matched to their corresponding came from.[51]
gene in the genome. If a
reference genome is unavailable, the tags can be directly used as diagnostic markers if found to be
differentially expressed in a disease state.[21]

The cap analysis gene expression (CAGE) method is a variant of SAGE that sequences tags from the 5’
end of an mRNA transcript only.[52] Therefore, the transcriptional start site of genes can be identified
when the tags are aligned to a reference genome. Identifying gene start sites is of use for promoter
analysis and for the cloning of full-length cDNAs.

SAGE and CAGE methods produce information on more genes than was possible when sequencing
single ESTs, but sample preparation and data analysis are typically more labour-intensive.[52]

Microarrays

Principles and advances

Microarrays usually consist of a
grid of short nucleotide
oligomers, known as "probes",
typically arranged on a glass
slide.[53] Transcript abundance
is determined by hybridisation
of fluorescently labelled
transcripts to these probes.[54]
The fluorescence intensity at
each probe location on the array
indicates the transcript
abundance for that probe
sequence.[54] Groups of probes
designed to measure the same
transcript (i.e., hybridizing a
specific transcript in different Summary of DNA Microarrays. Within the organisms, genes are
positions) are usually referred to transcribed and spliced (in eukaryotes) to produce mature mRNA
as "probesets". transcripts (red). The mRNA is extracted from the organism and reverse
transcriptase is used to copy the mRNA into stable ds-cDNA (blue). In
Microarrays require some microarrays, the ds-cDNA is fragmented and fluorescently labelled
genomic knowledge from the (orange). The labelled fragments bind to an ordered array of
organism of interest, for complementary oligonucleotides, and measurement of fluorescent
intensity across the array indicates the abundance of a predetermined set
example, in the form of an
of sequences. These sequences are typically specifically chosen to report
annotated genome sequence, or
on genes of interest within the organism's genome.[51]
a library of ESTs that can be
used to generate the probes for
the array.[36]
Methods
Microarrays for transcriptomics typically fall into one of two broad categories: low-density spotted arrays
or high-density short probe arrays. Transcript abundance is inferred from the intensity of fluorescence
derived from fluorophore-tagged transcripts that bind to the array.[36]

Spotted low-density arrays typically feature picolitre[note 2] drops of a range of purified cDNAs arrayed
on the surface of a glass slide.[55] These probes are longer than those of high-density arrays and cannot
identify alternative splicing events. Spotted arrays use two different fluorophores to label the test and
control samples, and the ratio of fluorescence is used to calculate a relative measure of abundance.[56]
High-density arrays use a single fluorescent label, and each sample is hybridised and detected
individually.[57] High-density arrays were popularised by the Affymetrix GeneChip array, where each
transcript is quantified by several short 25-mer probes that together assay one gene.[58]

NimbleGen arrays were a high-density array produced by a maskless-photochemistry method, which

permitted flexible manufacture of arrays in small or large numbers. These arrays had 100,000s of 45 to
85-mer probes and were hybridised with a one-colour labelled sample for expression analysis.[59] Some
designs incorporated up to 12 independent arrays per slide.

RNA-Seq

Principles and advances

RNA-Seq refers to the
combination of a high-
throughput sequencing
methodology with
computational methods to
capture and quantify transcripts
present in an RNA extract.[10]
The nucleotide sequences
generated are typically around
100 bp in length, but can range
from 30 bp to over 10,000 bp
depending on the sequencing
method used. RNA-Seq
leverages deep sampling of the
transcriptome with many short Summary of RNA-Seq. Within the organisms, genes are transcribed and
fragments from a transcriptome spliced (in eukaryotes) to produce mature mRNA transcripts (red). The
to allow computational mRNA is extracted from the organism, fragmented, and copied into stable
reconstruction of the original ds-cDNA (blue). The ds-cDNA is sequenced using high-throughput, short-
RNA transcript by aligning read sequencing methods. These sequences can then be aligned to a
reference genome sequence to reconstruct which genome regions were
reads to a reference genome or
being transcribed. This data can be used to annotate where expressed
to each other (de novo genes are, their relative expression levels, and any alternative splice
assembly).[9] Both low- variants.[51]
abundance and high-abundance
RNAs can be quantified in an
RNA-Seq experiment (dynamic range of 5 orders of magnitude)—a key advantage over microarray
transcriptomes. In addition, input RNA amounts are much lower for RNA-Seq (nanogram quantity)
compared to microarrays (microgram quantity), which allow examination of the transcriptome even at a
single-cell resolution when combined with amplification of cDNA.[25][60] Theoretically, there is no upper
limit of quantification in RNA-Seq, and background noise is very low for 100 bp reads in non-repetitive
regions.[10]

