Public peer review comments can be seen here
- 1 History
- 2 Data gathering
- 3 Data analysis
- 4 Applications
- 5 Transcriptome databases
- 6 Conclusions
- 7 See also
- 8 Pages that should link here
- 9 References
Public peer review comments will be posted here
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.
The first attempts to study the whole transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a widespread discipline. Transcriptomics has been defined by repeated technological innovations that transform the field. 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 capture all sequences.
Measuring the expression of an organism’s genes in different tissues, conditions, or time points gives information on how genes are regulated and reveal details of an organism’s biology. It can also help to infer the functions of previously unannotated genes. Transcriptomic 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.
Transcriptomics has been characterised by the development of new techniques which have redefined what is possible every decade or so and render previous technologies obsolete (figure 1). The first attempt at capturing a partial human transcriptome was published in 1991 and reported 609 mRNA sequences from the human brain. 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 states, tissues or even single cells are now routinely generated. This explosion in transcriptomics has been driven by the rapid development of new technologies with improved sensitivity and economy (table 1).
Studies of individual transcripts were being performed several decades before any transcriptomics approaches were available. Libraries of silkmoth mRNAs were collected and converted to complementary DNA (cDNA) for storage using reverse transcriptase in the late 1970s. In the 1980s, low-throughput Sanger sequencing began to be used to sequence random individual transcripts from these libraries, called Expressed Sequence Tags (ESTs). The Sanger method of sequencing was predominant until the advent of high-throughput methods such as sequencing by synthesis (Solexa/Illumina). ESTs came to prominence during the 1990’s as an efficient method to determine the gene content of an organism without sequencing the entire genome. Quantification of individual transcripts by Northern blotting, nylon membrane arrays, and later Reverse Transcriptase quantitative PCR (RT-qPCR) were also popular, 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.
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 (SAGE), which worked by Sanger sequencing 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 e.g. splice variants.
Development of contemporary techniques
|Input RNA amount||Low ~ 1 ng total RNA||High ~ 1 μg mRNA|
|Labour intensity||High (sample preparation and data analysis)||Low|
|Prior knowledge||None required, though genome sequence useful||Reference transcripts required for probes|
|Quantitation accuracy||~90% (limited by sequence coverage)||>90% (limited by fluorescence detection accuracy)|
|Sequence resolution||Can detect SNPs and splice variants (limited by sequencing accuracy of ~99%)||Dedicated arrays can detect splice variants (limited by probe design and cross-hybridisation)|
|Sensitivity||10-6 (limited by sequence coverage)||10-3 (limited by fluorescence detection)|
|Dynamic range||>105 (limited by sequence coverage)||103-104 (limited by fluorescence saturation)|
The dominant contemporary techniques, microarrays and RNA-Seq, 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 probes were first published in 1995. Microarray technology allowed the assay of 1000s of transcripts simultaneously, at a greatly reduced cost per gene and labour saving. Both spotted oligonucleotide arrays and Affymetrix 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 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.
RNA-Seq refers to the sequencing of transcript cDNAs, where 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 (MPSS) was an early example based on generating 16-20 bp sequences via a complex series of hybridisations, and was used in 2004 to validate the expression of 104 genes in Arabidopsis thaliana. The earliest RNA-Seq work was published in 2006 with 105 transcripts sequenced using the 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 109 transcript sequences to be recorded. This yield is now sufficient for accurate quantitation of entire human transcriptomes.
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 techniques are broadly similar and involve: mechanical disruption of cells or tissues, disruption of RNase with chaotropic salts, 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. Isolated RNA may additionally be treated with DNase to digest any traces of DNA. It is necessary to enrich messenger RNA as total RNA extracts are typically 98% ribosomal RNA. 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 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 (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. The Sanger method of sequencing was predominant until the advent of high-throughput methods such as sequencing by synthesis (Solexa/Illumina). Because ESTs don't require prior knowledge of the organism from which they come, they can also 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 GeneChip was designed from 350,000 previously sequenced ESTs.
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 (figure 2). cDNA is generated from the RNA but is then digested into 11 bp ‘tag’ fragments using restriction enzymes that cut at a specific sequence, and 11 base pairs along from that sequence. These cDNA tags are then concatenated head-to-tail into long strands (>500 bp) and sequenced using low-throughput, but long read length methods such as Sanger sequencing. Once the sequences are deconvoluted into their original 11 bp tags. If a reference genome is available, these tags can sometimes be aligned to identify their corresponding gene. If a reference genome is unavailable, the tags can simply be directly used as diagnostic markers if found to be differentially expressed in a disease state.
The Cap Analysis of 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 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.
Principles and advances
Microarrays consist of short nucleotide oligomers, known as "probes", which are arrayed on a solid substrate (e.g. glass). Transcript abundance is determined by hybridisation of fluorescently labelled transcripts to these probes (figure 3). The fluorescence intensity at each probe location on the array indicates the transcript abundance for that probe sequence.
Microarrays require some prior knowledge of the organism of interest, for example, in the form of an annotated genome sequence, or a library of ESTs that can be used to generate the probes for the array.
The manufacture of microarrays relies on micro and nanofabrication techniques. Microarrays for transcriptomics typically fall into one of two broad categories: low-density spotted arrays or high-density short probe arrays. Transcript presence may be recorded with single- or dual-channel detection of fluorescent tags.
Spotted low-density arrays typically feature picolitre drops of a range of purified cDNAs arrayed on the surface of a glass slide. The probes are longer than those of high-density arrays and typically lack the transcript resolution of high-density arrays. Spotted arrays use different fluorophores for test and control samples, and the ratio of fluorescence is used to calculate a relative measure of abundance. High-density arrays use single channel detection, 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 one gene.
