Transcriptomics technologies are the techniques used to study an organism's
transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...
, the sum of all of its
RNA transcripts. The information content of an organism is recorded in the DNA of its
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
and
expressed through
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
. Here,
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
serves as a transient intermediary molecule in the information network, whilst
non-coding RNA
A non-coding RNA (ncRNA) is a functional RNA molecule that is not translated into a protein. The DNA sequence from which a functional non-coding RNA is transcribed is often called an RNA gene. Abundant and functionally important types of non-c ...
s perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
. 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:
microarray
A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silicon t ...
s, which quantify a set of predetermined sequences, and
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
, which uses
high-throughput sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...
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 getting 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
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s in different
tissues or
conditions, or at different times, gives information on how genes are
regulated
Regulation is the management of complex systems according to a set of rules and trends. In systems theory, these types of rules exist in various fields of biology and society, but the term has slightly different meanings according to context. Fo ...
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
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted
assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...
s.
History
![Transcriptomics technique publications over time](https://upload.wikimedia.org/wikipedia/commons/6/6a/Transcriptomics_technique_publications_over_time.svg)
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
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
sequences from the
human brain
The human brain is the central organ of the human nervous system, and with the spinal cord makes up the central nervous system. The brain consists of the cerebrum, the brainstem and the cerebellum. It controls most of the activities of the ...
.
In 2008, two human transcriptomes, composed of millions of transcript-derived sequences covering 16,000 genes, were published,
and by 2015 transcriptomes had been published for hundreds of individuals.
Transcriptomes of different
disease
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
states,
tissues, or even single
cells
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...
are now routinely generated.
This explosion in transcriptomics has been driven by the rapid development of new technologies with improved sensitivity and economy.
Before transcriptomics
Studies of individual
transcripts were being performed several decades before any transcriptomics approaches were available.
Libraries
A library is a collection of materials, books or media that are accessible for use and not just for display purposes. A library provides physical (hard copies) or digital access (soft copies) materials, and may be a physical location or a vir ...
of
silkmoth mRNA transcripts were collected and converted to
complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
(cDNA) for storage using
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
in the late 1970s.
In the 1980s, low-throughput sequencing using the
Sanger method was used to sequence random transcripts, producing
expressed sequence tag In genetics, an expressed sequence tag (EST) is a short sub-sequence of a cDNA sequence. ESTs may be used to identify gene transcripts, and were instrumental in gene discovery and in gene-sequence determination. The identification of ESTs has proce ...
s (ESTs).
The
Sanger method of sequencing was predominant until the advent of
high-throughput methods such as
sequencing by synthesis Illumina dye sequencing is a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. The reversible terminated chemistry concept was invented by Bruno Canard and Simon Sarfati at the Pasteur Institute in Paris. I ...
(Solexa/Illumina).
ESTs came to prominence during the 1990s as an efficient method to determine the
gene content of an organism without
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
the entire
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
.
Amounts of individual transcripts were quantified using
Northern blotting
The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.K ...
,
nylon membrane arrays, and later
reverse transcriptase quantitative PCR (RT-qPCR) methods,
but these methods are laborious and can only capture a tiny subsection of a transcriptome.
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.
In 1995, one of the earliest sequencing-based transcriptomic methods was developed,
serial analysis of gene expression
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those tra ...
(SAGE), which worked by
Sanger sequencing
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederi ...
of concatenated random transcript fragments.
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.
However, these methods were largely overtaken by high throughput sequencing of entire transcripts, which provided additional information on transcript structure such as
splice variants.
Development of contemporary techniques
The dominant contemporary techniques,
microarrays
A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silicon t ...
and
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
, were developed in the mid-1990s and 2000s.
Microarrays that measure the abundances of a defined set of transcripts via their
hybridisation to an array of
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
probes were first published in 1995.
Microarray technology allowed the assay of thousands of transcripts simultaneously and at a greatly reduced cost per gene and labour saving.
Both
spotted oligonucleotide arrays and
Affymetrix
Affymetrix is now Applied Biosystems, a brand of DNA microarray products sold by Thermo Fisher Scientific that originated with an American biotechnology research and development and manufacturing company of the same name. The Santa Clara, Califor ...
high-density arrays were the method of choice for transcriptional profiling until the late 2000s.