RNA-Seq may be used to identify genes within a genome, or identify which genes are active at a
particular point in time, and read counts can be used to accurately model the relative gene expression
level. RNA-Seq methodology has constantly improved, primarily through the development of DNA
sequencing technologies to increase throughput, accuracy, and read length.[61] Since the first descriptions
in 2006 and 2008,[40][62] RNA-Seq has been rapidly adopted and overtook microarrays as the dominant
transcriptomics technique in 2015.[63]

The quest for transcriptome data at the level of individual cells has driven advances in RNA-Seq library
preparation methods, resulting in dramatic advances in sensitivity. Single-cell transcriptomes are now
well described and have even been extended to in situ RNA-Seq where transcriptomes of individual cells
are directly interrogated in fixed tissues.[64]

Methods
RNA-Seq was established in concert with the rapid development of a range of high-throughput DNA
sequencing technologies.[65] However, before the extracted RNA transcripts are sequenced, several key
processing steps are performed. Methods differ in the use of transcript enrichment, fragmentation,
amplification, single or paired-end sequencing, and whether to preserve strand information.[65]

The sensitivity of an RNA-Seq experiment can be increased by enriching classes of RNA that are of
interest and depleting known abundant RNAs. The mRNA molecules can be separated using
oligonucleotides probes which bind their poly-A tails. Alternatively, ribo-depletion can be used to
specifically remove abundant but uninformative ribosomal RNAs (rRNAs) by hybridisation to probes
tailored to the taxon's specific rRNA sequences (e.g. mammal rRNA, plant rRNA). However, ribo-
depletion can also introduce some bias via non-specific depletion of off-target transcripts.[66] Small
RNAs, such as micro RNAs, can be purified based on their size by gel electrophoresis and extraction.

Since mRNAs are longer than the read-lengths of typical high-throughput sequencing methods,
transcripts are usually fragmented prior to sequencing.[67] The fragmentation method is a key aspect of
sequencing library construction. Fragmentation may be achieved by chemical hydrolysis, nebulisation,
sonication, or reverse transcription with chain-terminating nucleotides.[67] Alternatively, fragmentation
and cDNA tagging may be done simultaneously by using transposase enzymes.[68]

During preparation for sequencing, cDNA copies of transcripts may be amplified by PCR to enrich for
fragments that contain the expected 5’ and 3’ adapter sequences.[69] Amplification is also used to allow
sequencing of very low input amounts of RNA, down to as little as 50 pg in extreme applications.[70]
Spike-in controls of known RNAs can be used for quality control assessment to check library preparation
and sequencing, in terms of GC-content, fragment length, as well as the bias due to fragment position
within a transcript.[71] Unique molecular identifiers (UMIs) are short random sequences that are used to
individually tag sequence fragments during library preparation so that every tagged fragment is
unique.[72] UMIs provide an absolute scale for quantification, the opportunity to correct for subsequent
amplification bias introduced during library construction, and accurately estimate the initial sample size.
UMIs are particularly well-suited to single-cell RNA-Seq transcriptomics, where the amount of input
RNA is restricted and extended amplification of the sample is required.[73][74][75]

Once the transcript molecules have been prepared they can be sequenced in just one direction (single-
end) or both directions (paired-end). A single-end sequence is usually quicker to produce, cheaper than
paired-end sequencing and sufficient for quantification of gene expression levels. Paired-end sequencing
produces more robust alignments/assemblies, which is beneficial for gene annotation and transcript
isoform discovery.[10] Strand-specific RNA-Seq methods preserve the strand information of a sequenced
transcript.[76] Without strand information, reads can be aligned to a gene locus but do not inform in which
direction the gene is transcribed. Stranded-RNA-Seq is useful for deciphering transcription for genes that
overlap in different directions and to make more robust gene predictions in non-model organisms.[76]

