{"id":1125,"date":"2016-12-05T21:53:16","date_gmt":"2016-12-05T20:53:16","guid":{"rendered":"http:\/\/www.newslab.sk\/2016\/12\/05\/odysea-citania-dna\/"},"modified":"2017-10-03T14:57:02","modified_gmt":"2017-10-03T12:57:02","slug":"the-odyssey-of-dna-reading","status":"publish","type":"post","link":"https:\/\/www.newslab.sk\/en\/the-odyssey-of-dna-reading\/","title":{"rendered":"The Odyssey of DNA reading"},"content":{"rendered":"
*All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf <\/span><\/strong>\r\n attachment at the end of the paper.<\/span> <\/strong><\/pre>\n
 <\/p>\n
Klasick\u00e9 met\u00f3dy sekvenovania<\/strong><\/p>\n
Prv\u00e9 pokusy sekvenovania sa datuj\u00fa do 70. rokov minul\u00e9ho storo\u010dia, kedy boli realizovan\u00e9 nepriamo – sekvenovan\u00edm molek\u00fal RNA alebo prote\u00ednov. Prv\u00fa sekvenciu DNA z\u00edskal v\u00a0roku 1968 Ray Wu z\u00a0Cornellskej univerzity.\u00a0Bol to re\u0165azec z\u00a0okrajovej oblasti gen\u00f3mu f\u00e1ga Lamda dlh\u00fd 12 nukleotidov. V\u00a0 roku 1977 dva\u00a0 pracovn\u00e9 t\u00edmy nez\u00e1visle na sebe predstavili dva odli\u0161n\u00e9 pr\u00edstupy sekvenovania, \u010do znamenalo prelom vtedaj\u0161\u00edch techn\u00edk. Boli to: chemick\u00e1 Maxam-Gilbertova met\u00f3da a\u00a0enzymatick\u00e1 met\u00f3da pop\u00edsan\u00e1 Frederickom Sangerom.<\/p>\n
Maxam-Gilbertova met\u00f3da funguje na princ\u00edpe chemick\u00fdch reakci\u00ed \u0161tiepiacich termin\u00e1lne zna\u010den\u00e9 nukleotidov\u00e9 b\u00e1zy,\u00a0n\u00e1slednom rozdelen\u00ed vzniknut\u00fdch zna\u010den\u00fdch DNA fragmentov v polyakrylamidovom g\u00e9li a\u00a0ich autor\u00e1diografickej vizualiz\u00e1cii.<\/p>\n
Sangerova enzymatick\u00e1 met\u00f3da vyu\u017e\u00edva modifikovan\u00e9 (ddNTP, 2\u00b4,3\u00b4-dideoxynukleozid-trifosf\u00e1t) anal\u00f3gy norm\u00e1nych stavebn\u00fdch jednotiek (dNTP, 2′-deoxyribonukleozid-trifosf\u00e1t) p\u00f4sobiacich ako \u0161peci\u00e1lne inhib\u00edtory DNA polymer\u00e1zy. Prvou priekopn\u00edckou technikou tohto sekvenovania DNA bola v\u00a0roku 1975 met\u00f3da \u201eplus-m\u00ednus\u201c, ktorou bol o\u00a0dva roky nesk\u00f4r kompletne pre\u010d\u00edtan\u00fd cel\u00fd gen\u00f3m bakteriof\u00e1ga \u0444X174 s 5386 nukleotidmi. Sangerovo sekvenovanie bolo postupne modifikovan\u00e9, a\u00a0tak p\u00f4vodn\u00e9 radioakt\u00edvne zna\u010denie ddNTP vystriedalo fluorescen\u010dn\u00e9 zna\u010denie, polyakrylamidov\u00e1 elektrofor\u00e9za na rozdelenie fragmentov nov\u00fdch molek\u00fal DNA bola nahraden\u00e1 kapil\u00e1rovou a\u00a0manu\u00e1lne od\u010d\u00edtavanie poradia b\u00e1z vystriedali sofistikovan\u00e9 automatizovan\u00e9 po\u010d\u00edta\u010dov\u00e9 softv\u00e9ry. Zdokona\u013eovan\u00edm technol\u00f3gi\u00ed sa proces sekvenovania plne zautomatizoval. Sangerova met\u00f3da sa stala zlat\u00fdm \u0161tandardom a\u00a0aj napriek svojej pr\u00e1cnosti a\u00a0\u010dasovej n\u00e1ro\u010dnosti desa\u0165ro\u010dia patrila k\u00a0najpou\u017e\u00edvanej\u0161\u00edm a\u00a0najspo\u013eahlivej\u0161\u00edm sekvena\u010dn\u00fdm met\u00f3dam [1].<\/p>\n
Tejto popularite do ve\u013ekej miery napomohol\u00a0Projekt sekvenovania \u013eudsk\u00e9ho gen\u00f3mu<\/strong>\u00a0(HGP, Human Genome Project) (1990-2003) s\u00a0cie\u013eom ur\u010di\u0165 kompletn\u00fa nukleotidov\u00fa sekvenciu \u013eudskej DNA. Projekt trval 13 rokov a st\u00e1l takmer 3 miliardy dol\u00e1rov. Bola to zd\u013ahav\u00e1 mrav\u010dia pr\u00e1ca. V\u00fdsledkom bolo osekvenovanie 99% \u013eudsk\u00e9ho\u00a0gen\u00f3mu<\/a>\u00a0s\u00a0ve\u013ekos\u0165ou 3,3 miliardy b\u00e1zov\u00fdch p\u00e1rov (bp). Pou\u017eit\u00e9 technol\u00f3gie vych\u00e1dzali z\u00a0metodick\u00e9ho princ\u00edpu pr\u00e1ve Sangerovej met\u00f3dy, av\u0161ak boli doplnen\u00e9 pr\u00edstupmi umo\u017e\u0148uj\u00facimi analyzova\u0165 dlh\u0161ie \u00faseky DNA [1]. Jednou z\u00a0mo\u017enost\u00ed bolo tzv. \u201eshotgun\u201c \u00a0sekvenovanie, pri ktorom je DNA n\u00e1hodne \u0161tiepen\u00e1 na kr\u00e1tke \u00faseky, klonovan\u00e1 do vektorov a\u00a0potom\u00a0n\u00e1sledne osekvenovan\u00e1 z\u00a0oboch koncov. Z\u00edskan\u00e9 \u00faseky sa vz\u00e1jomne prekr\u00fdvaj\u00fa, a t\u00fdm umo\u017e\u0148uj\u00fa zostavenie celej sekvencie. \u010eal\u0161\u00ed rozvoj sekvenovania individu\u00e1lnych \u013eudsk\u00fdch gen\u00f3mov bol nutne podmienen\u00fd vznikom nov\u00fdch lacnej\u0161\u00edch a\u00a0v\u00fdkonnej\u0161\u00edch technol\u00f3gi\u00ed [2]:<\/p>\n