NimbleGen arrays are a high-density array produced by a maskless-photochemistry method, which permits flexible manufacture of arrays in small or large numbers. These arrays have 100,000s of 45 to 85-mer probes and are hybridised with a one-colour labelled sample for expression analysis. Some designs incorporate up to 12 independent arrays per slide.
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 (figure 4). 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). The typical dynamic range of 5 orders of magnitude for RNA-Seq is 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 allowed 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 signal is very low for 100 bp reads in non-repetitive regions.
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. 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 RNA-Seq where transcriptomes of individual cells are directly interrogated in fixed tissues.
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 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. 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. The fragmentation method is a key aspect of sequencing library construction. It may incorporate chemical hydrolysis, nebulisation, or sonication of RNA, or utilise simultaneous fragmentation and tagging of cDNA by 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 can be used to provide quality control assessment of library preparation and sequencing, in terms of GC-content, fragment length, as well as the bias due to fragment position within a transcript. 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. UMIs provide an absolute scale for quantification and 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 discovery. Strand-specific RNA-Seq methods preserve the strand 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.
|Platform||Commercial release||Typical read length||Maximum throughput per run||Single read accuracy||RNA-Seq runs deposited in the NCBI SRA (Oct 2016)|
|454 Life Sciences||2005||700 bp||0.7 Gbp||99.9%||3548|
|Illumina||2006||50-300 bp||900 Gbp||99.9%||362903|
|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|
Currently, RNA-Seq relies on copying of RNA molecules into cDNA molecules prior to sequencing, hence the subsequent platforms are the same for transcriptomic and genomic data (table 2). Consequently, the development of DNA sequencing technologies has been a defining feature of RNA-Seq. Direct sequencing of RNA using nanopore sequencing represents a current state-of-the-art RNA-Seq technique in its infancy (in pre-release beta testing as of 2016). However, 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 and therefore requires a tailored sequence yield for an effective transcriptome. Early studies determined suitable thresholds empirically, but as the technology matured, suitable coverage is 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.
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 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-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 comprised 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. 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). RNA-Seq datasets can be uploaded via the Gene Expression Omnibus.
Microarray image processing must correctly identify the regular grid of features within an image and independently quantify the fluorescence intensity for each feature (figure 5). 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 a random or ordered 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’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.
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 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, either in a Unix environment or within the R/Bioconductor statistical environment.
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 for: high quality scores for base calls, GC content matches the expected distribution, the over representation of particular short sequence motifs (k-mers), and an unexpectedly high read duplication rate. Several software options exist for sequence quality analysis, including FastQC and FaQCs. Abnormalities identified may be removed by trimming, or tagged for special treatment during later processes.
In order to link sequence read abundance to 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 are further reducing multimapping reads. A list of currently available high-throughput sequence aligners is maintained by the EBI.
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. 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 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.
De novo assembly can be used to align reads to one another to construct full-length transcript sequences without use of a reference genome (table 3). Challenges particular to de novo assembly include larger computational requirements compared to a reference-based transcriptome, additional validation of gene variants or fragments, additional annotation of assembled transcripts. The first metrics used to describe transcriptome assemblies, such as N50, have been shown to be misleading and subsequently improved evaluation methods are now available. 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.
|Software||Released||Last Updated||Resource load||Strengths and weaknesses|
|Velvet-Oases||2008||2011||Heavy||The original short read assembler, now largely superseded.|
|SOAPdenovo-trans||2011||2015||Moderate||Early short read assembler, updated for transcript assembly|
|Trans-ABySS||2010||2016||Moderate||Short reads, large genomes, MPI-parallel version available|
|Trinity||2011||2017||Moderate||Short reads, large genomes, memory intensive.|
|miraEST||1999||2016||Moderate||Repetitive sequences, hybrid data input, wide range of sequence platforms accepted.|
|Newbler||2004||2012||Heavy||Specialised for Roche 454 sequence, homo-polymer error handling|
|CLC genomics workbench||2008||2014||Light||Graphical user interface, hybrid data|
Quantification of sequence alignments may be performed at the gene, exon, or transcript level. Typical outputs include a table of reads 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. 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 all together. The kallisto method combines pseudoalignment and quantification into a single step that runs 2 orders of magnitude faster than comparable methods such as tophat/cufflinks, with less computational burden.
Once quantitative counts of each transcript are available, differential gene expression is measured by normalising, modelling, and statistically analysing the data (figure 6). Examples of dedicated software are described in table 4. Most read a table of genes and read counts as their input, but some, 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.
|Cuffdiff2||Unix-based||Transcript analysis at isoform-level|
|EdgeR||R/Bioconductor||Any count-based genomic data|
|DEseq2||R/Bioconductor||Flexible data types, low replication|
|Limma/Voom||R/Bioconductor||Microarray or RNA-Seq data, isoform analysis, flexible experiment design|
Transcriptomic analyses may be validated using an independent technique, for example, quantitative PCR (qPCR), which is recognisable and statistically assessable. 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. 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 by an independent knock-down/rescue study in the organism of interest.
Diagnostics and disease profiling
Transcriptomic strategies have seen broad application across diverse areas of biomedical research, including disease diagnosis and profiling. 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. RNA-Seq can also identify disease-associated single nucleotide polymorphisms (SNP), allele-specific expression and gene fusions contributing to our understanding of disease causal variants.
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. 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.
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. A primary aim of this technology is to develop optimised infection control measures and targeted individualised treatment.
Transcriptomic analysis has predominantly focused on either the host or the pathogen. Dual RNA-Seq has recently 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.
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 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. 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.
Transcriptomic profiling also provides crucial information on mechanisms of drug resistance. Analysis of over 1000 Plasmodium falciparum isolates 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.
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. Integration of RNA-Seq datasets across different tissues has been used to improve annotation of gene functions in commercially important organisms (e.g. cucumber) or threatened species (e.g. koala).
