RNA sequencing: advances, challenges and opportunities

“The reference sequence used for alignment was supplemented with 'artificial' sequences that surround all possible splice junctions between the annotated exons of genes, allowing the reads to be aligne”

“This development eliminated several challenges posed by microarray technologies, including the limited dynamic range of detectio”

“The introduction of high-throughput next-generation DNA sequencing (NGS) technologie”

“The reference sequence used for alignment was supplemented with 'artificial' sequences that surround all possible splice junctions between the annotated exons of genes, allowing the reads to be aligne”

“These strategies have already begun to contribute to our understanding of transcriptomes, including mapping of translation states of RNAs (for example, polysome profiling”

“sequencing [9,13].”

“With these new technologies comes a new set of challenges, such as optimizing library construction, amplification strategies, read length bias, and, importantly, data analysis and storage (Pop and Salzberg, 2008; Ozsolak and Milos, 2011).”

“75-th percentile) of all genes for each individual (91); (4) relative log expression normalization (RLE) (10); (5) trimmed mean of Mvalues (TMM) (34); and (6) VOOM normalization (27).”

“This is largely due to the presence of read-through events, reverse transcriptase template switching artifacts, and different systematic errors in the analysis of the reads [21].”

“Compared with microarray technology, conventional EST or other tag-based sequencing, it has less sampling bias, higher resolution and much broader expression range coverage (Marioni et al., 2008; Ozsolak and Milos, 2011).”

“For vaccine re-search, NGS has numerous applications ( Figure 1 ), including sequencing of host and pathogen genomes [6] and their transcriptomes [7, 8] , as well as studies of the diversity in host immune responses, in both T and B cells [9] [10] [11] .”

“Also, a certain of dynamicity by post-transcriptional and translational regulations of gene expression can be estimated by measuring its proteome (expressed protein set from genome) [9].”

“New methods of RNA sequencing (RNA-seq) have illuminated an ever-increasing diversity of RNA types, but challenges associated with library generation from non-coding RNA (ncRNA) limit what is known about specifically processed forms of ncRNA in cells and in extracellular vesicles (EVs) (11-14).”

“seq[1,2].”

“The single cell RNA-seq revolution RNA-seq on tissue/organ samples such as the brain, liver, pancreas, testes or blood samples results in data that represent the average transcriptome ofmany different kinds of cell types, thus not faithfully characterizing any unique cell type (Kolodziejczyk et al., 2015a; Melé et al.…”

“High-throughput sequencing instruments have enabled the rapid profiling of transcriptomes (RNA expression of genes) [1–4], proteomes (proteins) [5–7] and other ‘omics’ data [8–10].”

“Since Leishmania exhibit an unusual mechanism of gene regulation, whereby the majority of the leishmanial genome is constitutively transcribed and changes in protein abundance are directed by post-transcriptional mechanisms [36,37], we have profiled post-transcriptional changes in mRNA stability by Whole Transcriptome…”

“Recently, whole transcriptome sequencing (RNA-Seq) has enabled the measurement of abundance of tens of thousands of RNA species in a given biological sample (Wang et al., 2009; Ozsolak and Milos, 2011).”

“Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier .com/locate /ybbrc A simple strand-specific RNA-Seq library preparation protocol combining the Illumina TruSeq RNA and the dUTP methods Marc Sultan a,⇑, Simon Dökel…”

“Recent reviews describe the technological foundations and working principles of these technologies [13,14].”

“In the field of gene expression analysis, NGS sequencing facilitates a robust quantitative analysis of RNA molecules through digitally counting sequence reads obtained from the cDNA population of a tissue [18,19].”

“, 2011), RNAseq analysis (Ozsolak and Milos, 2011; Van Vliet, 2010) and ChIP-Seq (Schmidt”

“Key methods in transcriptomics include RNA Sequencing (RNA-seq [73]) for global gene expression profiling, Long Non-Coding RNA (lncRNA-seq [74]) for the study of non-coding RNAs involved in gene regulation, MicroRNA (miRNA-seq [75]) to identify small RNAs regulating mRNA stability and translation,…”

“, long non-coding RNA transcripts), and splice variants that are rarely quantifiable using microarray technologies (15, 44).”

“(5) Libraries were sequenced using the 5’ adapter only, resulting in the sequencing of the first 36 nucleotides of the inserts that represented the 5’ ends of the original RNAs.”