Sequencing technology platforms commonly used for RNA-Seq[77][78]

Typical Maximum RNA-Seq runs deposited in
Commercial Single read
Platform read throughput per
release
length run
accuracy the NCBI SRA (Oct 2016)[79]

454 Life
2005 700 bp 0.7 Gbp 99.9% 3548
Sciences

50–300
Illumina 2006 900 Gbp 99.9% 362903
bp

SOLiD 2008 50 bp 320 Gbp 99.9% 7032

Ion Torrent 2010 400 bp 30 Gbp 98% 1953

PacBio 2011 10,000 bp 2 Gbp 87% 160

Legend: NCBI SRA – National center for biotechnology information sequence read archive.

Currently RNA-Seq relies on copying RNA molecules into cDNA molecules prior to sequencing;
therefore, the subsequent platforms are the same for transcriptomic and genomic data. Consequently, the
development of DNA sequencing technologies has been a defining feature of RNA-Seq.[78][80][81] Direct
sequencing of RNA using nanopore sequencing represents a current state-of-the-art RNA-Seq
technique.[82][83] Nanopore sequencing of RNA can detect modified bases that would be otherwise
masked when sequencing cDNA and also eliminates amplification steps that can otherwise introduce
bias.[11][84]

The sensitivity and accuracy of an RNA-Seq experiment are dependent on the number of reads obtained
from each sample.[85][86] A large number of reads are needed to ensure sufficient coverage of the
transcriptome, enabling detection of low abundance transcripts. Experimental design is further
complicated by sequencing technologies with a limited output range, the variable efficiency of sequence
creation, and variable sequence quality. Added to those considerations is that every species has a different
number of genes and therefore requires a tailored sequence yield for an effective transcriptome. Early
studies determined suitable thresholds empirically, but as the technology matured suitable coverage was
predicted computationally by transcriptome saturation. Somewhat counter-intuitively, the most effective
way to improve detection of differential expression in low expression genes is to add more biological
replicates rather than adding more reads.[87] The current benchmarks recommended by the Encyclopedia
of DNA Elements (ENCODE) Project are for 70-fold exome coverage for standard RNA-Seq and up to
500-fold exome coverage to detect rare transcripts and isoforms.[88][89][90]
Data analysis
Transcriptomics methods are highly parallel and require significant computation to produce meaningful
data for both microarray and RNA-Seq experiments.[91][92][93][94][95] Microarray data is recorded as
high-resolution images, requiring feature detection and spectral analysis.[96] Microarray raw image files
are each about 750 MB in size, while the processed intensities are around 60 MB in size. Multiple short
probes matching a single transcript can reveal details about the intron-exon structure, requiring statistical
models to determine the authenticity of the resulting signal. RNA-Seq studies produce billions of short
DNA sequences, which must be aligned to reference genomes composed of millions to billions of base
pairs. De novo assembly of reads within a dataset requires the construction of highly complex sequence
graphs.[97] RNA-Seq operations are highly repetitious and benefit from parallelised computation but
modern algorithms mean consumer computing hardware is sufficient for simple transcriptomics
experiments that do not require de novo assembly of reads.[98] A human transcriptome could be
accurately captured using RNA-Seq with 30 million 100 bp sequences per sample.[85][86] This example
would require approximately 1.8 gigabytes of disk space per sample when stored in a compressed fastq
format. Processed count data for each gene would be much smaller, equivalent to processed microarray
intensities. Sequence data may be stored in public repositories, such as the Sequence Read Archive
(SRA).[99] RNA-Seq datasets can be uploaded via the Gene Expression Omnibus.[100]