Assembly of RNA-Seq reads is not dependent on a reference genome and so 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. Similarly, genes that function in the development of cardiac, muscle and nervous tissue in lobster 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 regions in existing sequenced genomes.
Transcriptomics is most commonly applied to the mRNA content of the cell. However, the same techniques are equally applicable to non-coding RNAs 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). Many of these ncRNAs affect disease states, including cancer, cardiovascular and neurological diseases.
Transcriptomics studies generate large amounts of data that has 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 (table 5). For example, as of 2016, the Gene Expression Omnibus contained millions of experiments.
|Gene Expression Omnibus||NCBI||Microarray RNA-Seq||First transcriptomics database to accept data from any source. Introduced MIAME and MINSEQE community standards that define necessary experiment metadata to ensure effective interpretation and repeatability.|
|ArrayExpress||ENA||Microarray||Imports datasets from the Gene Expression Omnibus and accepts direct submissions. Processed data and experiment metadata is stored at ArrayExpress, while the raw sequence reads are held at the ENA. Complies with MIAME and MINSEQE standards.|
|Expression Atlas||EBI||Microarray RNA-Seq||Tissue-specific gene expression database for animals and plants. Displays secondary analyses and visualisation, such as functional enrichment of Gene Ontology terms, InterPro domains, or pathways. Links to protein abundance data where available.|
|Genevestigator||Privately curated||Microarray RNA-Seq||Contains manual curations of public transcriptome datasets, focusing on medical and plant biology data. Individual experiments are normalised across the full database, to allow comparison of gene expression across diverse experiments. Full functionality requires licence purchase, with free access to a limited functionality.|
|RefEx||DDBJ||All||Human, mouse, and rat transcriptomes from 40 different organs. Gene expression visualised as heatmaps projected onto 3D representations of anatomical structures.|
|NONCODE||noncode.org||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
- Journal version only
Transcriptomics has revolutionised our understanding of how genomes are expressed. Over the last three decades, new technologies have redefined what is possible to investigate, and integration with other -omics technologies is giving an increasingly integrated view of the complexities of cellular life. The plummeting cost of transcriptomics studies have made them possible for small laboratories, and large-scale transcriptomics consortia are able to undertake experiments comparing transcriptomes of thousands of organisms, tissues, or environmental conditions. This trend is likely to continue as sequencing technologies improve.
- DNA Microarray
- Expressed sequence tag
- Gene expression profiling
- Systems biology
- Gene prediction
- Primary transcript
- Microarray databases
- De novo transcriptome assembly
- Exome sequencing
- List of RNA-Seq bioinformatics tools
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ a b Adams MD, Kelley JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B & Moreno RF (1991) Complementary DNA sequencing: expressed sequence tags and human genome project Science 252:1651-6 [PMID: 2047873]
- ^ Pan Q, Shai O, Lee LJ, Frey BJ & Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing Nat. Genet. 40:1413-5 [PMID: 18978789][DOI]
- ^ a b Sultan M, Schulz MH, Richard H, Magen A, Klingenhoff A, Scherf M, Seifert M, Borodina T, Soldatov A, Parkhomchuk D, Schmidt D, O'Keeffe S, Haas S, Vingron M, Lehrach H & Yaspo ML (2008) A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome Science 321:956-60 [PMID: 18599741][DOI]
- ^ Lappalainen T, Sammeth M, Friedländer MR, 't Hoen PA, Monlong J, Rivas MA, Gonzàlez-Porta M, Kurbatova N, Griebel T, Ferreira PG, Barann M, Wieland T, Greger L, van Iterson M, Almlöf J, Ribeca P, Pulyakhina I, Esser D, Giger T, Tikhonov A, Sultan M, Bertier G, MacArthur DG, Lek M, Lizano E, Buermans HP, Padioleau I, Schwarzmayr T, Karlberg O, Ongen H, Kilpinen H, Beltran S, Gut M, Kahlem K, Amstislavskiy V, Stegle O, Pirinen M, Montgomery SB, Donnelly P, McCarthy MI, Flicek P, Strom TM, Lehrach H, Schreiber S, Sudbrak R, Carracedo A, Antonarakis SE, Häsler R, Syvänen AC, van Ommen GJ, Brazma A, Meitinger T, Rosenstiel P, Guigó R, Gut IG, Estivill X & Dermitzakis ET (2013) Transcriptome and genome sequencing uncovers functional variation in humans Nature 501:506-11 [PMID: 24037378][DOI]
- ^ a b Melé M, Ferreira PG, Reverter F, DeLuca DS, Monlong J, Sammeth M, Young TR, Goldmann JM, Pervouchine DD, Sullivan TJ, Johnson R, Segrè AV, Djebali S, Niarchou A, Wright FA, Lappalainen T, Calvo M, Getz G, Dermitzakis ET, Ardlie KG & Guigó R (2015) Human genomics. The human transcriptome across tissues and individuals Science 348:660-5 [PMID: 25954002][DOI]
- ^ Sandberg R (2014) Entering the era of single-cell transcriptomics in biology and medicine Nat. Methods 11:22-4 [PMID: 24524133]
- ^ Kolodziejczyk AA, Kim JK, Svensson V, Marioni JC & Teichmann SA (2015) The technology and biology of single-cell RNA sequencing Mol. Cell 58:610-20 [PMID: 26000846][DOI]
- ^ a b c d e f McGettigan PA (2013) Transcriptomics in the RNA-seq era Curr Opin Chem Biol 17:4-11 [PMID: 23290152][DOI]
- ^ a b c d e f g h i j k Wang Z, Gerstein M & Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics Nat. Rev. Genet. 10:57-63 [PMID: 19015660][DOI]
- ^ a b c Ozsolak F & Milos PM (2011) RNA sequencing: advances, challenges and opportunities Nat. Rev. Genet. 12:87-98 [PMID: 21191423][DOI]
