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<small>Legend: NCBI SRA – National center for biotechnology information sequence read archive.</small>
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.<ref name="#22829749">{{cite journal | vauthors = Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M | title = Comparison of next-generation sequencing systems | journal = Journal of Biomedicine & Biotechnology | volume = 2012 | pages = 251364 | date = 2012 | pmid = 22829749 | pmc = 3398667 | doi = 10.1155/2012/251364 | doi-access = free }}</ref><ref name="#22522955">{{cite journal | vauthors = Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ | title = Performance comparison of benchtop high-throughput sequencing platforms | journal = Nature Biotechnology | volume = 30 | issue = 5 | pages = 434–9 | date = May 2012 | pmid = 22522955 | doi = 10.1038/nbt.2198 | s2cid = 5300923 }}</ref><ref name="#27184599">{{cite journal | vauthors = Goodwin S, McPherson JD, McCombie WR | title = Coming of age: ten years of next-generation sequencing technologies | journal = Nature Reviews Genetics | volume = 17 | issue = 6 | pages = 333–51 | date = May 2016 | pmid = 27184599 | doi = 10.1038/nrg.2016.49 | s2cid = 8295541 | pmc = 10373632 }}</ref> Direct sequencing of RNA using [[nanopore sequencing]] represents a current state-of-the-art RNA-Seq technique.<ref>{{cite journal | vauthors = Garalde DR, Snell EA, Jachimowicz D, Sipos B, Lloyd JH, Bruce M, Pantic N, Admassu T, James P, Warland A, Jordan M, Ciccone J, Serra S, Keenan J, Martin S, McNeill L, Wallace EJ, Jayasinghe L, Wright C, Blasco J, Young S, Brocklebank D, Juul S, Clarke J, Heron AJ, Turner DJ | title = Highly parallel direct RNA sequencing on an array of nanopores | language = En | journal = Nature Methods | volume = 15 | issue = 3 | pages = 201–206 | date = March 2018 | pmid = 29334379 | doi = 10.1038/nmeth.4577 | s2cid = 3589823 }}</ref><ref name="#26076426">{{cite journal | vauthors = Loman NJ, Quick J, Simpson JT | title = A complete bacterial genome assembled de novo using only nanopore sequencing data | journal = Nature Methods | volume = 12 | issue = 8 | pages = 733–5 | date = August 2015 | pmid = 26076426 | doi = 10.1038/nmeth.3444 | s2cid = 15053702 }}</ref> Nanopore sequencing of RNA can detect [[RNA#Structure|modified bases]] that would be otherwise masked when sequencing cDNA and also eliminates [[DNA replication|amplification]] steps that can otherwise introduce bias.<ref name="#21191423" /><ref name="#19776739">{{cite journal | vauthors = Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, Thompson JF, Bowers J, Jarosz M, Milos PM | title = Direct RNA sequencing | journal = Nature | volume = 461 | issue = 7265 | pages = 814–8 | date = October 2009 | pmid = 19776739 | doi = 10.1038/nature08390 | bibcode = 2009Natur.461..814O | s2cid = 4426760 }}</ref>
The sensitivity and accuracy of an RNA-Seq experiment are dependent on the [[sequencing depth|number of reads]] obtained from each sample.<ref name="#23961961" /><ref name="#26813401" /> A large number of reads are needed to ensure sufficient coverage of the transcriptome, enabling detection of low abundance transcripts. Experimental design is further complicated by sequencing technologies with a limited output range, the variable efficiency of sequence creation, and variable sequence quality. Added to those considerations is that every species has a different [[number of genes]] and therefore requires a tailored sequence yield for an effective transcriptome. Early studies determined suitable thresholds empirically, but as the technology matured suitable coverage was predicted computationally by transcriptome saturation. Somewhat counter-intuitively, the most effective way to improve detection of differential expression in low expression genes is to add more [[Replicate (biology)|biological replicates]] rather than adding more reads.<ref name="#24020486">{{cite journal | vauthors = Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND, Betel D | title = Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data | journal = Genome Biology | volume = 14 | issue = 9 | pages = R95 | date = 2013 | pmid = 24020486 | pmc = 4054597 | doi = 10.1186/gb-2013-14-9-r95 }}</ref> 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.<ref name=pmid22955616>{{cite journal | title = An integrated encyclopedia of DNA elements in the human genome | journal = Nature | volume = 489 | issue = 7414 | pages = 57–74 | date = September 2012 | pmid = 22955616 | pmc = 3439153 | doi = 10.1038/nature11247 | bibcode = 2012Natur.489...57T | author1 = ENCODE Project Consortium | last2 = Aldred | first2 = Shelley F. | last3 = Collins | first3 = Patrick J. | last4 = Davis | first4 = Carrie A. | last5 = Doyle | first5 = Francis | last6 = Epstein | first6 = Charles B. | last7 = Frietze | first7 = Seth | last8 = Harrow | first8 = Jennifer | last9 = Kaul | first9 = Rajinder | last10 = Khatun | first10 = Jainab | last11 = Lajoie | first11 = Bryan R. | last12 = Landt | first12 = Stephen G. | last13 = Lee | first13 = Bum-Kyu | last14 = Pauli | first14 = Florencia | last15 = Rosenbloom | first15 = Kate R. | last16 = Sabo | first16 = Peter | last17 = Safi | first17 = Alexias | last18 = Sanyal | first18 = Amartya | last19 = Shoresh | first19 = Noam | last20 = Simon | first20 = Jeremy M. | last21 = Song | first21 = Lingyun | last22 = Altshuler | first22 = Robert C. | last23 = Birney | first23 = Ewan | last24 = Brown | first24 = James B. | last25 = Cheng | first25 = Chao | last26 = Djebali | first26 = Sarah | last27 = Dong | first27 = Xianjun | last28 = Dunham | first28 = Ian | last29 = Ernst | first29 = Jason | last30 = Furey | first30 = Terrence S. | display-authors = 29 }}</ref><ref name="#26527727">{{cite journal | vauthors = 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 | display-authors = 6 | title = ENCODE data at the ENCODE portal | journal = Nucleic Acids Research | volume = 44 | issue = D1 | pages = D726–32 | date = January 2016 | pmid = 26527727 | pmc = 4702836 | doi = 10.1093/nar/gkv1160 }}</ref><ref>{{Cite web|url=http://www.encodeproject.org|title=ENCODE: Encyclopedia of DNA Elements|website=encodeproject.org}}</ref>
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