 <\/p>\n
Sekvenovanie novej gener\u00e1cie (NGS; next generation sequencing)<\/strong><\/p>\n
Dopyt po n\u00edzkon\u00e1kladovom sekvenovan\u00ed mal za n\u00e1sledok tlak na v\u00fdvoj vysoko v\u00fdkonn\u00fdch tzv. \u201ehigh-throughput\u201c technol\u00f3gi\u00ed. Princ\u00edp spo\u010d\u00edva v\u00a0paraleliz\u00e1cii tohto procesu a\u00a0produkcii tis\u00edcov a\u017e mili\u00f3nov sekvenci\u00ed s\u00fa\u010dasne, preto sa NGS ozna\u010duje aj ako mas\u00edvne paraleln\u00e9 sekvenovanie. Sekven\u00e1tory prvej gener\u00e1cie detegovali jednotliv\u00e9 DNA b\u00e1zy v rade jednu za druhou a zv\u00fd\u0161enie sekvena\u010dnej kapacity dosahovali v\u00a0z\u00e1sade iba prid\u00e1van\u00edm kapil\u00e1r v\u00a0jednom pr\u00edstroji.<\/p>\n
V\u00fdstupom NGS je obrovsk\u00fd objem d\u00e1t, ktor\u00e9 je nutn\u00e9 roztriedi\u0165 a\u00a0spracova\u0165. Probl\u00e9m teda nie je (sn\u00e1\u010f prv\u00fdkr\u00e1t v\u00a0hist\u00f3rii) v z\u00edskan\u00ed dostato\u010dn\u00e9ho mno\u017estva \u00fadajov, ale v\u00a0ich zmysluplnej interpret\u00e1cii. \u0160tatistick\u00e9 modely a v\u00fdpo\u010dtov\u00e9 algoritmy, ktor\u00e9 mali doteraz viac-menej akademick\u00fd v\u00fdznam, t\u00fdm z\u00edskali praktick\u00fd rozmer. Nastal \u010das na etablovanie bioinformatiky.<\/p>\n
V\u00a0s\u00fa\u010dasnosti m\u00f4\u017eu by\u0165\u00a0 NGS technol\u00f3gie rozdelen\u00e9 do dvoch z\u00e1kladn\u00fdch kateg\u00f3ri\u00ed. Prv\u00fa skupinu predstavuj\u00fa platformy zalo\u017een\u00e9 na PCR amplifik\u00e1cii templ\u00e1tu (PCR-based technologies), naz\u00fdvaj\u00fa sa aj sekven\u00e1tory druhej gener\u00e1cie. Druh\u00fa skupinu tvoria technol\u00f3gie bez nutnosti amplifika\u010dn\u00e9ho kroku pred vlastnou sekven\u00e1ciou, ktor\u00e9 vyu\u017e\u00edvaj\u00fa tzv. Single-molecule sequencing a\u00a0s\u00fa ozna\u010dovan\u00e9 za sekven\u00e1tory tretej gener\u00e1cie [3, 4].<\/p>\n
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Sekven\u00e1tory druhej gener\u00e1cie<\/strong><\/p>\n
V\u00a0priebehu posledn\u00fdch rokov sa objavuj\u00fa na trhu r\u00f4zne pr\u00edstupy NGS technol\u00f3gi\u00ed pon\u00fakan\u00e9 nieko\u013ek\u00fdmi\u00a0 firmami, ktor\u00e9 si navz\u00e1jom konkuruj\u00fa. V d\u00f4sledku patentovej ochrany zvolili navz\u00e1jom mierne odli\u0161n\u00e9 pr\u00edstupy av\u0161ak so\u00a0v\u0161eobecne podobn\u00fdm princ\u00edpom. V\u00e4\u010d\u0161ina vych\u00e1dza z\u00a0pomerne kr\u00e1tkych fragmentov DNA, z pr\u00edpravy templ\u00e1tu a\u00a0vytvorenia kni\u017en\u00edc amplik\u00f3nov emulznou PCR (emPCR) alebo PCR v\u00a0zhlukoch (cluster PCR), naz\u00fdvanou tie\u017e tzv. \u201ebridge\u201c PCR. Stru\u010dn\u00fd popis princ\u00edpu oboch met\u00f3d PCR zn\u00e1zor\u0148uje obr\u00e1zok \u010d\u00edslo 1. Nasleduje samotn\u00e9 sekvenovanie zalo\u017een\u00e9 na synt\u00e9ze alebo lig\u00e1cii s\u00a0detekciou inkorporovan\u00fdch nukleotidov a\u00a0anal\u00fdza z\u00edskan\u00fdch d\u00e1t [5].<\/p>\n
Ka\u017ed\u00fd v\u00fdrobca rie\u0161i tieto z\u00e1kladn\u00e9 kroky inou kombin\u00e1ciou uveden\u00fdch mo\u017enost\u00ed, z\u00a0\u010doho vypl\u00fdva rozdielna \u0161pecificita, senzitivita a r\u00f4zna miera chybovosti. Pri porovn\u00e1van\u00ed rovnakej sekvencie tak m\u00f4\u017eu vznikn\u00fa\u0165 rozdiely v type a mno\u017estve produkovan\u00fdch d\u00e1t a ich r\u00f4znej interpret\u00e1cii [6]. Hlavn\u00fdmi protagonistami NGS revol\u00facie s\u00fa \u0161tyri platformy.<\/p>\n