- ^ a b c Morozova O, Hirst M & Marra MA (2009) Applications of new sequencing technologies for transcriptome analysis Annu Rev Genomics Hum Genet 10:135-51 [PMID: 19715439][DOI]
- ^ Sim GK, Kafatos FC, Jones CW, Koehler MD, Efstratiadis A & Maniatis T (1979) Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families Cell 18:1303-16 [PMID: 519770]
- ^ Sutcliffe JG, Milner RJ, Bloom FE & Lerner RA (1982) Common 82-nucleotide sequence unique to brain RNA Proc. Natl. Acad. Sci. U.S.A. 79:4942-6 [PMID: 6956902]
- ^ Putney SD, Herlihy WC & Schimmel P (1983) A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing Nature 302:718-21 [PMID: 6687628]
- ^ a b c d Marra MA, Hillier L & Waterston RH (1998) Expressed sequence tags--ESTablishing bridges between genomes Trends Genet. 14:4-7 [PMID: 9448457][DOI]
- ^ Alwine JC, Kemp DJ & Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes Proc. Natl. Acad. Sci. U.S.A. 74:5350-4 [PMID: 414220]
- ^ Becker-André M & Hahlbrock K (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY) Nucleic Acids Res. 17:9437-46 [PMID: 2479917]
- ^ Piétu G, Mariage-Samson R, Fayein NA, Matingou C, Eveno E, Houlgatte R, Decraene C, Vandenbrouck Y, Tahi F, Devignes MD, Wirkner U, Ansorge W, Cox D, Nagase T, Nomura N & Auffray C (1999) The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics Genome Res. 9:195-209 [PMID: 10022985]
- ^ Velculescu VE, Zhang L, Zhou W, Vogelstein J, Basrai MA, Bassett DE, Hieter P, Vogelstein B & Kinzler KW (1997) Characterization of the yeast transcriptome Cell 88:243-51 [PMID: 9008165]
- ^ a b c Velculescu VE, Zhang L, Vogelstein B & Kinzler KW (1995) Serial analysis of gene expression Science 270:484-7 [PMID: 7570003]
- ^ Audic S & Claverie JM (1997) The significance of digital gene expression profiles Genome Res. 7:986-95 [PMID: 9331369]
- ^ a b c d e Mantione KJ, Kream RM, Kuzelova H, Ptacek R, Raboch J, Samuel JM & Stefano GB (2014) Comparing bioinformatic gene expression profiling methods: microarray and RNA-Seq Med Sci Monit Basic Res 20:138-42 [PMID: 25149683][DOI]
- ^ Zhao S, Fung-Leung WP, Bittner A, Ngo K & Liu X (2014) Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells PLoS ONE 9:e78644 [PMID: 24454679][DOI]
- ^ a b Hashimshony T, Wagner F, Sher N & Yanai I (2012) CEL-Seq: single-cell RNA-Seq by multiplexed linear amplification Cell Rep 2:666-73 [PMID: 22939981][DOI]
- ^ Stears RL, Getts RC & Gullans SR (2000) A novel, sensitive detection system for high-density microarrays using dendrimer technology Physiol. Genomics 3:93-9 [PMID: 11015604]
- ^ a b c d e f Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ a b Black MB, Parks BB, Pluta L, Chu TM, Allen BC, Wolfinger RD & Thomas RS (2014) Comparison of microarrays and RNA-seq for gene expression analyses of dose-response experiments Toxicol. Sci. 137:385-403 [PMID: 24194394][DOI]
- ^ Marioni JC, Mason CE, Mane SM, Stephens M & Gilad Y (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays Genome Res. 18:1509-17 [PMID: 18550803][DOI]
- ^ SEQC/MAQC-III Consortium (2014) A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium Nat. Biotechnol. 32:903-14 [PMID: 25150838][DOI]
- ^ Chen JJ, Hsueh HM, Delongchamp RR, Lin CJ & Tsai CA (2007) Reproducibility of microarray data: a further analysis of microarray quality control (MAQC) data BMC Bioinformatics 8:412 [PMID: 17961233][DOI]
- ^ Larkin JE, Frank BC, Gavras H, Sultana R & Quackenbush J (2005) Independence and reproducibility across microarray platforms Nat. Methods 2:337-44 [PMID: 15846360][DOI]
- ^ a b Nelson NJ (2001) Microarrays have arrived: gene expression tool matures J. Natl. Cancer Inst. 93:492-4 [PMID: 11287436]
- ^ Schena M, Shalon D, Davis RW & Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray Science 270:467-70 [PMID: 7569999]
- ^ a b Pozhitkov AE, Tautz D & Noble PA (2007) Oligonucleotide microarrays: widely applied--poorly understood Brief Funct Genomic Proteomic 6:141-8 [PMID: 17644526][DOI]
- ^ a b Heller MJ (2002) DNA microarray technology: devices, systems, and applications Annu Rev Biomed Eng 4:129-53 [PMID: 12117754][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ Brenner S, Johnson M, Bridgham J, Golda G, Lloyd DH, Johnson D, Luo S, McCurdy S, Foy M, Ewan M, Roth R, George D, Eletr S, Albrecht G, Vermaas E, Williams SR, Moon K, Burcham T, Pallas M, DuBridge RB, Kirchner J, Fearon K, Mao J & Corcoran K (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays Nat. Biotechnol. 18:630-4 [PMID: 10835600][DOI]
- ^ Meyers BC, Vu TH, Tej SS, Ghazal H, Matvienko M, Agrawal V, Ning J & Haudenschild CD (2004) Analysis of the transcriptional complexity of Arabidopsis thaliana by massively parallel signature sequencing Nat. Biotechnol. 22:1006-11 [PMID: 15247925][DOI]
- ^ a b Bainbridge MN, Warren RL, Hirst M, Romanuik T, Zeng T, Go A, Delaney A, Griffith M, Hickenbotham M, Magrini V, Mardis ER, Sadar MD, Siddiqui AS, Marra MA & Jones SJ (2006) Analysis of the prostate cancer cell line LNCaP transcriptome using a sequencing-by-synthesis approach BMC Genomics 7:246 [PMID: 17010196][DOI]
- ^ Mortazavi A, Williams BA, McCue K, Schaeffer L & Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq Nat. Methods 5:621-8 [PMID: 18516045][DOI]
- ^ Wilhelm BT, Marguerat S, Watt S, Schubert F, Wood V, Goodhead I, Penkett CJ, Rogers J & Bähler J (2008) Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution Nature 453:1239-43 [PMID: 18488015][DOI]
- ^ a b Chomczynski P & Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal. Biochem. 162:156-9 [PMID: 2440339][DOI]
- ^ a b Chomczynski P & Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on Nat Protoc 1:581-5 [PMID: 17406285][DOI]
- ^ Grillo M & Margolis FL (1990) Use of reverse transcriptase polymerase chain reaction to monitor expression of intronless genes BioTechniques 9:262, 264, 266-8 [PMID: 1699561]