“Studies on plants’ genetics and functional genomics have been transformed by transcriptome sequencing, especially for non-model species lacking sequenced genomes [67].”

“RNA-Seq has allowed many advances in the characterization and quantification of the transcriptome [36], and has been widely used for gene discovery in soybean [37–41].”

“RNA-seq has emerged as a powerful technology to measure gene expression and tissue specificity at the whole-genome level 39,40 .”

“Transcriptomics, for instance, uses RNA-sequencing to simultaneously quantify tens of thousands of RNA species [1, 2].”

“In particular, RNA sequencing (RNA-Seq) has been used to identify gene fusions that are transcribed into RNAs within various cells [5].”

“Background Sequencing of RNA using next generation sequencing technology (RNA-seq) has become the standard approach for analyzing the transcriptomic landscape of a cell [1,2].”

“The most popular method in this field is gene chip / microarray; however, as sequencing costs decrease, NGS will likely replace microarrays for gene expression analysis in the future, mainly due to improved dynamic range, and additional capabilities for detecting expressed single nucleotide variants, and transcript is…”

“RNA-Seq has many exciting applications [8, 9], including the phylogenomic example mentioned above, single nucleotide polymorphism (SNP) discovery, small RNA profiling, novel transcript or splice variant discovery and comparison of global transcriptional mRNA profiles under distinct environmental conditions, e.”

“However, routine RT-PCR tests do not differentiate among SARS-CoV-2 variants or do so only by producing negative assay results (18), which still require sequence confirmation (19).”

“Compared with hybridization-based tool, such as microarray, RNA-Seq emerges as higher sensitivity, greater dynamic range of expression and base-pair resolution for transcription profiling [15–17].”

“Identification of PtNcl transcripts is often severely hampered by false positives arising from sequencing errors, alignment errors, genetic rearrangements and experimental artifacts (27,30,35,36).”

“Nowadays, RNA-seq and ChIP-seq are widely used to measure gene expression and to obtain genome-wide maps of transcription factor (TF) occupancies and epigenetic signatures (Park, 2009; Wang et al., 2009; Costa et al., 2010; Ozsolak and Milos, 2011; Furey, 2012).”

“Some of the other transcriptomes of these crops have also been generated in response to specific biological questions, and RNA sequencing (RNA-seq) has become the method of choice.(83) Researchers identified at least 2416 differentially expressed genes while profiling for response against salt stress in quinoa (Chenop…”

“As sequencing cost decreases and access to instrumentation increases, NGS will likely replace microarrays for gene expression analysis due to added benefits such as quantitation of transcripts, improved dynamic range, and additional capabilities for detecting expressed single nucleotide variants (SNVs), translocations…”

“Unmapped reads can arise due to technical sequencing artifacts that were produced by low quality and error prone copies of the nascent RNA sequence being sampled [11].”

“…of BZLF1 reads (and low mRNA counts generally) ostensibly may result from factors including naturally low transcript abundance, reduced transcript capture efficiency, and/or reduced efficiency of reverse transcription to cDNA owing to RNA secondary structural motifs ( Ozsolak and Milos, 2011 ).”

“This phenomenon could be explained by the potentially higher variance of genes expressed at low levels [58] or by the fact that microarrays have a limited dynamic range compared to RNA-Seq [28-32].”

“Recently, as a result of the low cost of next generation sequencing technologies [6], transcriptome profiling by RNA sequencing (RNA-Seq) is becoming the method of choice because of its unprecedented sensitivity and accuracy (reviewed in [7,8]).”

“Omics approaches involve high-throughput technologies that enable the measurement of global changes in the abundance of mRNA transcripts (transcriptomic), proteins (proteomic), and other biomolecular components (metabolomic) in complex biological systems as a result of chemical perturbation or transition of developmen…”

“High throughput sequencing technology including RNA sequencing (RNA-seq) has become a mature and efficient tool for deeper understanding the underlying molecular mechanism of complex trait such as RFI [7].”

“RNA-seq is performed by isolating RNA, converting it to cDNA, deep sequencing the pool, and then mapping the reads to the genome to create a transcript pro fi le 36 .”

“High-throughput next-generation sequencing is a revolutionary tool in transcriptomics that can overcome the limitations of array-based approaches because it can capture all sequences [143,149] and its popularity grew surprisingly after 2008 when more advanced Illumina technologies recorded 109 transcript sequences wit…”

“RNA-seq analyzes the full transcriptome, indicating which of the genes encoded in the DNA are turned on or off and to what extent [19].”