Obr. 1 (A) Emulzn\u00e1 PCR (emPCR, emulsionPCR): Amplifik\u00e1cia DNA fragmentov na partikul\u00e1ch (beads). K fragmentom DNA sa z oboch str\u00e1n liguj\u00fa adapt\u00e9ry. Jeden obsahuje v\u00e4zobn\u00e9 miesto pre primer a\u00a0druh\u00fd\u00a0 biot\u00ednov\u00fa zna\u010dku, ktorou sa DNA via\u017ee k povrchu partikuly. Amplifik\u00e1cia prebieha v\u00a0emulzii vody a\u00a0oleja s\u00a0obsahom potrebn\u00fdch PCR reagenci\u00ed, \u010do zabezpe\u010duje optim\u00e1lne prostredie. V\u00a0ka\u017edej kvapke tejto emulzie sa tak nach\u00e1dza v\u017edy len jedna partikula s\u00a0nieko\u013eko sto k\u00f3piami rovnak\u00e9ho DNA fragmentu. Pri tejto met\u00f3de doch\u00e1dza k paralelnej amplifik\u00e1cii mili\u00f3nov jednotliv\u00fdch fragmentov na partikul\u00e1ch. V\u00fdsledkom je DNA kni\u017enica pripraven\u00e1 pre sekvenovanie.<\/p>\n
(B) PCR v\u00a0zhlukoch (cluster PCR, bridge PCR): DNA fragment sa oboma koncami pomocou adapt\u00e9rov komplement\u00e1rne via\u017ee na povrch prietokovej kom\u00f4rky (Flow cell), ktor\u00e1 je pokryt\u00e1 oligonukleotidmi. Doch\u00e1dza k\u00a0jeho ohybu \/pripom\u00edna most\/ a\u00a0k\u00a0jednovl\u00e1knov\u00e9mu fragmentu je syntetizovan\u00e9 druh\u00e9 vl\u00e1kno, n\u00e1sledne je dvojvl\u00e1knov\u00e1 DNA denaturovan\u00e1. Proces amplifik\u00e1cie sa opakuje a\u017e k\u00fdm nevznikne pribli\u017ene 1000 k\u00f3pi\u00ed dan\u00e9ho DNA fragmentu v tesnej bl\u00edzkosti, \u010d\u00edm sa vytvor\u00ed zhluk\/klaster funk\u010dne ekvivalentn\u00fd jednej partikule (bead). T\u00fato techniku pou\u017e\u00edvaj\u00fa v\u00fdhradne sekven\u00e1tory Illuminy [5].<\/p>\n
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454 LifeScience \/ Roche<\/strong><\/p>\n
Prv\u00e1 NGS platforma bola komer\u010dne dostupn\u00e1 v\u00a0roku 2005. Princ\u00edpom technol\u00f3gie je kombin\u00e1cia emPCR a\u00a0pyrosekvenovania. Pyrosekvenovanie mo\u017eno zjednodu\u0161ene definova\u0165 ako real-time sekvenovanie, pri ktorom sa sledom enzymatick\u00fdch reakci\u00ed deteguje sveteln\u00fd sign\u00e1l uvo\u013enen\u00fd pri zabudovan\u00ed dNTP do vznikaj\u00faceho re\u0165azca DNA. Mno\u017estvo uvo\u013enen\u00e9ho svetla je \u00famern\u00e9 po\u010dtu za\u010dlenen\u00fdch nukleotidov. V\u00fdhodou tejto met\u00f3dy je r\u00fdchlos\u0165 a\u00a0d\u013a\u017eka pre\u010d\u00edtanej sekvencie. Technol\u00f3gia 454 za\u010d\u00ednala s\u00a0d\u013a\u017ekou sekvencie 400 bp a\u00a0v\u00a0roku 2013 sa pohybuje na \u00farovni 1000 bp. Optim\u00e1lna d\u013a\u017eka \u010d\u00edtanej sekvencie je 700 bp, \u010do je porovnate\u013en\u00e9 so Sangerovou met\u00f3dou. V\u00a0roku 2007 bol touto NGS technol\u00f3giou prv\u00fdkr\u00e1t pre\u010d\u00edtan\u00fd gen\u00f3m Jamesa Watsona a\u00a0trvalo to dva mesiace [7].<\/p>\n
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Illumina (Solexa)<\/strong><\/p>\n
Druhou NGS platformou uvedenou na verejnos\u0165 v\u00a0roku 2006 bol sekven\u00e1tor firmy Solexa, ktor\u00fd o\u00a0rok nesk\u00f4r odk\u00fapila spolo\u010dnos\u0165 Illumina. Met\u00f3da je zalo\u017een\u00e1 na\u00a0 sekvena\u010dnej reakcii\u00a0 synt\u00e9zou s\u00a0vyu\u017eit\u00edm fluorescen\u010dne zna\u010den\u00fdch reverzibiln\u00fdch termin\u00e1torov a\u00a0bridge PCR. Pri sekvenovan\u00ed mo\u017eno postupova\u0165 dvoma sp\u00f4sobmi, bu\u010f sa sekvencia pre\u010d\u00edta v\u00a0jednom smere (single long reed) alebo sa postupuje proti sebe z\u00a0oboch koncov (pair end read). Spolo\u010dnos\u0165 Illumina v\u00a0dne\u0161nej dobe dominuje na NGS poli a\u00a0 pon\u00faka nieko\u013eko pr\u00edstrojov r\u00f4znej v\u00fdkonnosti: MiSeq (2×300 bp), MiSeqDx (2×125 bp), NextSeq 500 (2×150 bp), HiSeq2500 (2×250 bp) a HiSeq X Ten. HiSeq X Ten\u00a0 sa sklad\u00e1 z 10 ultra vysokokapacitn\u00fdch sekven\u00e1torov a\u00a0v\u00a0s\u00fa\u010dasnosti je najv\u00fdkonnej\u0161ou sekvena\u010dnou platformou, ktor\u00e1\u00a0 prekon\u00e1va cenov\u00fa bari\u00e9ru 1 000 $ dol\u00e1rov za osekvenovanie jedn\u00e9ho \u013eudsk\u00e9ho gen\u00f3mu.\u00a0 Zameran\u00e1 je najm\u00e4 na popula\u010dn\u00e9 \u0161t\u00fadie. Illumina platformy ovl\u00e1daj\u00fa trh najm\u00e4 v\u010faka vysokej v\u00fdkonnosti sekven\u00e1torov, robustnosti prevedenia, kapacite a\u00a0priaznivej cenovej rel\u00e1cii prev\u00e1dzky [8].<\/p>\n