- ^ Bryant S & Manning DL (1998) Isolation of messenger RNA Methods Mol. Biol. 86:61-4 [PMID: 9664454][DOI]
- ^ Zhao W, He X, Hoadley KA, Parker JS, Hayes DN & Perou CM (2014) Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling BMC Genomics 15:419 [PMID: 24888378][DOI]
- ^ Close TJ, Wanamaker SI, Caldo RA, Turner SM, Ashlock DA, Dickerson JA, Wing RA, Muehlbauer GJ, Kleinhofs A & Wise RP (2004) A new resource for cereal genomics: 22K barley GeneChip comes of age Plant Physiol. 134:960-8 [PMID: 15020760][DOI]
- ^ Shiraki T, Kondo S, Katayama S, Waki K, Kasukawa T, Kawaji H, Kodzius R, Watahiki A, Nakamura M, Arakawa T, Fukuda S, Sasaki D, Podhajska A, Harbers M, Kawai J, Carninci P & Hayashizaki Y (2003) Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage Proc. Natl. Acad. Sci. U.S.A. 100:15776-81 [PMID: 14663149][DOI]
- ^ Romanov V, Davidoff SN, Miles AR, Grainger DW, Gale BK & Brooks BD (2014) A critical comparison of protein microarray fabrication technologies Analyst 139:1303-26 [PMID: 24479125][DOI]
- ^ a b Barbulovic-Nad I, Lucente M, Sun Y, Zhang M, Wheeler AR & Bussmann M (2006) Bio-microarray fabrication techniques--a review Crit. Rev. Biotechnol. 26:237-59 [PMID: 17095434][DOI]
- ^ Auburn RP, Kreil DP, Meadows LA, Fischer B, Matilla SS & Russell S (2005) Robotic spotting of cDNA and oligonucleotide microarrays Trends Biotechnol. 23:374-9 [PMID: 15978318][DOI]
- ^ Shalon D, Smith SJ & Brown PO (1996) A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization Genome Res. 6:639-45 [PMID: 8796352]
- ^ Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H & Brown EL (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays Nat. Biotechnol. 14:1675-80 [PMID: 9634850][DOI]
- ^ Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B & Speed TP (2003) Summaries of Affymetrix GeneChip probe level data Nucleic Acids Res. 31:e15 [PMID: 12582260]
- ^ Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, Nair P, Brothman AR & Stallings RL (2005) Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH Genes Chromosomes Cancer 44:305-19 [PMID: 16075461][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ a b Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M & Snyder M (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing Science 320:1344-9 [PMID: 18451266][DOI]
- ^ Su Z, Fang H, Hong H, Shi L, Zhang W, Zhang W, Zhang Y, Dong Z, Lancashire LJ, Bessarabova M, Yang X, Ning B, Gong B, Meehan J, Xu J, Ge W, Perkins R, Fischer M & Tong W (2014) An investigation of biomarkers derived from legacy microarray data for their utility in the RNA-seq era Genome Biol. 15:523 [PMID: 25633159][DOI]
- ^ Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SS, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J & Church GM (2014) Highly multiplexed subcellular RNA sequencing in situ Science 343:1360-3 [PMID: 24578530][DOI]
- ^ Shendure J & Ji H (2008) Next-generation DNA sequencing Nat. Biotechnol. 26:1135-45 [PMID: 18846087][DOI]
- ^ Lahens NF, Kavakli IH, Zhang R, Hayer K, Black MB, Dueck H, Pizarro A, Kim J, Irizarry R, Thomas RS, Grant GR & Hogenesch JB (2014) IVT-seq reveals extreme bias in RNA sequencing Genome Biol. 15:R86 [PMID: 24981968][DOI]
- ^ Knierim E, Lucke B, Schwarz JM, Schuelke M & Seelow D (2011) Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing PLoS ONE 6:e28240 [PMID: 22140562][DOI]
- ^ Parekh S, Ziegenhain C, Vieth B, Enard W & Hellmann I (2016) The impact of amplification on differential expression analyses by RNA-seq Sci Rep 6:25533 [PMID: 27156886][DOI]
- ^ Shanker S, Paulson A, Edenberg HJ, Peak A, Perera A, Alekseyev YO, Beckloff N, Bivens NJ, Donnelly R, Gillaspy AF, Grove D, Gu W, Jafari N, Kerley-Hamilton JS, Lyons RH, Tepper C & Nicolet CM (2015) Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA J Biomol Tech 26:4-18 [PMID: 25649271][DOI]
- ^ Jiang L, Schlesinger F, Davis CA, Zhang Y, Li R, Salit M, Gingeras TR & Oliver B (2011) Synthetic spike-in standards for RNA-seq experiments Genome Res. 21:1543-51 [PMID: 21816910][DOI]
- ^ Kivioja T, Vähärautio A, Karlsson K, Bonke M, Enge M, Linnarsson S & Taipale J (2011) Counting absolute numbers of molecules using unique molecular identifiers Nat. Methods 9:72-4 [PMID: 22101854][DOI]
- ^ Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K & Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell Nat. Methods 6:377-82 [PMID: 19349980][DOI]
- ^ Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, Lönnerberg P & Linnarsson S (2014) Quantitative single-cell RNA-seq with unique molecular identifiers Nat. Methods 11:163-6 [PMID: 24363023][DOI]
- ^ Jaitin DA, Kenigsberg E, Keren-Shaul H, Elefant N, Paul F, Zaretsky I, Mildner A, Cohen N, Jung S, Tanay A & Amit I (2014) Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types Science 343:776-9 [PMID: 24531970][DOI]
- ^ a b Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A & Regev A (2010) Comprehensive comparative analysis of strand-specific RNA sequencing methods Nat. Methods 7:709-15 [PMID: 20711195][DOI]
- ^ Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP & Gu Y (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers BMC Genomics 13:341 [PMID: 22827831][DOI]
- ^ a b Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L & Law M (2012) Comparison of next-generation sequencing systems J. Biomed. Biotechnol. 2012:251364 [PMID: 22829749][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).The NCBI Sequence Read Archive (SRA) was searched using “RNA-Seq[Strategy]” and one of "LS454[Platform]”, “Illumina[platform]”, "ABI Solid[Platform]”, "Ion Torrent[Platform]”, "PacBio SMRT"[Platform]” to report the number of RNA-Seq runs deposited for each platform.