Image processing
Microarray image processing must correctly identify
the regular grid of features within an image and
independently quantify the fluorescence intensity for
each feature. Image artefacts must be additionally
identified and removed from the overall analysis.
Fluorescence intensities directly indicate the
abundance of each sequence, since the sequence of
each probe on the array is already known.[102]
Microarray and sequencing flow cell. Microarrays
The first steps of RNA-seq also include similar image and RNA-seq rely on image analysis in different
processing; however, conversion of images to ways. In a microarray chip, each spot on a chip is
sequence data is typically handled automatically by a defined oligonucleotide probe, and fluorescence
intensity directly detects the abundance of a
the instrument software. The Illumina sequencing-by-
specific sequence (Affymetrix). In a high-
synthesis method results in an array of clusters
throughput sequencing flow cell, spots are
distributed over the surface of a flow cell.[103] The sequenced one nucleotide at a time, with the
flow cell is imaged up to four times during each colour at each round indicating the next
sequencing cycle, with tens to hundreds of cycles in nucleotide in the sequence (Illumina Hiseq). Other
total. Flow cell clusters are analogous to microarray variations of these techniques use more or fewer
spots and must be correctly identified during the early colour channels.[51][101]
stages of the sequencing process. In Roche’s
pyrosequencing method, the intensity of emitted light
determines the number of consecutive nucleotides in a homopolymer repeat. There are many variants on
these methods, each with a different error profile for the resulting data.[104]
RNA-Seq data analysis
RNA-Seq experiments generate a large volume of raw sequence reads which have to be processed to
yield useful information. Data analysis usually requires a combination of bioinformatics software tools
(see also List of RNA-Seq bioinformatics tools) that vary according to the experimental design and goals.
The process can be broken down into four stages: quality control, alignment, quantification, and
differential expression.[105] Most popular RNA-Seq programs are run from a command-line interface,
either in a Unix environment or within the R/Bioconductor statistical environment.[94]

Quality control
Sequence reads are not perfect, so the accuracy of each base in the sequence needs to be estimated for
downstream analyses. Raw data is examined to ensure: quality scores for base calls are high, the GC
content matches the expected distribution, short sequence motifs (k-mers) are not over-represented, and
the read duplication rate is acceptably low.[86] Several software options exist for sequence quality
analysis, including FastQC and FaQCs.[106][107] Abnormalities may be removed (trimming) or tagged for
special treatment during later processes.

Alignment
In order to link sequence read abundance to the expression of a particular gene, transcript sequences are
aligned to a reference genome or de novo aligned to one another if no reference is available.[108][109][110]
The key challenges for alignment software include sufficient speed to permit billions of short sequences
to be aligned in a meaningful timeframe, flexibility to recognise and deal with intron splicing of
eukaryotic mRNA, and correct assignment of reads that map to multiple locations. Software advances
have greatly addressed these issues, and increases in sequencing read length reduce the chance of
ambiguous read alignments. A list of currently available high-throughput sequence aligners is maintained
by the EBI.[111][112]

Alignment of primary transcript mRNA sequences derived from eukaryotes to a reference genome
requires specialised handling of intron sequences, which are absent from mature mRNA.[113] Short read
aligners perform an additional round of alignments specifically designed to identify splice junctions,
informed by canonical splice site sequences and known intron splice site information. Identification of
intron splice junctions prevents reads from being misaligned across splice junctions or erroneously
discarded, allowing more reads to be aligned to the reference genome and improving the accuracy of gene
expression estimates. Since gene regulation may occur at the mRNA isoform level, splice-aware
alignments also permit detection of isoform abundance changes that would otherwise be lost in a bulked
analysis.[114]

De novo assembly can be used to align reads to one another to construct full-length transcript sequences
without use of a reference genome.[115] Challenges particular to de novo assembly include larger
computational requirements compared to a reference-based transcriptome, additional validation of gene
variants or fragments, and additional annotation of assembled transcripts. The first metrics used to
describe transcriptome assemblies, such as N50, have been shown to be misleading[116] and improved
evaluation methods are now available.[117][118] Annotation-based metrics are better assessments of
assembly completeness, such as contig reciprocal best hit count. Once assembled de novo, the assembly
can be used as a reference for subsequent sequence alignment methods and quantitative gene expression
analysis.
 RNA-Seq de novo assembly software
Last Computational
Software Released Strengths and weaknesses
updated efficiency

Velvet- Low, single-threaded,

The original short read assembler. It is
2008 2011 high RAM
Oases[119][120] requirement
now largely superseded.