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SOLiD System (Life Technologies)<\/strong><\/p>\n
Teret\u00ed typ sekven\u00e1tora predstavila v\u00a0roku 2007 vtedy e\u0161te spolo\u010dnos\u0165 Applied Biosystems (dnes Life Technologies). SOLiD syst\u00e9m (Sequencing by Oligonucleotide Ligation and Detection) pracuje na princ\u00edpe emPCR a sekvenovan\u00ed DNA lig\u00e1zou. Vyu\u017e\u00edvaj\u00fa sa fluorescen\u010dne zna\u010den\u00e9 oktam\u00e9rov\u00e9\u00a0 \u00faseky s\u00a0dvoma definovan\u00fdmi b\u00e1zami [9]. Tento dvojb\u00e1zov\u00fd k\u00f3dovac\u00ed syst\u00e9m zaru\u010duje pre\u010d\u00edtanie ka\u017ed\u00e9ho nukleotidu dvakr\u00e1t, \u010d\u00edm sa zvy\u0161uje presnos\u0165, s akou je ur\u010den\u00e9 poradie nukleotidov danej sekvencie. Medzi vy\u0161\u0161ie uveden\u00fdmi NGS platformami m\u00e1 teda SOLiD syst\u00e9m najni\u017e\u0161iu chybovos\u0165. V s\u00fa\u010dasnosti s\u00fa v ponuke dva varianty sekven\u00e1tora: 5500 System a 5500xl System, ktor\u00fd v\u00fdrazne zv\u00fd\u0161il d\u013a\u017eku \u010d\u00edtania z\u00a0p\u00f4vodn\u00fdch 35 bp na 75 bp a tie\u017e zlep\u0161il presnos\u0165 meran\u00ed na 99,99%. Z\u00a0prv\u00fdch troch spomenut\u00fdch NGS technol\u00f3gii Illumina HiSeq generuje najviac d\u00e1t za najni\u017e\u0161iu cenu, SOLiD System m\u00e1 najvy\u0161\u0161iu presnos\u0165 a Roche 454 poskytuje najv\u00e4\u010d\u0161iu d\u013a\u017eku \u010d\u00edtania.<\/p>\n
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Ion Torrent (Life Technologies)<\/strong><\/p>\n
Technol\u00f3gia Ion Torrent sa objavila na sc\u00e9ne v\u00a0roku 2010 a ako jedin\u00e1 vyu\u017e\u00edva namiesto optick\u00e9ho\/sveteln\u00e9ho sp\u00f4sobu zaznamen\u00e1vania jednotliv\u00fdch nukleotidov detekciu elektrochemick\u00e9ho sign\u00e1lu. Inkorpor\u00e1cia nukleotidu do rast\u00faceho re\u0165azca DNA sp\u00f4sob\u00ed uvo\u013enenie vod\u00edkov\u00e9ho i\u00f3nu (H+), \u010d\u00edm d\u00f4jde k zmene pH. Proces prebieha na polovodi\u010dovom \u010dipe husto pokrytom mikrojamkami, pod ktor\u00fdmi je umiestnen\u00fd senzor citliv\u00fd na zmenu pH. Samotn\u00e1 pr\u00edprava kni\u017enice prebieha podobne ako v\u00a0predch\u00e1dzaj\u00facich met\u00f3dach napojen\u00edm adaporov s\u00a0n\u00e1slednou emPCR a\u00a0sekvena\u010dnou reakciou syn\u00e9zou.<\/p>\n
Ion Torrent aktu\u00e1lne pon\u00faka Ion PGM Sequencer (314\/316\/318 chips) a Ion Proton System (I, II, III), pri\u010dom\u00a0 model Ion Proton Sequencer s\u00a0mikro\u010dipom Ion PII Chip je schopn\u00fd pre\u010d\u00edta\u0165 cel\u00fd gen\u00f3m \u010dloveka v\u00a0priebehu jedn\u00e9ho d\u0148a. Celkov\u00e9 mno\u017estvo produkovan\u00fdch d\u00e1t je ur\u010den\u00e9 hustotou jamiek na \u010dipe. Prv\u00fd \u013eudsk\u00fd gen\u00f3m osekvenovan\u00fd technol\u00f3giou Ion Torrent bol gen\u00f3m Gordona Moora [9, 10].<\/p>\n
 <\/p>\n
Sekven\u00e1tory tretej gener\u00e1cie.<\/strong><\/p>\n
V\u00fdrazn\u00fa zmenu predstavuj\u00fa met\u00f3dy, ktor\u00e9 nie s\u00fa zalo\u017een\u00e9 na nutnom namno\u017een\u00ed sekvenovan\u00fdch \u00fasekov. Nevyu\u017e\u00edvaj\u00fa tak amplifika\u010dn\u00fd krok, \u010do zni\u017euje v\u00fdskyt spont\u00e1nnych mut\u00e1ci\u00ed vzniknut\u00fdch chybovos\u0165ou DNA-polymer\u00e1zy. Pri klonovan\u00ed DNA pomocou PCR m\u00f4\u017ee d\u00f4js\u0165 k z\u00e1mene niektor\u00fdch b\u00e1z a t\u00fdm aj k\u00a0r\u00f4znemu po\u010dtu v\u00fdskytu jednotliv\u00fdch fragmentov, \u010do m\u00f4\u017ee ovplyvni\u0165 v\u00fdsledok experimentu.<\/p>\n
Prv\u00fd pr\u00edstroj, ktor\u00fd st\u00e1l na prahu tre\u0165ogenera\u010dnej technol\u00f3gie sekvenovania predstavila firma Helicos Bioscience v\u00a0roku 2007. Met\u00f3da vyu\u017e\u00edva jednotliv\u00e9 individu\u00e1lne molekuly DNA (tSMS, True Single Molecule Sequencing, tSMS) pevne fixovan\u00e9 k\u00a0povrchu. Sekvena\u010dn\u00e1 reakcia prebieha synt\u00e9zou s\u00a0fluorescen\u010dne zna\u010den\u00fdmi nukleotidmi. Zavedeniu tohto analzy\u00e1tora do praxe br\u00e1nila vysok\u00e1 cena pr\u00edstrojov a d\u013a\u017eka \u010d\u00edtanej sekvencie (30-35 bp). V\u00a0s\u00fa\u010dasnosti nie je technol\u00f3gia \u010falej vyv\u00edjan\u00e1 [11].<\/p>\n