- ^ Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J & Pallen MJ (2012) Performance comparison of benchtop high-throughput sequencing platforms Nat. Biotechnol. 30:434-9 [PMID: 22522955][DOI]
- ^ Goodwin S, McPherson JD & McCombie WR (2016) Coming of age: ten years of next-generation sequencing technologies Nat. Rev. Genet. 17:333-51 [PMID: 27184599][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ Loman NJ, Quick J & Simpson JT (2015) A complete bacterial genome assembled de novo using only nanopore sequencing data Nat. Methods 12:733-5 [PMID: 26076426][DOI]
- ^ Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, Thompson JF, Bowers J, Jarosz M & Milos PM (2009) Direct RNA sequencing Nature 461:814-8 [PMID: 19776739][DOI]
- ^ Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND & Betel D (2013) Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data Genome Biol. 14:R95 [PMID: 24020486][DOI]
- ^ ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome Nature 489:57-74 [PMID: 22955616][DOI]
- ^ Sloan CA, Chan ET, Davidson JM, Malladi VS, Strattan JS, Hitz BC, Gabdank I, Narayanan AK, Ho M, Lee BT, Rowe LD, Dreszer TR, Roe G, Podduturi NR, Tanaka F, Hong EL & Cherry JM (2016) ENCODE data at the ENCODE portal Nucleic Acids Res. 44:D726-32 [PMID: 26527727][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ Hart SN, Therneau TM, Zhang Y, Poland GA & Kocher JP (2013) Calculating sample size estimates for RNA sequencing data J. Comput. Biol. 20:970-8 [PMID: 23961961][DOI]
- ^ a b Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, Szcześniak MW, Gaffney DJ, Elo LL, Zhang X & Mortazavi A (2016) A survey of best practices for RNA-seq data analysis Genome Biol. 17:13 [PMID: 26813401][DOI]
- ^ Kodama Y, Shumway M & Leinonen R (2012) The Sequence Read Archive: explosive growth of sequencing data Nucleic Acids Res. 40:D54-6 [PMID: 22009675][DOI]
- ^ Lua error: bad argument #3 to 'gsub' (function or table or string expected, got nil).
- ^ Nakamura K, Oshima T, Morimoto T, Ikeda S, Yoshikawa H, Shiwa Y, Ishikawa S, Linak MC, Hirai A, Takahashi H, Altaf-Ul-Amin M, Ogasawara N & Kanaya S (2011) Sequence-specific error profile of Illumina sequencers Nucleic Acids Res. 39:e90 [PMID: 21576222][DOI]
- ^ Van Verk MC, Hickman R, Pieterse CM & Van Wees SC (2013) RNA-Seq: revelation of the messengers Trends Plant Sci. 18:175-9 [PMID: 23481128][DOI]
- ^ Huber W, Carey VJ, Gentleman R, Anders S, Carlson M, Carvalho BS, Bravo HC, Davis S, Gatto L, Girke T, Gottardo R, Hahne F, Hansen KD, Irizarry RA, Lawrence M, Love MI, MacDonald J, Obenchain V, Oleś AK, Pagès H, Reyes A, Shannon P, Smyth GK, Tenenbaum D, Waldron L & Morgan M (2015) Orchestrating high-throughput genomic analysis with Bioconductor Nat. Methods 12:115-21 [PMID: 25633503][DOI]
- ^ Andrews S. (2010). FastQC: a quality control tool for high throughput sequence data. Available online at:http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
- ^ Lo CC & Chain PS (2014) Rapid evaluation and quality control of next generation sequencing data with FaQCs BMC Bioinformatics 15:366 [PMID: 25408143][DOI]
- ^ HTS Mappers. http://www.ebi.ac.uk/~nf/hts_mappers/
- ^ Fonseca NA, Rung J, Brazma A & Marioni JC (2012) Tools for mapping high-throughput sequencing data Bioinformatics 28:3169-77 [PMID: 23060614][DOI]
- ^ a b Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ & Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation Nat. Biotechnol. 28:511-5 [PMID: 20436464][DOI]
- ^ Miller JR, Koren S & Sutton G (2010) Assembly algorithms for next-generation sequencing data Genomics 95:315-27 [PMID: 20211242][DOI]
- ^ O'Neil ST & Emrich SJ (2013) Assessing De Novo transcriptome assembly metrics for consistency and utility BMC Genomics 14:465 [PMID: 23837739][DOI]
- ^ Smith-Unna R, Boursnell C, Patro R, Hibberd JM & Kelly S (2016) TransRate: reference-free quality assessment of de novo transcriptome assemblies Genome Res. 26:1134-44 [PMID: 27252236][DOI]
- ^ Li B, Fillmore N, Bai Y, Collins M, Thomson JA, Stewart R & Dewey CN (2014) Evaluation of de novo transcriptome assemblies from RNA-Seq data Genome Biol. 15:553 [PMID: 25608678][DOI]
- ^ Zerbino DR & Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs Genome Res. 18:821-9 [PMID: 18349386][DOI]
- ^ Schulz MH, Zerbino DR, Vingron M & Birney E (2012) Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels Bioinformatics 28:1086-92 [PMID: 22368243][DOI]
- ^ Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S, Zhou X, Lam TW, Li Y, Xu X, Wong GK & Wang J (2014) SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads Bioinformatics 30:1660-6 [PMID: 24532719][DOI]