Over this period, a range of microarrays were produced to cover known genes in
model
A model is an informative representation of an object, person or system. The term originally denoted the Plan_(drawing), plans of a building in late 16th-century English, and derived via French and Italian ultimately from Latin ''modulus'', a mea ...
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
A fluorometer, fluorimeter or fluormeter is a device used to measure parameters of visible spectrum fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters ...
increased the sensitivity and measurement accuracy for low abundance transcripts.
RNA-Seq is accomplished by reverse transcribing RNA ''in vitro'' and sequencing the resulting
cDNAs
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
.
Transcript abundance is derived from the number of counts from each transcript. The technique has therefore been heavily influenced by the development of
high-throughput sequencing technologies.
Massively parallel signature sequencing
Massive parallel signature sequencing (MPSS) is a procedure that is used to identify and quantify mRNA transcripts, resulting in data similar to serial analysis of gene expression (SAGE), although it employs a series of biochemical and sequencing ...
(MPSS) was an early example based on generating 16–20
bp sequences via a complex series of
hybridisations,
[In molecular biology, hybridisation is a phenomenon in which single-stranded deoxyribonucleic acid ( DNA) or ribonucleic acid (]RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
) molecules anneal to complementary DNA or RNA. and was used in 2004 to validate the expression of ten thousand genes in ''
Arabidopsis thaliana
''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land.
A winter a ...
''.
The earliest RNA-Seq work was published in 2006 with one hundred thousand transcripts sequenced using
454 technology.
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.
This yield now allows for the
quantification and comparison of human transcriptomes.
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).
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
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology t ...
techniques are broadly similar and involve mechanical
disruption of cells or tissues, disruption of
RNase
Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the ...
with
chaotropic salts,
disruption of macromolecules and nucleotide complexes, separation of RNA from undesired
biomolecule
A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include large ...
s including DNA, and concentration of the RNA via
precipitation
In meteorology, precipitation is any product of the condensation of atmospheric water vapor that falls under gravitational pull from clouds. The main forms of precipitation include drizzle, rain, sleet, snow, ice pellets, graupel and hail. ...
from solution or
elution from a solid matrix.
Isolated RNA may additionally be treated with
DNase Deoxyribonuclease (DNase, for short) refers to a group of glycoprotein endonucleases which are enzymes that catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. The role of the DNase enzyme in cells ...
to digest any traces of DNA.
It is necessary to enrich messenger RNA as total RNA extracts are typically 98%
ribosomal RNA
Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
.
Enrichment for transcripts can be performed by
poly-A affinity methods or by depletion of ribosomal RNA using sequence-specific probes.
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.
Expressed sequence tags
An
expressed sequence tag In genetics, an expressed sequence tag (EST) is a short sub-sequence of a cDNA sequence. ESTs may be used to identify gene transcripts, and were instrumental in gene discovery and in gene-sequence determination. The identification of ESTs has proce ...
(EST) is a short nucleotide sequence generated from a single RNA transcript. RNA is first copied as
complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
(cDNA) by a
reverse transcriptase
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
enzyme before the resultant cDNA is sequenced.
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.
Although higher-throughput methods are now used,
EST libraries commonly provided sequence information for early microarray designs; for example, a
barley
Barley (''Hordeum vulgare''), a member of the grass family, is a major cereal grain grown in temperate climates globally. It was one of the first cultivated grains, particularly in Eurasia as early as 10,000 years ago. Globally 70% of barley pr ...
microarray was designed from 350,000 previously sequenced ESTs.
Serial and cap analysis of gene expression (SAGE/CAGE)
Serial analysis of gene expression
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular biologists to produce a snapshot of the messenger RNA population in a sample of interest in the form of small tags that correspond to fragments of those tra ...
(SAGE) was a development of EST methodology to increase the throughput of the tags generated and allow some quantitation of transcript abundance.
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
is generated from the
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
but is then digested into 11 bp "tag" fragments using
restriction enzyme
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
s 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 methods such as
Sanger sequencing
Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. After first being developed by Frederi ...
. The sequences are then divided back into their original 11 bp tags using computer software in a process called
deconvolution
In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deco ...
.
If a high-quality
reference genome
A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species. As they are assemble ...
is available, these tags may be matched to their corresponding 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.