 <\/p>\n
SMRT (Pacific Bioscience)<\/strong><\/p>\n
V\u00a0roku 2009 prich\u00e1dza na trh nov\u00e1 technol\u00f3gia, ktor\u00e1 sekvenuje jednotliv\u00e9 molekuly DNA v\u00a0re\u00e1lnom \u010dase (SMRT, single-molecule real-time). Syst\u00e9m vyu\u017e\u00edva \u010dipy pokryt\u00e9 nano\u0161trukt\u00farov\u00fdm materi\u00e1lom, ktor\u00fd vytv\u00e1ra jamky (ZMW, Zero Mode Waveguide) na dne s DNA polymer\u00e1zou. Sekvena\u010dn\u00fd proces prebieha synt\u00e9zou fluorescen\u010dne zna\u010den\u00fdch nukleotidov. Vzh\u013eadom na to, \u017ee tento pr\u00edstup nevy\u017eaduje prem\u00fdvacie kroky pri za\u010dle\u0148ovan\u00ed jednotliv\u00fdch typov nukleotidov (wash-and-scan), nevyhnutn\u00fdch pri analyz\u00e1toroch druhej gener\u00e1cie, cel\u00fd proces sekvenovania sa zr\u00fdch\u013euje. S\u00fa\u010dasn\u00fdm l\u00eddrom v tejto oblasti je syst\u00e9m PacBio RS a\u00a0v\u00a0porovnan\u00ed so sekven\u00e1tormi druhej gener\u00e1cie dosahuje priemern\u00fa d\u013a\u017eku \u010d\u00edtania 5500-8500 bp. Okrem toho m\u00f4\u017ee tie\u017e priamo detegova\u0165 epigenetick\u00e9 modifik\u00e1cie, ako je 4-metylcytoz\u00edn, 5-metylcytoz\u00edn a 6-metyladen\u00edn [12].<\/p>\n
 <\/p>\n
Nanopor (Oxford Nanopor)<\/strong><\/p>\n
Sekvenovanie pomocou nanop\u00f3rov je zalo\u017een\u00e9 na meran\u00ed elektrick\u00e9ho pr\u00fadu prech\u00e1dzaj\u00faceho cez biologick\u00fd prote\u00edn tvoriaci p\u00f3r v nevodivej membr\u00e1ne. Analytom je jednore\u0165azov\u00e1 molekula DNA, pri jej prechode nanop\u00f3rom d\u00f4jde k detekcii jednotliv\u00fdch nukleotidov, pri\u010dom pre ka\u017ed\u00fd typ nukleotidu je vopred ur\u010den\u00e1 modul\u00e1cia pr\u00fadu. Technol\u00f3gia m\u00e1 minim\u00e1lne po\u017eiadavky na reagencie i pr\u00edpravu vzorky, je lacn\u00e1, r\u00fdchla a pon\u00faka anal\u00fdzu DNA v re\u00e1lnom \u010dase. V s\u00fa\u010dasnosti firma Oxford Nanopor\u00a0 pon\u00faka analyz\u00e1tory MinION\u2122 , PromethION\u2122 a GridION\u2122 [12, 13].<\/p>\n
NGS\u00a0technol\u00f3gie sekvenovania sa neust\u00e1le modifikuj\u00fa a prudko napreduj\u00fa. Dnes vieme s\u00a0istotou poveda\u0165, \u017ee s\u00fa v\u00fdrazne r\u00fdchlej\u0161ie ako v\u00a0roku minulom a\u00a0pomal\u0161ie ako v\u00a0roku bud\u00facom.<\/p>\n
 <\/p>\n
Aplik\u00e1cia a\u00a0vyu\u017eitie NGS technol\u00f3gi\u00ed<\/strong><\/p>\n
Vysok\u00fd v\u00fdkon s\u00fa\u010dasn\u00fdch sekven\u00e1torov je mo\u017en\u00e9 pou\u017ei\u0165 v \u0161irokom spektre aplik\u00e1ci\u00ed, od pokrytia rozsiahlej oblasti\u00a0 cel\u00e9ho gen\u00f3mu (ultra\u0161irok\u00e9 sekvenovanie), po sekvenovanie iba jednej oblasti s\u00a0vysok\u00fdm po\u010dtom \u010d\u00edtan\u00ed (ultrahlbok\u00e9 sekvenovanie). V\u00fdhoda ultrahlbok\u00e9ho sekvenovania je detekcia variantov, ktor\u00e9 sa vo vzorke vyskytuj\u00fa s n\u00edzkou frekvenciou a pr\u00ednos ultra\u0161irok\u00e9ho sekvenovania spo\u010d\u00edva v\u00a0 anal\u00fdze vzoriek viacero pacientov v krat\u0161om \u010dase. Pre \u0161pecifick\u00e9 \u00fa\u010dely m\u00f4\u017eeme teda vyu\u017ei\u0165 celogen\u00f3mov\u00e9 sekvenovanie zah\u0155\u0148aj\u00face de novo<\/em> sekvenovanie a\u00a0resekvenovanie, exomov\u00e9 sekvenovanie k\u00f3duj\u00facich \u010dast\u00ed DNA, sekvenovanie transkriptomu, ktor\u00fd predstavuje s\u00fabor v\u0161etk\u00fdch molek\u00fal RNA (mRNA, rRNA, tRNA a \u010fal\u0161ie nek\u00f3duj\u00faca RNA molekuly) a cielen\u00e9 sekvenovanie (targeted sequencing) umo\u017e\u0148uje sekvenova\u0165 iba vybran\u00e9 g\u00e9ny alebo vymedzen\u00fa oblas\u0165 gen\u00f3mu [14].<\/p>\n