- ^ Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K, Lee S, Okada HM, Qian JQ, Griffith M, Raymond A, Thiessen N, Cezard T, Butterfield YS, Newsome R, Chan SK, She R, Varhol R, Kamoh B, Prabhu AL, Tam A, Zhao Y, Moore RA, Hirst M, Marra MA, Jones SJ, Hoodless PA & Birol I (2010) De novo assembly and analysis of RNA-seq data Nat. Methods 7:909-12 [PMID: 20935650][DOI]
- ^ a b Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N & Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome Nat. Biotechnol. 29:644-52 [PMID: 21572440][DOI]
- ^ Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, Macmanes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N & Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis Nat Protoc 8:1494-512 [PMID: 23845962][DOI]
- ^ Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Müller WE, Wetter T & Suhai S (2004) Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs Genome Res. 14:1147-59 [PMID: 15140833][DOI]
- ^ Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF & Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors Nature 437:376-80 [PMID: 16056220][DOI]
- ^ Kumar S & Blaxter ML (2010) Comparing de novo assemblers for 454 transcriptome data BMC Genomics 11:571 [PMID: 20950480][DOI]
- ^ Anders S, Pyl PT & Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data Bioinformatics 31:166-9 [PMID: 25260700][DOI]
- ^ Bray NL, Pimentel H, Melsted P & Pachter L (2016) Near-optimal probabilistic RNA-seq quantification Nat. Biotechnol. 34:525-7 [PMID: 27043002][DOI]
- ^ Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL & Pachter L (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq Nat. Biotechnol. 31:46-53 [PMID: 23222703][DOI]
- ^ Robinson MD, McCarthy DJ & Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data Bioinformatics 26:139-40 [PMID: 19910308][DOI]
- ^ Love MI, Huber W & Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Genome Biol. 15:550 [PMID: 25516281][DOI]
- ^ Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W & Smyth GK (2015) limma powers differential expression analyses for RNA-sequencing and microarray studies Nucleic Acids Res. 43:e47 [PMID: 25605792][DOI]
- ^ Fang Z & Cui X (2011) Design and validation issues in RNA-seq experiments Brief. Bioinformatics 12:280-7 [PMID: 21498551][DOI]
- ^ Ramsköld D, Wang ET, Burge CB & Sandberg R (2009) An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data PLoS Comput. Biol. 5:e1000598 [PMID: 20011106][DOI]
- ^ Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A & Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol. 3:RESEARCH0034 [PMID: 12184808]
- ^ Core LJ, Waterfall JJ & Lis JT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters Science 322:1845-8 [PMID: 19056941][DOI]
- ^ Camarena L, Bruno V, Euskirchen G, Poggio S & Snyder M (2010) Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing PLoS Pathog. 6:e1000834 [PMID: 20368969][DOI]
- ^ Costa V, Aprile M, Esposito R & Ciccodicola A (2013) RNA-Seq and human complex diseases: recent accomplishments and future perspectives Eur. J. Hum. Genet. 21:134-42 [PMID: 22739340][DOI]
- ^ Khurana E, Fu Y, Chakravarty D, Demichelis F, Rubin MA & Gerstein M (2016) Role of non-coding sequence variants in cancer Nat. Rev. Genet. 17:93-108 [PMID: 26781813][DOI]
- ^ Slotkin RK & Martienssen R (2007) Transposable elements and the epigenetic regulation of the genome Nat. Rev. Genet. 8:272-85 [PMID: 17363976][DOI]
- ^ Proserpio V & Mahata B (2016) Single-cell technologies to study the immune system Immunology 147:133-40 [PMID: 26551575][DOI]
- ^ a b Byron SA, Van Keuren-Jensen KR, Engelthaler DM, Carpten JD & Craig DW (2016) Translating RNA sequencing into clinical diagnostics: opportunities and challenges Nat. Rev. Genet. 17:257-71 [PMID: 26996076][DOI]
- ^ Wu HJ, Wang AH & Jennings MP (2008) Discovery of virulence factors of pathogenic bacteria Curr Opin Chem Biol 12:93-101 [PMID: 18284925][DOI]
- ^ Suzuki S, Horinouchi T & Furusawa C (2014) Prediction of antibiotic resistance by gene expression profiles Nat Commun 5:5792 [PMID: 25517437][DOI]
- ^ Westermann AJ, Gorski SA & Vogel J (2012) Dual RNA-seq of pathogen and host Nat. Rev. Microbiol. 10:618-30 [PMID: 22890146][DOI]
- ^ Durmuş S, Çakır T, Özgür A & Guthke R (2015) A review on computational systems biology of pathogen-host interactions Front Microbiol 6:235 [PMID: 25914674][DOI]
- ^ Garg R, Shankar R, Thakkar B, Kudapa H, Krishnamurthy L, Mantri N, Varshney RK, Bhatia S & Jain M (2016) Transcriptome analyses reveal genotype- and developmental stage-specific molecular responses to drought and salinity stresses in chickpea Sci Rep 6:19228 [PMID: 26759178][DOI]
