1 research outputs found
Long read sequencing ā the next level in genomic research
Sekvenciranje dugih fragmenata ili treÄa generacija sekvenciranja u realnom vremenu produkuje oÄitavanja
pojedinaÄnih molekula DNK dužine od 1 kb do nekoliko Mb sa oÄuvanim epigenetiÄkim oznakama.
Dostupne tehnologije su sekvenciranje pojedinaÄnih 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 pojedinaÄnog molekula DNK
polimeraze u realnom vremenu, koriÅ”Äenjem fluoresecencije kao surogat markera. PacBio HiFi oÄitavanja
su dužine ~15 kb sa taÄnoÅ”Äu >99,9%. Oxford Nanopore tehnologija izvodi sekvencu nukleotida iz promena
u intenzitetu jonske struje dok DNK prolazi kroz stohastiÄki senzor ā proteinsku nanoporu.Može sekvencirati
fragmente DNK u rasponu od pet redova veliÄina (20 bp do nekoliko Mb) sa taÄnoÅ”Äu dupleks
oÄitavanja >99,9% kada se koriste R10.4.1 nanopore. Sa elektronskim āÄitanjemā nukleinskih kiselina, inovacije
kao Å”to su minijaturni ureÄaj veliÄine dlana sa cenom <1000 dolara, sekvenciranje na terenu, digitalno
obogaÄivanje ciljnih sekvenci (adaptivno uzorkovanje) i direktno sekvenciranje RNK, postali su
stvarnost. Sekvenciranje dugih fragmenata omoguÄilo je kompletiranje sekvence genoma Äoveka, objavljivanje
drafta ljudskog pangenoma i ubrzalo je sekvenciranje genoma eukariota. Od uvoÄenja metode
2011. godine sekvencirano je ~1000 od 1065 genoma deponovanih u NCBI bazi. Puni potencijal metode
u izuÄavanju transkriptoma i epigenoma biÄe vidljiv u godinama koje slede. Sekvenciranje dugih fragmenata
postaje osnova precizne medicine efikasne za sve ljudske populacije i oÄuvanja biodiverziteta, i zavredelo
je da bude metoda 2022. godine prema Äasopisu Nature Methods.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 ā 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 āreadingā 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