NGS technol\u00f3gie\u00a0pon\u00fakaj\u00fa cel\u00fa plej\u00e1du mo\u017enost\u00ed ich vyu\u017eitia nielen v\u00a0medic\u00edne, ale aj v\u00a0r\u00f4znych vedn\u00fdch odboroch. V\u00fdznamn\u00e9 uplatnenie nach\u00e1dzaj\u00fa napr\u00edklad v\u00a0metagenomike zaoberaj\u00facej sa genomick\u00fdmi anal\u00fdzami mikroorganizmov, kde umo\u017e\u0148uje definova\u0165 napr. mikrobiom \u010dloveka, stanovi\u0165 v\u0161etky druhy bakt\u00e9ri\u00ed vyskytuj\u00facich sa vo vzork\u00e1ch p\u00f4dy, vody. Vo forenznej genetike rezonuje potenci\u00e1lny pr\u00ednos epigenetick\u00fdch \u0161t\u00fadi\u00ed zameran\u00fdch na odl\u00ed\u0161enie monozygotn\u00fdch (jednovaje\u010dn\u00fdch) dvoj\u010diat, ktor\u00e9 maj\u00fa rovnak\u00fa sekvenciu DNA a klasick\u00fdmi met\u00f3dami sa nedali doposia\u013e rozozna\u0165. Vo fylogenetike NGS umo\u017e\u0148uje \u0161tudova\u0165 evolu\u010dn\u00fd v\u00fdvoj a\u00a0vz\u0165ahy medzi organizmami. Antropol\u00f3gia zase vyu\u017e\u00edva porovn\u00e1vanie DNA k zis\u0165ovaniu migr\u00e1cie \u013eudsk\u00fdch r\u00e1s (pod\u013ea mitochondri\u00e1lnej DNA a Y-chromoz\u00f3movej DNA) [1-5, 15-17].<\/p>\n
Odborom, do ktor\u00e9ho NGS sekvenovanie prinieslo doslova revol\u00faciu, je n\u00e1dorov\u00e1 genomika. Realizovali sa rozsiahle vedeck\u00e9 projekty, ktor\u00e9 prispeli ku komplexnej\u0161ej a podrobnej\u0161ej charakteristike molekulovej podstaty n\u00e1dorov\u00fdch ochoren\u00ed. Tieto \u0161t\u00fadie dopomohli k objaveniu nov\u00fdch g\u00e9nov asociovan\u00fdch s\u00a0dan\u00fdm ochoren\u00edm a k stanoveniu genetick\u00e9ho profilu n\u00e1doru [18].\u00a0\u00a0 Obr\u00e1zok \u010d. 2 zn\u00e1zor\u0148uje preh\u013ead novoobjaven\u00fdch somatick\u00fdch mut\u00e1ci\u00ed v\u00a0niektor\u00fdch onkohematologick\u00fdch malignit\u00e1ch.<\/p>\n
Obr. 2 Naj\u010dastej\u0161ie somatick\u00e9 mut\u00e1cie de novo<\/em> detegovan\u00e9 met\u00f3dami NGS v\u00a0g\u00e9noch hematologick\u00fdch malign\u00edt.<\/p>\n
(HCL-vlasatobunkov\u00e1 leuk\u00e9mia, WM-Waldenstr\u00f6mova makroglobulin\u00e9mia, FL-folikulov\u00fd lymf\u00f3m, MCL-lymf\u00f3m z\u00a0pl\u00e1\u0161\u0165ov\u00fdch buniek, CLL-chronick\u00e1 lymfocytov\u00e1 leuk\u00e9mia, MM-mnohopo\u010detn\u00fd myel\u00f3m, ETP ALL- prekurzorov\u00e1 T-bunkov\u00e1 \u00a0ak\u00fatna lymfoblastov\u00e1 leuk\u00e9mia, DLBCL-dif\u00fazny ve\u013ekobunkov\u00fd B-lymf\u00f3m) [18].<\/p>\n
Z\u00a0klinick\u00e9ho h\u013eadiska s\u00fa NGS technol\u00f3gie s\u013eubn\u00fdm n\u00e1strojom s priamym dopadom pri nahraden\u00ed alebo doplnen\u00ed existuj\u00facich laborat\u00f3rnych algoritmov: napr\u00edklad detekcia chromoz\u00f3mov\u00fdch aneuploidi\u00ed DNA plodu pri neinv\u00e1zivnej prenat\u00e1lnej diagnostike, \u00a0identifik\u00e1cia vz\u00e1cnych genetick\u00fdch variantov ochoren\u00ed s mendelovskou dedi\u010dnos\u0165ou, pri identifik\u00e1cii z\u00e1rodo\u010dn\u00fdch mut\u00e1ci\u00ed pri famili\u00e1rnych syndr\u00f3moch. Potenci\u00e1lne vyu\u017eitie je aj pri v\u010dasnej diagnostike \u0161irok\u00e9ho spektra tumorov, anal\u00fdzou vo\u013ene cirkuluj\u00facej n\u00e1dorovej DNA v plazme napr. detekcia mut\u00e1ci\u00ed pri kolorekt\u00e1lnom karcin\u00f3me, muta\u010dn\u00fd status tumor- supresorov\u00fdch g\u00e9nov a chromoz\u00f3mov\u00fdch prestavieb pri lymf\u00f3moch alebo leuk\u00e9mi\u00e1ch, detekcia DNA v\u00edrusov asociovan\u00fdch s\u00a0ur\u010dit\u00fdm typom n\u00e1doru (EBV\u2013nazofarynge\u00e1lny karcin\u00f3m, HPV- cervik\u00e1lne l\u00e9zie) [16, 18-20].<\/p>\n
 <\/p>\n
Z\u00e1ver<\/strong><\/p>\n
Technol\u00f3gie NGS od svojho zavedenia takmer ovl\u00e1dli oblas\u0165 z\u00e1kladn\u00e9ho i aplikovan\u00e9ho v\u00fdskumu a\u00a0za\u010d\u00ednaj\u00fa vstupova\u0165 na p\u00f4du klinickej diagnostiky. Pokia\u013e skuto\u010dne dospejeme k\u00a0rutinn\u00e9mu pou\u017e\u00edvaniu NGS, bude nevyhnutn\u00e9 vyrie\u0161i\u0165 nieko\u013eko prek\u00e1\u017eok a ot\u00e1zok. Vygenerovan\u00e9 mno\u017estvo prim\u00e1rnych d\u00e1t je nutn\u00e9 spr\u00e1vnym sp\u00f4sobom uchova\u0165 a\u00a0anotova\u0165, efekt\u00edvne analyzova\u0165, ale predov\u0161etk\u00fdm spr\u00e1vne interpretova\u0165. Ur\u010denie kvality sekvena\u010dn\u00fdch v\u00fdstupov komplikuje aj absencia medzin\u00e1rodn\u00fdch \u0161tandardov a\u00a0s\u00fa\u010dasn\u00e1 r\u00fdchlos\u0165 regula\u010dn\u00fdch org\u00e1nov, ktor\u00e9 s\u00fa prekonan\u00e9 tempom v\u00fdvoja NGS technol\u00f3gi\u00ed. Mus\u00edme akt\u00edvne prist\u00fapi\u0165 k\u00a0eduk\u00e1cii nielen lek\u00e1rskej obce, ale aj \u0161irokej verejnosti, s cie\u013eom spr\u00e1vne pochopi\u0165, vysvetli\u0165 a\u00a0pou\u017ei\u0165 z\u00edskan\u00e9 inform\u00e1cie.