- ^ García-Sánchez S, Aubert S, Iraqui I, Janbon G, Ghigo JM & d'Enfert C (2004) Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns Eukaryotic Cell 3:536-45 [PMID: 15075282]
- ^ Mok S, Ashley EA, Ferreira PE, Zhu L, Lin Z, Yeo T, Chotivanich K, Imwong M, Pukrittayakamee S, Dhorda M, Nguon C, Lim P, Amaratunga C, Suon S, Hien TT, Htut Y, Faiz MA, Onyamboko MA, Mayxay M, Newton PN, Tripura R, Woodrow CJ, Miotto O, Kwiatkowski DP, Nosten F, Day NP, Preiser PR, White NJ, Dondorp AM, Fairhurst RM & Bozdech Z (2015) Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance Science 347:431-5 [PMID: 25502316][DOI]
- ^ Verbruggen N, Hermans C & Schat H (2009) Molecular mechanisms of metal hyperaccumulation in plants New Phytol. 181:759-76 [PMID: 19192189][DOI]
- ^ Li Z, Zhang Z, Yan P, Huang S, Fei Z & Lin K (2011) RNA-Seq improves annotation of protein-coding genes in the cucumber genome BMC Genomics 12:540 [PMID: 22047402][DOI]
- ^ Hobbs M, Pavasovic A, King AG, Prentis PJ, Eldridge MD, Chen Z, Colgan DJ, Polkinghorne A, Wilkins MR, Flanagan C, Gillett A, Hanger J, Johnson RN & Timms P (2014) A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity BMC Genomics 15:786 [PMID: 25214207][DOI]
- ^ Howe GT, Yu J, Knaus B, Cronn R, Kolpak S, Dolan P, Lorenz WW & Dean JF (2013) A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation BMC Genomics 14:137 [PMID: 23445355][DOI]
- ^ McGrath LL, Vollmer SV, Kaluziak ST & Ayers J (2016) De novo transcriptome assembly for the lobster Homarus americanus and characterization of differential gene expression across nervous system tissues BMC Genomics 17:63 [PMID: 26772543][DOI]
- ^ Noller HF (1991) Ribosomal RNA and translation Annu. Rev. Biochem. 60:191-227 [PMID: 1883196][DOI]
- ^ Christov CP, Gardiner TJ, Szüts D & Krude T (2006) Functional requirement of noncoding Y RNAs for human chromosomal DNA replication Mol. Cell. Biol. 26:6993-7004 [PMID: 16943439][DOI]
- ^ Kishore S & Stamm S (2006) The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C Science 311:230-2 [PMID: 16357227][DOI]
- ^ Hüttenhofer A, Schattner P & Polacek N (2005) Non-coding RNAs: hope or hype? Trends Genet. 21:289-97 [PMID: 15851066][DOI]
- ^ Esteller M (2011) Non-coding RNAs in human disease Nat. Rev. Genet. 12:861-74 [PMID: 22094949][DOI]
- ^ Legend: NCBI - National Center for Biotechnology Informationl; EBI - European Bioinformatics Institute; DDBY - 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
- ^ Edgar R, Domrachev M & Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository Nucleic Acids Res. 30:207-10 [PMID: 11752295]
- ^ a b Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J, Ansorge W, Ball CA, Causton HC, Gaasterland T, Glenisson P, Holstege FC, Kim IF, Markowitz V, Matese JC, Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J, Taylor R, Vilo J & Vingron M (2001) Minimum information about a microarray experiment (MIAME)-toward standards for microarray data Nat. Genet. 29:365-71 [PMID: 11726920][DOI]
- ^ a b Brazma A (2009) Minimum Information About a Microarray Experiment (MIAME)--successes, failures, challenges ScientificWorldJournal 9:420-3 [PMID: 19484163][DOI]
- ^ Kolesnikov N, Hastings E, Keays M, Melnichuk O, Tang YA, Williams E, Dylag M, Kurbatova N, Brandizi M, Burdett T, Megy K, Pilicheva E, Rustici G, Tikhonov A, Parkinson H, Petryszak R, Sarkans U & Brazma A (2015) ArrayExpress update--simplifying data submissions Nucleic Acids Res. 43:D1113-6 [PMID: 25361974][DOI]
- ^ Petryszak R, Keays M, Tang YA, Fonseca NA, Barrera E, Burdett T, Füllgrabe A, Fuentes AM, Jupp S, Koskinen S, Mannion O, Huerta L, Megy K, Snow C, Williams E, Barzine M, Hastings E, Weisser H, Wright J, Jaiswal P, Huber W, Choudhary J, Parkinson HE & Brazma A (2016) Expression Atlas update--an integrated database of gene and protein expression in humans, animals and plants Nucleic Acids Res. 44:D746-52 [PMID: 26481351][DOI]
- ^ Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W & Zimmermann P (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes Adv Bioinformatics 2008:420747 [PMID: 19956698][DOI]
- ^ Mitsuhashi N, Fujieda K, Tamura T, Kawamoto S, Takagi T & Okubo K (2009) BodyParts3D: 3D structure database for anatomical concepts Nucleic Acids Res. 37:D782-5 [PMID: 18835852][DOI]
- ^ Zhao Y, Li H, Fang S, Kang Y, Wu W, Hao Y, Li Z, Bu D, Sun N, Zhang MQ & Chen R (2016) NONCODE 2016: an informative and valuable data source of long non-coding RNAs Nucleic Acids Res. 44:D203-8 [PMID: 26586799][DOI]