“Sequenced transcriptomes reveal the expression levels of gene sets for different cells, tissues and organs at different developmental stages and under different physiological states [15,16].”

“Gene expression can be surveyed at an unprecedented depth because of the precision and high resolution of this technology and its ability to capture weakly expressed RNAs as well as RNA species that are usually not represented in commonmicroarrays (20).”

“It has been employed widely in exploiting physiological dynamics of model as well as non-model crop plants (Ozsolak and Milos, 2011).”

“Third, the biological importance of non-coding RNA, including microRNA and long non-coding RNA, are also well recognized.(107) Therefore, the ability to accurately detect PC-relevant gene fusions and non-coding RNA, and to quantify the expression of disease driving genes, holds great promise for functional disease pro…”

“It enables us to investigate alternative splicing patterns without demand for knowledge of transcript annotations, and also has a merit of gene fusion identification due to the increased physical coverage it offers [26].”

“…sequencing (NGS) and by the relative ease of sequencing a collection of cDNAs as opposed to predicting gene models in plant chromosomes (Ozsolak and Milos, 2011), means that many transcript profiles are now publicly available, and individual laboratories can afford to generate new…”

“Fortunately, new highthroughput sequencing technologies, such as those based on the transcriptome, avoid the cloning process and are quick and efficient (Ozsolak and Milos 2011).”

“Furthermore, deep-sequencing methods such as RNA-seq can discover novel transcripts not anticipated in the design of the microarray, and also detect and quantify low-abundance transcripts below the detection threshold of microarray analysis [11].”

“RNA-Seq has recently emerged as a complementary technique to microarray, bringing advantages such as low background noise, capable of detecting novel transcripts and alternative splicing forms, exon and nucleotide resolution, high dynamic range in detection, high precision, and high reproducibility [51–54].”

“外还有一些新的针对低数量细胞进行转录组研究的 技术正在不断被开发([20])。最后, 标准的RNA-Seq技 术不能提供序列转录的方向信息, 而这对于转录组注 释尤为重要, 采用single-strand sequencing([70])和strandspecific sequencing([71])技术能很好的解决这一问题 , 或将成为RNA-Seq技术发展的一个重要方向。 虽然 RNA-Seq技术还面临着种种困难, 但作为 一个刚刚起步的新技术RNA-Seq已经显示出其他转”

“Emerging high-throughput sequencing technologies has changed the pace of DNA sequencing in plants and animals which perform comprehensive profiling of RNA expression [11, 12] other than model plant organisms were constrained molecular genetics applications have been implemented.”

“RNA sequencing (RNA-seq) (Wang et al., 2009; Ozsolak and Milos, 2010) has become a standard tool for studying gene expression due to its ability to detect novel transcriptional activity without relying on previously defined gene sequence.”

“In recent years, RNA sequencing (RNA-Seq) technology has emerged as a powerful and revolutionary approach to quantify gene expression levels and survey detailed transcriptome profiling at unprecedented resolution and sensitivity [16, 17].”

“It can also detect rare transcripts of low abundance [16].”

“In recent years, significant advances in sequencing technology have expanded the RNA world even further [3].”

“Typically, studies have used microarrays for discovery likely due to familiarity and cost issues although next-generation sequencing is arguably the state of the art technique for microRNA profiling [44, 45].”

“…no restriction to known genes, an unlimited dynamic range in quantitation, a more accurate expression level assessment, less sophisticated normalization needed and no problem with cross-hybridization of similar transcripts (Wang et al. , 2009; Ozsolak & Milos, 2011; Van Verk et al. , 2013).”

“unprecedented capability to discover novel genes, alternative transcript variants, chimeric transcripts, expressed sequence variants and allele-specific expressions (19-22).”

“Although high-density or custom- made microarrays can be constructed, these approaches have several limitations, including high background levels owing to cross-hybridization, and the requirement for genomic sequence information (Wang et al., 2009; Ozsolak and Milos, 2011).”

“the quantification of expression, RNA-seq offers other advantages, such as the possibility to detect novel transcripts, splice variants or allele-specific expression [5].”

“This disparity has been exacerbated with the development and popularity of next-generation technologies, such as RNA-seq, which enable genome-wide measurements at unprecedented resolution and cost (1).”