\u00a0 Sn\u00e1\u010f do doby, kedy sa t\u00e1to vyhliadka stane re\u00e1lnou, budeme ma\u0165 aj n\u00e1le\u017eit\u00fd progres\u00edvny z\u00e1kon o\u00a0DNA, nako\u013eko ide o\u00a0d\u00e1ta nielen citliv\u00e9, ale najm\u00e4 cenn\u00e9 z\u00a0viacer\u00fdch aspektov.<\/p>\n
 <\/p>\n
Literat\u00fara<\/strong><\/p>\n
    \n
  1. Hutchison, C. A. (2007) DNA sequencing: bench to bedside and beyond., Nucleic Acids Res.,\u00a0 35(18): 6227\u20136237.<\/li>\n
  2. Posp\u00ed\u0161ilov\u00e1 \u0160., Tich\u00fd B., Mayer J. (2009) Sekvenov\u00e1n\u00ed lidsk\u00e9ho genomu \u2013 technologie nov\u00e9 generace aneb budeme rutinn\u011b sekvenovat lidsk\u00e9 genomy?, \u010cas L\u00e9k \u010des., 148: 296-302.<\/li>\n
  3. Metzker M. L. (2010) Sequencing technologies \u2013 the next generation.,\u00a0 Nat Rew Genet., 11: 31-46.<\/li>\n
  4. Anderson M., Schrijver I. (2010) Next Generation DNA Sequencing and the Future of Genomic Medicine., Genes,<\/em> 1<\/em>(1): 38-69.<\/li>\n
  5. Shendure J., Ji H. (2008) Next-generation DNA sequencing., Nature Biotechnology,<\/em>\u00a026: 1135 \u2013 114.<\/li>\n
  6. Liu L., Li Y., Li S. et al. (2012) Comparison of next-generation sequencing systems., J Biomed Biotechnol., 251364. doi: 10.1155\/2012\/251364.<\/li>\n
  7. www.roche.com<\/a><\/li>\n
  8. \u00a0www.illumina.com<\/a><\/li>\n
  9. \u00a0www.lifetechnologies.com<\/a><\/li>\n
  10. Rothberg J. M., Hinz W., Rearick T. M., et al.(2011)An integrated semiconductor device enabling non-optical genome sequencing., Nature, 457: 348-352.<\/li>\n
  11. Schadt E. E. et al. (2010) A window into third-generation sequencing., Hum Mol Genet.,\u00a0 19(R2): R227.<\/li>\n
  12. van Dijk . E. L.,. Auger H., Jaszczyszyn Y., C. Thermes C. (2014) Ten years of next-generation sequencing technology., Trends Genet, 30 (2014), pp. 418\u2013426<\/li>\n
  13. www.nanoprotech.com<\/a><\/li>\n
  14. Travis C., Glenn. (2011) Field guide to next-generation DNA sequencers., Molecular Ecology Resources., 11: 759\u2013769.<\/li>\n
  15. Yang Y., Xie B., Yan J. (2014) Application of Next-generation Sequencing Technology in Forensic Science., Genomics Proteomics Bioinformatics, 12: 190\u2013197.<\/li>\n
  16. Desai A. N., Jere A. (2012) Next-generation sequencing: ready for the clinics?, Clin Genet., 81: 503-510.<\/li>\n
  17. \u00a0www.sciencedaily.com<\/a><\/li>\n
  18. Braggio E., Egan J. B., Fonseca R., Stewart A., K. (2013) Lessons from next-generation sequencing analysis in hematological malignancies., 3, e127; doi:10.1038\/bcj.2013.26<\/li>\n
  19. Yu J., Gu\u00a0 G., Ju S. (2014) Recent Advances in Clinical Applications of Circulating Cell-free DNA Integrity., Lab Med., 45(1): 6-12.<\/li>\n
  20. Chan M., , Lee CH. W-H., Wu M. (2013) Integrating Next-Generation Sequencing Into Clinical Cancer Diagnostics,. Expert Rev Mol Diagn.,\u00a013(7): 647-650.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"
    *All tables, charts, graphs and pictures that are featured in this article can be found in the .pdf attachment at the end of the paper.   Klasick\u00e9 met\u00f3dy sekvenovania Prv\u00e9 pokusy sekvenovania sa datuj\u00fa do 70. rokov minul\u00e9ho storo\u010dia, kedy boli realizovan\u00e9 nepriamo – sekvenovan\u00edm molek\u00fal RNA alebo prote\u00ednov. Prv\u00fa sekvenciu DNA z\u00edskal v\u00a0roku 1968<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_mi_skip_tracking":false,"footnotes":""},"categories":[290],"tags":[728,612,179,729],"class_list":["post-1125","post","type-post","status-publish","format-standard","hentry","category-genetics","tag-dna-sequencing","tag-massive-parallel-sequencing","tag-next-generation-sequencing","tag-ngs-applications","typ_clanku-review-article"],"acf":{"abstrakt":"
