@inbook{
author = "Savić-Pavićević, Dušanka and Radenković, Lana and Velimirov, Luka and Radovanović, Nemanja and Ninković, Anastasija and Garai, Nemanja and Brkušanin, Miloš and Panić, Marko and Pešović, Jovan",
year = "2023",
abstract = "Long read or third-generation sequencing produces reads from 1 kb to several Mb in length with preserved epigenetic marks, at the single-molecule level and in real-time. Single-molecule real-time sequencing (PacBio) and protein nanopore sequencing (Oxford Nanopore Technologies) are available technologies. PacBio technology is based on monitoring the nucleotide incorporation by a single DNA polymerase molecule in real time using fluorescence as a surrogate marker. PacBio HiFi reads are ~15 kb in length with >99.9% accuracy. Oxford Nanopore sequencing infers nucleotide sequence from the changes in ion current intensity while DNA passes through a stochastic sensor – a protein nanopore. It can sequence DNA fragments ranging in five orders of magnitude (20 bp to several Mb), with duplex read accuracy >99.9% when using R10.4.1 nanopores. Innovations such as a miniature device of the palm-size with a price <1000 dollars, sequencing in the field, digital enrichment of target sequences (adaptive sampling) and direct RNA sequencing have become a reality with the electronic „reading“ of nucleic acids. Long read sequencing enabled completing the human genome sequence and releasing a draft of the human pangenome reference. It has also accelerated genome sequencing of eukaryotic species. Out of 1065 genomes deposited in the NCBI database, ~1000 were sequenced since its development. The full potential of the method in studying transcriptome and epigenome will be visible in the years to come. Long read sequencing is becoming the basis of precision medicine effective for all human populations and biodiversity conservation and was announced as the method of the year 2022 according to Nature Methods., Sekvenciranje dugih fragmenata ili treća generacija sekvenciranja u realnom vremenu produkuje očitavanja pojedinačnih molekula DNK dužineod 1 kb do nekoliko Mbsa očuvanim epigenetičkim oznakama. Dostupne tehnologije su sekvenciranje pojedinačnih molekula u realnom vremenu (eng. single-molecule real-time sequencing, PacBio) i sekvenciranje kroz proteinske nanopore (Oxford Nanopore Technologies). PacBio tehnologija zasnovana je na detekciji ugradnje nukelotida od strane pojedinačnog molekula DNK polimeraze u realnom vremenu, korišćenjem fluoresecencije kao surogat markera. PacBio HiFi očitavanja su dužine ~15 kb sa tačnošću >99,9%. Oxford Nanopore tehnologija izvodi sekvencu nukleotida iz promena u intenzitetu jonske struje dok DNK prolazi kroz stohastički senzor – proteinsku nanoporu.Može sekvencirati fragmente DNK u rasponu od pet redova veličina (20 bp do nekoliko Mb) sa tačnošću dupleks očitavanja >99,9% kada se koriste R10.4.1 nanopore. Sa elektronskim „čitanjem” nukleinskih kiselina, inovacije kao što su minijaturni uređaj veličine dlana sa cenom <1000 dolara, sekvenciranje na terenu, digitalno obogaćivanje ciljnih sekvenci (adaptivno uzorkovanje) i direktno sekvenciranje RNK, postali su stvarnost. Sekvenciranje dugih fragmenata omogućilo je kompletiranje sekvence genoma čoveka, objavljivanje drafta ljudskog pangenoma i ubrzalo je sekvenciranje genoma eukariota. Od uvođenja metode 2011. godine sekvencirano je ~1000 od 1065 genoma deponovanih u NCBI bazi. Puni potencijal metode u izučavanju transkriptoma i epigenoma biće vidljiv u godinama koje slede. Sekvenciranje dugih fragmenata postaje osnova precizne medicine efikasne za sve ljudske populacije i očuvanja biodiverziteta, i zavredelo je da bude metoda 2022. godine prema časopisu Nature Methods.",
publisher = "IMGGI",
booktitle = "Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja, Long read sequencing – the next level in genomic research",
url = "https://hdl.handle.net/21.15107/rcub_intor_937"
}