SOAPdenovo- Moderate, multi- An early example of a short read

2011 2014 thread, medium RAM assembler. It has been updated for
trans[109] requirement transcriptome assembly.
Suited to short reads, can handle
Moderate, multi-
[121] complex transcriptomes, and an MPI-
Trans-ABySS 2010 2016 thread, medium RAM
parallel version is available for
requirement
computing clusters.

Moderate, multi- Suited to short reads. It can handle

Trinity[122][97] 2011 2017 thread, medium RAM complex transcriptomes but is memory
requirement intensive.

Can process repetitive sequences,

Moderate, multi-
[123] combine different sequencing formats,
miraEST 1999 2016 thread, medium RAM
and a wide range of sequence platforms
requirement
are accepted.
Low, single-thread, Specialised to accommodate the homo-
Newbler[124] 2004 2012 high RAM polymer sequencing errors typical of
requirement Roche 454 sequencers.

Has a graphical user interface, can

CLC genomics combine diverse sequencing
High, multi-thread,
2008 2014 technologies, has no transcriptome-
workbench[125] low RAM requirement
specific features, and a licence must be
purchased before use.

High, multi-thread, Used for transcriptomics experiments on

SPAdes[126] 2012 2017
low RAM requirement single cells.
High, multi-thread, Can estimate frequency of alternatively
RSEM[127] 2011 2017
low RAM requirement spliced transcripts. User friendly.

Can use a combination of reference-

High, multi-thread,
StringTie[98][128] 2015 2019
low RAM requirement
guided and de novo assembly methods
to identify transcripts.

Legend: RAM – random access memory; MPI – message passing interface; EST – expressed sequence tag.

Quantification
Quantification of sequence alignments may be performed at the gene, exon, or transcript level.[91][87]
Typical outputs include a table of read counts for each feature supplied to the software; for example, for
genes in a general feature format file. Gene and exon read counts may be calculated quite easily using
HTSeq, for example.[130] Quantitation at the transcript level is more complicated and requires
probabilistic methods to estimate transcript isoform abundance from short read information; for example,
using cufflinks software.[114] Reads that align equally well to multiple locations must be identified and
either removed, aligned to one of the possible locations, or aligned to the most probable location.

Some quantification methods can circumvent the need for an exact alignment of a read to a reference
sequence altogether. The kallisto software method combines pseudoalignment and quantification into a
single step that runs 2 orders of magnitude faster than contemporary methods such as those used by
tophat/cufflinks software, with less
computational burden.[131]

Differential expression
Once quantitative counts of each transcript are
available, differential gene expression is
measured by normalising, modelling, and
statistically analysing the data.[108] Most tools
will read a table of genes and read counts as their
input, but some programs, such as cuffdiff, will
accept binary alignment map format read
alignments as input. The final outputs of these
analyses are gene lists with associated pair-wise
tests for differential expression between
treatments and the probability estimates of those
differences.[132]

Heatmap identification of gene co-expression patterns

across different samples. Each column contains the
measurements for gene expression change for a single
sample. Relative gene expression is indicated by
colour: high-expression (red), median-expression
(white) and low-expression (blue). Genes and samples
with similar expression profiles can be automatically
grouped (left and top trees). Samples may be different
individuals, tissues, environments or health conditions.
In this example, expression of gene set 1 is high and
expression of gene set 2 is low in samples 1, 2, and
3.[51][129]

RNA-Seq differential gene expression software

Software Environment Specialisation

Cuffdiff2[108] Unix-based Transcript analysis that tracks alternative splicing of mRNA

EdgeR[93] R/Bioconductor Any count-based genomic data

DEseq2[133] R/Bioconductor Flexible data types, low replication

Limma/Voom[92] R/Bioconductor Microarray or RNA-Seq data, flexible experiment design

Ballgown[134] R/Bioconductor Efficient and sensitive transcript discovery, flexible.

Legend: mRNA - messenger RNA.