    DNA sequencing is a process of determining the precise sequence of nucleotides in the DNA molecule. It includes all methods or technologies that are used to determine the order of the four bases – adenine (A), guanine (G), cytosine (C) and thymine (T) in a strand of DNA. In the 21st<\/sup> century, a new technology titled next generation sequencing (NGS) was developed. In many aspects, it exceeds the conventional sequencing methods. New technology is specified by high performance, speed and low cost per base. Currently, NGS is one of the most rapidly advancing methods in molecular genetics and has the potential to usher in a breakthrough in the field of personalized medicine in the near future. In this review, we briefly describe basic principles and the most common NGS technologies.<\/p>\n
    Key words<\/strong>: DNA sequencing, next generation sequencing, massive parallel sequencing, NGS applications.<\/p>\n","casopis":[{"ID":1000,"post_author":"7","post_date":"2015-11-21 14:33:24","post_date_gmt":"2015-11-21 13:33:24","post_content":"
    GENETICS<\/strong><\/h4>\r\n
      \r\n \t
    • The laboratory age<\/li>\r\n \t
    • The Odyssey of DNA reading<\/li>\r\n \t
    • Comparative genomic hybridisation: a methodological introduction<\/li>\r\n \t
    • Next generation sequencing and its application in clinical genetics<\/li>\r\n<\/ul>\r\n
      <\/h4>\r\n
      BIOCHEMISTRY<\/strong><\/h4>\r\n
        \r\n \t
      • Determining of the trace elements in blood serum<\/li>\r\n \t
      • Determining of \u03b1-tocopherol (vitamin E) in serum by way of liquid chromatography with tandem mass spectrometry (LC\/MS\/MS)<\/li>\r\n \t
      • Analysis of urinary calculi and its path to Europe<\/li>\r\n<\/ul>\r\n \r\n
        IMMUNOLOGY <\/strong><\/h4>\r\n
          \r\n \t
        • New autoantibodies in diagnosis of autoimmunity myopathies<\/li>\r\n \t
        • Taking advantage of flow-based cytometry in determining prognostic markers<\/li>\r\n<\/ul>\r\nCD38 and ZAP-70 in patients with B-CLL\r\n\r\n \r\n
          CYTOLOGY AND PATHOLOGY <\/strong><\/h4>\r\n
            \r\n \t
          • Mucinous ovarian carcinoma \u2013 news in diagnosis from the pathologist\u2019s perspective<\/li>\r\n<\/ul>","post_title":"newsLab","post_excerpt":"","post_status":"publish","comment_status":"closed","ping_status":"closed","post_password":"","post_name":"newslab-12015","to_ping":"","pinged":"","post_modified":"2017-08-16 21:43:10","post_modified_gmt":"2017-08-16 19:43:10","post_content_filtered":"","post_parent":0,"guid":"http:\/\/www.newslab.sk\/casopis\/newslab-12015\/","menu_order":0,"post_type":"casopis","post_mime_type":"","comment_count":"0","filter":"raw"}],"strana":"7","upload_clanok":{"ID":1126,"id":1126,"title":"medirex_1_2015-tomasova","filename":"MEDIREX_1_2015-\u2013-Tomasova.pdf","filesize":171743,"url":"https:\/\/www.newslab.sk\/wp-content\/uploads\/2016\/12\/MEDIREX_1_2015-\u2013-Tomasova.pdf","link":"https:\/\/www.newslab.sk\/en\/the-odyssey-of-dna-reading\/medirex_1_2015-tomasova\/","alt":"","author":"7","description":"","caption":"","name":"medirex_1_2015-tomasova","status":"inherit","uploaded_to":1125,"date":"2016-12-05 20:52:28","modified":"2016-12-05 20:52:28","menu_order":0,"mime_type":"application\/pdf","type":"application","subtype":"pdf","icon":"https:\/\/www.newslab.sk\/wp-includes\/images\/media\/document.png"}},"_links":{"self":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1125","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/comments?post=1125"}],"version-history":[{"count":0,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/posts\/1125\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/media?parent=1125"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/categories?post=1125"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newslab.sk\/en\/wp-json\/wp\/v2\/tags?post=1125"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}