The
cap analysis gene expression (CAGE) method is a variant of SAGE that sequences tags from the
5’ end of an mRNA transcript only.
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
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cl ...
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.
Microarrays
Principles and advances
Microarray
A microarray is a multiplex lab-on-a-chip. Its purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silicon t ...
s usually consist of a grid of short nucleotide
oligomers
In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomers.Quote: ''Oligomer molecule: A molecule of intermediate relative ...
, known as "
probes", typically arranged on a glass slide.
Transcript abundance is determined by hybridisation of
fluorescently labelled transcripts to these probes.
The
fluorescence intensity at each probe location on the array indicates the transcript abundance for that probe sequence.
Groups of probes designed to measure the same transcript (i.e., hybridizing a specific transcript in different positions) are usually referred to as "probesets".
Microarrays require some genomic knowledge from the organism of interest, for example, in the form of an
annotated
An annotation is extra information associated with a particular point in a document or other piece of information. It can be a note that includes a comment or explanation. Annotations are sometimes presented in the margin of book pages. For ann ...
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
sequence, or a
library
A library is a collection of materials, books or media that are accessible for use and not just for display purposes. A library provides physical (hard copies) or digital access (soft copies) materials, and may be a physical location or a vir ...
of ESTs that can be used to generate the probes for the array.
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.
[
Spotted low-density arrays typically feature ]picolitre
The litre (international spelling) or liter (American English spelling) (SI symbols L and l, other symbol used: ℓ) is a metric unit of volume. It is equal to 1 cubic decimetre (dm3), 1000 cubic centimetres (cm3) or 0.001 cubic metre (m3). ...
[One picolitre is about 30 million times smaller than a drop of water.] drops of a range of purified cDNAs
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
arrayed on the surface of a glass slide. These probes are longer than those of high-density arrays and cannot identify alternative splicing
Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be ...
events. Spotted arrays use two different fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
s to label the test and control samples, and the ratio of fluorescence is used to calculate a relative measure of abundance. High-density arrays use a single fluorescent label, and each sample is hybridised and detected individually. High-density arrays were popularised by the Affymetrix GeneChip array, where each transcript is quantified by several short 25 -mer probes that together assay
An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...
one gene.
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. Some designs incorporated up to 12 independent arrays per slide.
RNA-Seq
Principles and advances
RNA-Seq
RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...
refers to the combination of a high-throughput sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The ...
methodology with computational methods to capture and quantify transcripts present in an RNA extract. 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 fragments from a transcriptome to allow computational reconstruction of the original RNA transcript by aligning reads to a reference genome or to each other ( de novo assembly). Both low-abundance and high-abundance RNAs can be quantified in an RNA-Seq experiment (dynamic range
Dynamic range (abbreviated DR, DNR, or DYR) is the ratio between the largest and smallest values that a certain quantity can assume. It is often used in the context of signals, like sound and light. It is measured either as a ratio or as a base-1 ...
of 5 orders of magnitude
An order of magnitude is an approximation of the logarithm of a value relative to some contextually understood reference value, usually 10, interpreted as the base of the logarithm and the representative of values of magnitude one. Logarithmic dis ...
)—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 allows finer examination of cellular structures down to the single-cell level when combined with linear amplification of cDNA. 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.
RNA-Seq may be used to identify genes within a genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
, 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. Since the first descriptions in 2006 and 2008, RNA-Seq has been rapidly adopted and overtook microarrays as the dominant transcriptomics technique in 2015.
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
''In situ'' (; often not italicized in English) is a Latin phrase that translates literally to "on site" or "in position." It can mean "locally", "on site", "on the premises", or "in place" to describe where an event takes place and is used in ...
'' RNA-Seq where transcriptomes of individual cells are directly interrogated in fixed
Fixed may refer to:
* ''Fixed'' (EP), EP by Nine Inch Nails
* ''Fixed'', an upcoming 2D adult animated film directed by Genndy Tartakovsky
* Fixed (typeface), a collection of monospace bitmap fonts that is distributed with the X Window System
* ...
tissues.
Methods
RNA-Seq was established in concert with the rapid development of a range of high-throughput DNA sequencing technologies. 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.
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 RNA
Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosomal ...
s (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. Small RNAs, such as micro RNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
s, can be purified based on their size by gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
and extraction.