Validation
Transcriptomic analyses may be validated using an independent technique, for example, quantitative PCR
(qPCR), which is recognisable and statistically assessable.[135] Gene expression is measured against
defined standards both for the gene of interest and control genes. The measurement by qPCR is similar to
that obtained by RNA-Seq wherein a value can be calculated for the concentration of a target region in a
given sample. qPCR is, however, restricted to amplicons smaller than 300 bp, usually toward the 3’ end
of the coding region, avoiding the 3’UTR.[136] If validation of transcript isoforms is required, an
inspection of RNA-Seq read alignments should indicate where qPCR primers might be placed for
maximum discrimination. The measurement of multiple control genes along with the genes of interest
produces a stable reference within a biological context.[137] qPCR validation of RNA-Seq data has
generally shown that different RNA-Seq methods are highly correlated.[62][138][139]

Functional validation of key genes is an important consideration for post transcriptome planning.
Observed gene expression patterns may be functionally linked to a phenotype by an independent knock-
down/rescue study in the organism of interest.[140]

Applications

Diagnostics and disease profiling

Transcriptomic strategies have seen broad application across diverse areas of biomedical research,
including disease diagnosis and profiling.[10][141] RNA-Seq approaches have allowed for the large-scale
identification of transcriptional start sites, uncovered alternative promoter usage, and novel splicing
alterations. These regulatory elements are important in human disease and, therefore, defining such
variants is crucial to the interpretation of disease-association studies.[142] RNA-Seq can also identify
disease-associated single nucleotide polymorphisms (SNPs), allele-specific expression, and gene fusions,
which contributes to the understanding of disease causal variants.[143]

Retrotransposons are transposable elements which proliferate within eukaryotic genomes through a
process involving reverse transcription. RNA-Seq can provide information about the transcription of
endogenous retrotransposons that may influence the transcription of neighboring genes by various
epigenetic mechanisms that lead to disease.[144] Similarly, the potential for using RNA-Seq to understand
immune-related disease is expanding rapidly due to the ability to dissect immune cell populations and to
sequence T cell and B cell receptor repertoires from patients.[145][146]

Human and pathogen transcriptomes

RNA-Seq of human pathogens has become an established method for quantifying gene expression
changes, identifying novel virulence factors, predicting antibiotic resistance, and unveiling host-pathogen
immune interactions.[147][148] A primary aim of this technology is to develop optimised infection control
measures and targeted individualised treatment.[146]

Transcriptomic analysis has predominantly focused on either the host or the pathogen. Dual RNA-Seq has
been applied to simultaneously profile RNA expression in both the pathogen and host throughout the
infection process. This technique enables the study of the dynamic response and interspecies gene
regulatory networks in both interaction partners from initial contact through to invasion and the final
persistence of the pathogen or clearance by the host immune system.[149][150]

Responses to environment
Transcriptomics allows identification of genes and pathways that respond to and counteract biotic and
abiotic environmental stresses.[151][140] The non-targeted nature of transcriptomics allows the
identification of novel transcriptional networks in complex systems. For example, comparative analysis
of a range of chickpea lines at different developmental stages identified distinct transcriptional profiles
associated with drought and salinity stresses, including identifying the role of transcript isoforms of AP2-
EREBP.[151] Investigation of gene expression during biofilm formation by the fungal pathogen Candida
albicans revealed a co-regulated set of genes critical for biofilm establishment and maintenance.[152]

Transcriptomic profiling also provides crucial information on mechanisms of drug resistance. Analysis of
over 1000 isolates of Plasmodium falciparum, a virulent parasite responsible for malaria in humans,[153]
identified that upregulation of the unfolded protein response and slower progression through the early
stages of the asexual intraerythrocytic developmental cycle were associated with artemisinin resistance in
isolates from Southeast Asia.[154]

The use of transcriptomics is also important to investigate responses in the marine environment.[155] In
marine ecology, "stress" and "adaptation" have been among the most common research topics, especially
related to anthropogenic stress, such as global change and pollution.[155] Most of the studies in this area
have been done in animals, although invertebrates have been underrepresented.[155] One issue still is a
deficiency in functional genetic studies, which hamper gene annotations, especially for non-model
species, and can lead to vague conclusions on the effects of responses studied.[155]

Gene function annotation

All transcriptomic techniques have been particularly useful in identifying the functions of genes and
identifying those responsible for particular phenotypes. Transcriptomics of Arabidopsis ecotypes that
hyperaccumulate metals correlated genes involved in metal uptake, tolerance, and homeostasis with the
phenotype.[156] Integration of RNA-Seq datasets across different tissues has been used to improve
annotation of gene functions in commercially important organisms (e.g. cucumber)[157] or threatened
species (e.g. koala).[158]

Assembly of RNA-Seq reads is not dependent on a reference genome[122] and so is ideal for gene
expression studies of non-model organisms with non-existing or poorly developed genomic resources.
For example, a database of SNPs used in Douglas fir breeding programs was created by de novo
transcriptome analysis in the absence of a sequenced genome.[159] Similarly, genes that function in the
development of cardiac, muscle, and nervous tissue in lobsters were identified by comparing the
transcriptomes of the various tissue types without use of a genome sequence.[160] RNA-Seq can also be
used to identify previously unknown protein coding regions in existing sequenced genomes.
Non-coding RNA
Transcriptomics is most commonly applied to the mRNA content of the cell. However, the same
techniques are equally applicable to non-coding RNAs (ncRNAs) that are not translated into a protein,
but instead have direct functions (e.g. roles in protein translation, DNA replication, RNA splicing, and
transcriptional regulation).[161][162][163][164] Many of these ncRNAs affect disease states, including
cancer, cardiovascular, and neurological diseases.[165]

Transcriptome databases
Transcriptomics studies generate large amounts of data that have potential applications far beyond the
original aims of an experiment. As such, raw or processed data may be deposited in public databases to
ensure their utility for the broader scientific community. For example, as of 2018, the Gene Expression
Omnibus contained millions of experiments.[166]

Transcriptomic databases
Name Host Data Description

First transcriptomics database to accept data from any

Gene Expression Microarray source. Introduced MIAME and MINSEQE community
NCBI
Omnibus[100] RNA-Seq standards that define necessary experiment metadata to
ensure effective interpretation and repeatability.[167][168]
Imports datasets from the Gene Expression Omnibus
and accepts direct submissions. Processed data and
ArrayExpress[169] ENA Microarray experiment metadata is stored at ArrayExpress, while the
raw sequence reads are held at the ENA. Complies with
MIAME and MINSEQE standards.[167][168]
Tissue-specific gene expression database for animals
and plants. Displays secondary analyses and
Microarray
Expression Atlas[170] EBI
RNA-Seq
visualisation, such as functional enrichment of Gene
Ontology terms, InterPro domains, or pathways. Links to
protein abundance data where available.
Contains manual curations of public transcriptome
datasets, focusing on medical and plant biology data.
Privately Microarray Individual experiments are normalised across the full
Genevestigator[171] curated RNA-Seq database to allow comparison of gene expression across
diverse experiments. Full functionality requires licence
purchase, with free access to a limited functionality.

Human, mouse, and rat transcriptomes from 40 different

organs. Gene expression visualised as heatmaps
RefEx[172] DDBJ All
projected onto 3D representations of anatomical
structures.

NONCODE[173] [Link] RNA-Seq Non-coding RNAs (ncRNAs) excluding tRNA and rRNA.

Legend: NCBI – National Center for Biotechnology Information; EBI – European Bioinformatics Institute; DDBJ – DNA
Data Bank of Japan; ENA – European Nucleotide Archive; MIAME – Minimum Information About a Microarray
Experiment; MINSEQE – Minimum Information about a high-throughput nucleotide SEQuencing Experiment.
See also
omics
Genomics
Proteomics
Metabolomics
Interactomics

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Notes
1. In molecular biology, hybridisation is a phenomenon in which single-stranded
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary
DNA or RNA.
2. One picolitre is about 30 million times smaller than a drop of water.

Further reading
Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T (May 2017). "Transcriptomics
technologies" ([Link] PLOS
Computational Biology. 13 (5): e1005457. Bibcode:2017PLSCB..13E5457L ([Link]
[Link]/abs/2017PLSCB..13E5457L). doi:10.1371/[Link].1005457 ([Link]
g/10.1371%[Link].1005457). PMC 5436640 ([Link]
les/PMC5436640). PMID 28545146 ([Link]
Comparative Transcriptomics Analysis ([Link]
5) in Reference Module in Life Sciences ([Link]
 orks/9780128096338)
Software used in transcriptomics:
cufflinks ([Link]
kallisto ([Link]
tophat ([Link]

Retrieved from "[Link]