Since mRNAs are longer than the read-lengths of typical high-throughput sequencing methods, transcripts are usually fragmented prior to sequencing. The fragmentation method is a key aspect of sequencing library construction. Fragmentation may be achieved by chemical hydrolysis, nebulisation, sonication
A sonicator at the Weizmann Institute of Science during sonicationSonication is the act of applying sound energy to agitate particles in a sample, for various purposes such as the extraction of multiple compounds from plants, microalgae and seawe ...
, or reverse transcription
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
with chain-terminating nucleotides. Alternatively, fragmentation and cDNA tagging may be done simultaneously by using transposase enzymes.
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. Amplification is also used to allow sequencing of very low input amounts of RNA, down to as little as 50 pg in extreme applications. Spike-in controls of known RNAs can be used for quality control assessment to check library preparation and sequencing, in terms of GC-content
In molecular biology and genetics, GC-content (or guanine-cytosine content) is the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). This measure indicates the proportion of G and C bases out o ...
, fragment length, as well as the bias due to fragment position within a transcript. Unique molecular identifiers Unique molecular identifiers (UMIs), or molecular barcodes (MBC) are short sequences or molecular "tags" added to DNA fragments in some next generation sequencing library preparation protocols to identify the input DNA molecule. These tags are added ...
(UMIs) are short random sequences that are used to individually tag sequence fragments during library preparation so that every tagged fragment is unique. 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.
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
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isof ...
discovery. Strand-specific RNA-Seq methods preserve the strand
Strand may refer to:
Topography
*The flat area of land bordering a body of water, a:
** Beach
** Shoreline
* Strand swamp, a type of swamp habitat in Florida
Places Africa
* Strand, Western Cape, a seaside town in South Africa
* Strand Street ...
information of a sequenced transcript. 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.
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. Direct sequencing of RNA using nanopore sequencing
Nanopore sequencing is a third generation approach used in the sequencing of biopolymers — specifically, polynucleotides in the form of DNA or RNA.
Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the ne ...
represents a current state-of-the-art RNA-Seq technique. 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.
The sensitivity and accuracy of an RNA-Seq experiment are dependent on the number of reads obtained from each sample. 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
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
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. 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.
Data analysis
Transcriptomics methods are highly parallel and require significant computation to produce meaningful data for both microarray and RNA-Seq experiments. Microarray data is recorded as high-resolution
Image resolution is the detail an image holds. The term applies to digital images, film images, and other types of images. "Higher resolution" means more image detail.
Image resolution can be measured in various ways. Resolution quantifies how cl ...
images, requiring feature detection and spectral analysis. 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
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
-exon
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequen ...
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 genome
A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species. As they are assemble ...
s composed of millions to billions of base pairs. ''De novo'' assembly of reads within a dataset requires the construction of highly complex sequence graphs. 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. A human transcriptome could be accurately captured using RNA-Seq with 30 million 100 bp sequences per sample. This example would require approximately 1.8 gigabytes of disk space per sample when stored in a compressed fastq format
FASTQ format is a text-based format for storing both a biological sequence (usually nucleotide sequence) and its corresponding quality scores. Both the sequence letter and quality score are each encoded with a single ASCII character for brevity.
...
. 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
The Sequence Read Archive (SRA, previously known as the Short Read Archive) is a bioinformatics database that provides a public repository for DNA sequencing data, especially the "short reads" generated by high-throughput sequencing, which are typ ...
(SRA). RNA-Seq datasets can be uploaded via the Gene Expression Omnibus.
Image processing
Microarray image processing
An image is a visual representation of something. It can be two-dimensional, three-dimensional, or somehow otherwise feed into the visual system to convey information. An image can be an artifact, such as a photograph or other two-dimensiona ...
must correctly identify the regular grid
A regular grid is a tessellation of ''n''-dimensional Euclidean space by congruent parallelotopes (e.g. bricks).
Its opposite is irregular grid.
Grids of this type appear on graph paper and may be used in finite element analysis, finite volume ...
of features within an image and independently quantify the fluorescence intensity
Intensity may refer to:
In colloquial use
*Strength (disambiguation)
*Amplitude
* Level (disambiguation)
* Magnitude (disambiguation)
In physical sciences
Physics
*Intensity (physics), power per unit area (W/m2)
*Field strength of electric, ma ...
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.
The first steps of RNA-seq also include similar image processing; however, conversion of images to sequence data is typically handled automatically by the instrument software. The Illumina sequencing-by-synthesis method results in an array of clusters distributed over the surface of a flow cell. The flow cell is imaged up to four times during each sequencing cycle, with tens to hundreds of cycles in total. Flow cell clusters are analogous to microarray spots and must be correctly identified during the early stages of the sequencing process. In Roche
F. Hoffmann-La Roche AG, commonly known as Roche, is a Swiss multinational healthcare company that operates worldwide under two divisions: Pharmaceuticals and Diagnostics. Its holding company, Roche Holding AG, has shares listed on the SIX S ...
’s pyrosequencing
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase. Pyrosequ ...
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.
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 The list of bioinformatics software tools can be split up according to the license used:
*List of proprietary bioinformatics software
*List of open-source bioinformatics software
Alternatively, here is a categorization according to the respective b ...
tools (see also List of RNA-Seq bioinformatics tools
RNA-Seq is a technique that allows transcriptome studies (see also Transcriptomics technologies) based on next-generation sequencing technologies. This technique is largely dependent on bioinformatics tools developed to support the different steps ...
) 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. Most popular RNA-Seq programs are run from a command-line interface
A command-line interpreter or command-line processor uses a command-line interface (CLI) to receive commands from a user in the form of lines of text. This provides a means of setting parameters for the environment, invoking executables and pro ...
, either in a Unix
Unix (; trademarked as UNIX) is a family of multitasking, multiuser computer operating systems that derive from the original AT&T Unix, whose development started in 1969 at the Bell Labs research center by Ken Thompson, Dennis Ritchie, and ot ...
environment or within the R/Bioconductor
Bioconductor is a Free software, free, Open-source software, open source and Open source software development, open development software project for the analysis and comprehension of Genome, genomic data generated by Wet laboratory, wet lab experi ...
statistical environment.
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
In bioinformatics, ''k''-mers are substrings of length k contained within a biological sequence. Primarily used within the context of computational genomics and sequence analysis, in which ''k''-mers are composed of nucleotides (''i.e''. A, T, G, ...
) are not over-represented, and the read duplication rate is acceptably low. Several software options exist for sequence quality analysis, including FastQC and FaQCs. 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. 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
Ebrahim Hamedi ( fa, اِبراهیم حامدی, also Romanized as "Ebrāhim Hāmedi"; born 1949), better known by his stage name Ebi (Persian: ), is an Iranian pop singer who first started his career in Tehran, gaining fame as part of a ban ...
.
Alignment of primary transcript mRNA sequences derived from eukaryote
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
s to a reference genome requires specialised handling of intron
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
sequences, which are absent from mature mRNA. 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
Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA). Sophisticated programs of gene expression are wide ...
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.
''De novo'' assembly can be used to align reads to one another to construct full-length transcript sequences without use of a reference genome. 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 and improved evaluation methods are now available. Annotation-based metrics are better assessments of assembly completeness, such as contig
A contig (from ''contiguous'') is a set of overlapping DNA segments that together represent a consensus region of DNA.Gregory, S. ''Contig Assembly''. Encyclopedia of Life Sciences, 2005.
In bottom-up sequencing projects, a contig refers to ov ...
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.
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. Typical outputs include a table of read counts for each feature supplied to the software; for example, for genes in a general feature format
In bioinformatics, the general feature format (gene-finding format, generic feature format, GFF) is a file format used for describing genes and other features of DNA, RNA and protein sequences.
GFF Versions
The following versions of GFF exist: ...
file. Gene and exon read counts may be calculated quite easily using HTSeq, for example. 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. 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.
Differential expression
Once quantitative counts of each transcript are available, differential gene expression is measured by normalising, modelling, and statistically analysing the data. 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 Binary Alignment Map (BAM) is the comprehensive raw data of genome sequencing; it consists of the lossless, compressed binary
Binary may refer to:
Science and technology Mathematics
* Binary number, a representation of numbers using only tw ...
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.
Legend: mRNA - messenger RNA.
Validation
Transcriptomic analyses may be validated using an independent technique, for example, quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
(qPCR), which is recognisable and statistically assessable. Gene expression is measured against defined standards both for the gene of interest and control
Control may refer to:
Basic meanings Economics and business
* Control (management), an element of management
* Control, an element of management accounting
* Comptroller (or controller), a senior financial officer in an organization
* Controllin ...
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 amplicon
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification (molecular biology), amplification or DNA replication, replication events. It can be formed artificially, using various methods including ...
s smaller than 300 bp, usually toward the 3’ end of the coding region, avoiding the 3’UTR. 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. qPCR validation of RNA-Seq data has generally shown that different RNA-Seq methods are highly correlated.
Functional validation of key genes is an important consideration for post transcriptome planning. Observed gene expression patterns may be functionally linked to a phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
by an independent knock-down/rescue
Rescue comprises responsive operations that usually involve the saving of life, or the urgent treatment of injuries after an accident or a dangerous situation.
Tools used might include search and rescue dogs, mounted search and rescue ho ...
study in the organism of interest.
Applications
Diagnostics and disease profiling
Transcriptomic strategies have seen broad application across diverse areas of biomedical research, including disease diagnosis
Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine " cause and effect". In systems engin ...
and profiling. RNA-Seq approaches have allowed for the large-scale identification of transcriptional start sites
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called ...
, uncovered alternative promoter usage, and novel splicing alterations. These regulatory elements
A regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Regulation of gene expression is an essential feature of all living organisms and vi ...
are important in human disease and, therefore, defining such variants is crucial to the interpretation of disease-association studies. RNA-Seq can also identify disease-associated single nucleotide polymorphism
In genetics, a single-nucleotide polymorphism (SNP ; plural SNPs ) is a germline substitution of a single nucleotide at a specific position in the genome. Although certain definitions require the substitution to be present in a sufficiently larg ...
s (SNPs), allele-specific expression, and gene fusions, which contributes to the understanding of disease causal variants.
Retrotransposon
Retrotransposons (also called Class I transposable elements or transposons via RNA intermediates) are a type of genetic component that copy and paste themselves into different genomic locations (transposon) by converting RNA back into DNA through ...
s are transposable element
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transp ...
s which proliferate within eukaryotic genomes through a process involving reverse transcription
A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...
. RNA-Seq can provide information about the transcription of endogenous retrotransposons that may influence the transcription of neighboring genes by various epigenetic mechanisms
In biology, epigenetics is the study of stable phenotypic changes (known as ''marks'') that do not involve alterations in the DNA sequence. The Greek prefix '' epi-'' ( "over, outside of, around") in ''epigenetics'' implies features that are "o ...
that lead to disease. 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
A T cell is a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell r ...
and B cell receptor
The B cell receptor (BCR) is a transmembrane protein on the surface of a B cell. A B cell receptor is composed of a membrane-bound immunoglobulin molecule and a signal transduction moiety. The former forms a type 1 transmembrane receptor protein, ...
repertoires from patients.
Human and pathogen transcriptomes
RNA-Seq of human pathogen
In biology, a pathogen ( el, πάθος, "suffering", "passion" and , "producer of") in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a germ ...
s has become an established method for quantifying gene expression changes, identifying novel virulence factors
Virulence factors (preferably known as pathogenicity factors or effectors in plant science) are cellular structures, molecules and regulatory systems that enable microbial pathogens (bacteria, viruses, fungi, and protozoa) to achieve the followin ...
, predicting antibiotic resistance
Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. ...
, and unveiling host-pathogen immune interactions. A primary aim of this technology is to develop optimised infection control
Infection prevention and control is the discipline concerned with preventing healthcare-associated infections; a practical rather than academic sub-discipline of epidemiology. In Northern Europe, infection prevention and control is expanded from ...
measures and targeted individualised treatment.
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 network
A gene (or genetic) regulatory network (GRN) is a collection of molecular regulators that interact with each other and with other substances in the cell to govern the gene expression levels of mRNA and proteins which, in turn, determine the fun ...
s in both interaction partners from initial contact through to invasion and the final persistence of the pathogen or clearance by the host immune system.
Responses to environment
Transcriptomics allows identification of genes and pathways that respond to and counteract biotic and abiotic environmental stresses. 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
The chickpea or chick pea (''Cicer arietinum'') is an annual legume of the family Fabaceae, subfamily Faboideae. Its different types are variously known as gram" or Bengal gram, garbanzo or garbanzo bean, or Egyptian pea. Chickpea seeds are high ...
lines at different developmental stages identified distinct transcriptional profiles associated with drought
A drought is defined as drier than normal conditions.Douville, H., K. Raghavan, J. Renwick, R.P. Allan, P.A. Arias, M. Barlow, R. Cerezo-Mota, A. Cherchi, T.Y. Gan, J. Gergis, D. Jiang, A. Khan, W. Pokam Mba, D. Rosenfeld, J. Tierney, an ...
and salinity
Salinity () is the saltiness or amount of salt dissolved in a body of water, called saline water (see also soil salinity). It is usually measured in g/L or g/kg (grams of salt per liter/kilogram of water; the latter is dimensionless and equal ...
stresses, including identifying the role of transcript isoforms of AP2- EREBP. Investigation of gene expression during biofilm
A biofilm comprises any syntrophic consortium of microorganisms in which cells stick to each other and often also to a surface. These adherent cells become embedded within a slimy extracellular matrix that is composed of extracellular ...
formation by the fungal
A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately from th ...
pathogen ''Candida albicans
''Candida albicans'' is an opportunistic pathogenic yeast that is a common member of the human gut flora. It can also survive outside the human body. It is detected in the gastrointestinal tract and mouth in 40–60% of healthy adults. It is us ...
'' revealed a co-regulated set of genes critical for biofilm establishment and maintenance.
Transcriptomic profiling also provides crucial information on mechanisms of drug resistance
Drug resistance is the reduction in effectiveness of a medication such as an antimicrobial or an antineoplastic in treating a disease or condition. The term is used in the context of resistance that pathogens or cancers have "acquired", that is, ...
. Analysis of over 1000 isolates of ''Plasmodium falciparum
''Plasmodium falciparum'' is a Unicellular organism, unicellular protozoan parasite of humans, and the deadliest species of ''Plasmodium'' that causes malaria in humans. The parasite is transmitted through the bite of a female ''Anopheles'' mosqu ...
'', a virulent parasite responsible for malaria in humans, identified that upregulation of the unfolded protein response
The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum (ER) stress. It has been found to be conserved between all mammalian species, as well as yeast and worm organisms.
The UPR is activated in resp ...
and slower progression through the early stages of the asexual intraerythrocytic developmental cycle were associated with artemisinin resistance in isolates from Southeast Asia
Southeast Asia, also spelled South East Asia and South-East Asia, and also known as Southeastern Asia, South-eastern Asia or SEA, is the geographical United Nations geoscheme for Asia#South-eastern Asia, south-eastern region of Asia, consistin ...
.
The use of transcriptomics is also important to investigate responses in the marine environment.[ ] In marine ecology, "stress
Stress may refer to:
Science and medicine
* Stress (biology), an organism's response to a stressor such as an environmental condition
* Stress (linguistics), relative emphasis or prominence given to a syllable in a word, or to a word in a phrase ...
" and "adaptation
In biology, adaptation has three related meanings. Firstly, it is the dynamic evolutionary process of natural selection that fits organisms to their environment, enhancing their evolutionary fitness. Secondly, it is a state reached by the po ...
" have been among the most common research topics, especially related to anthropogenic stress, such as global change
Global change in broad sense refers to planetary-scale changes in the Earth system. It is most commonly use to encompass the variety of changes connected to the rapid increase in human activities which started around mid-20th century, i.e. the ...
and pollution
Pollution is the introduction of contaminants into the natural environment that cause adverse change. Pollution can take the form of any substance (solid, liquid, or gas) or energy (such as radioactivity, heat, sound, or light). Pollutants, the ...
. Most of the studies in this area have been done in animals
Animals are multicellular, eukaryotic organisms in the biological kingdom Animalia. With few exceptions, animals consume organic material, breathe oxygen, are able to move, can reproduce sexually, and go through an ontogenetic stage in ...
, although invertebrates
Invertebrates are a paraphyletic group of animals that neither possess nor develop a vertebral column (commonly known as a ''backbone'' or ''spine''), derived from the notochord. This is a grouping including all animals apart from the chordate ...
have been underrepresented. 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.
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'' ecotype
In evolutionary ecology, an ecotype,Greek: ''οίκος'' = home and ''τύπος'' = type, coined by Göte Turesson in 1922 sometimes called ecospecies, describes a genetically distinct geographic variety, population, or race within a species, ...
s that hyperaccumulate metals correlated genes involved in metal uptake, tolerance, and homeostasis
In biology, homeostasis (British English, British also homoeostasis) Help:IPA/English, (/hɒmɪə(ʊ)ˈsteɪsɪs/) is the state of steady internal, physics, physical, and chemistry, chemical conditions maintained by organism, living systems. Thi ...
with the phenotype. Integration of RNA-Seq datasets across different tissues has been used to improve annotation of gene functions in commercially important organisms (e.g. cucumber
Cucumber (''Cucumis sativus'') is a widely-cultivated Vine#Horticultural climbing plants, creeping vine plant in the Cucurbitaceae family that bears usually cylindrical Fruit, fruits, which are used as culinary vegetables. ) or threatened species (e.g. koala
The koala or, inaccurately, koala bear (''Phascolarctos cinereus''), is an arboreal herbivorous marsupial native to Australia. It is the only extant representative of the family Phascolarctidae and its closest living relatives are the womb ...
).
Assembly of RNA-Seq reads is not dependent on a reference genome
A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species. As they are assemble ...
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
The Douglas fir (''Pseudotsuga menziesii'') is an evergreen conifer species in the pine family, Pinaceae. It is native to western North America and is also known as Douglas-fir, Douglas spruce, Oregon pine, and Columbian pine. There are three va ...
breeding programs was created by ''de novo'' transcriptome analysis in the absence of a sequenced genome. 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. RNA-Seq can also be used to identify previously unknown protein coding region
The coding region of a gene, also known as the coding sequence (CDS), is the portion of a gene's DNA or RNA that codes for protein. Studying the length, composition, regulation, splicing, structures, and functions of coding regions compared to n ...
s in existing sequenced genomes.
A transcriptome based aging clock
Aging-related preventive interventions are not possible without personal aging speed measurement. The most up to date and complex way to measure aging rate is by using varying biomarkers of human aging is based on the utilization of deep neural networks which may be trained on any type of omics biological data to predict the subject's age. Aging has been shown to be a strong driver of transcriptome changes. Aging clocks based on transcriptomes have suffered from considerable variation in the data and relatively low accuracy. However an approach that uses temporal scaling and binarization of transcriptomes to define a gene set that predicts biological age with an accuracy allowed to reach an assessment close to the theoretical limit.
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
In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is ...
, DNA replication
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
, RNA splicing
RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns (non-coding regions of RNA) and ''splicing'' b ...
, and transcriptional regulation
In molecular biology and genetics, transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity. A single gene can be regulated in a range of ways, from alt ...
). Many of these ncRNAs affect disease states, including cancer, cardiovascular, and neurological diseases.
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.
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
The branches of science known informally as omics are various disciplines in biology whose names end in the suffix '' -omics'', such as genomics, proteomics, metabolomics, metagenomics, phenomics and transcriptomics. Omics aims at the collect ...
** Genomics
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
** Proteomics
Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
** Metabolomics
Metabolomics is the scientific study of chemical processes involving metabolites, the small molecule substrates, intermediates, and products of cell metabolism. Specifically, metabolomics is the "systematic study of the unique chemical fingerprin ...
** Venomics
Venomics is the large-scale study of proteins associated with venom. Venom is a toxic substance secreted by animals, which is typically injected either offensively or defensively into prey or aggressors, respectively.
Venom is produced in a specia ...
References
Notes
Further reading
* {{cite journal , vauthors = Lowe R, Shirley N, Bleackley M, Dolan S, Shafee T , title = Transcriptomics technologies , journal = PLOS Computational Biology , volume = 13 , issue = 5 , pages = e1005457 , date = May 2017 , pmid = 28545146 , pmc = 5436640 , doi = 10.1371/journal.pcbi.1005457 , bibcode = 2017PLSCB..13E5457L
Comparative Transcriptomics Analysis
i
Reference Module in Life Sciences
* Software used in transcriptomics:
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cufflinks
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kallisto
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tophat
Omics
Molecular biology