TY  - CHAP
AU  - Savić-Pavićević, Dušanka
AU  - Radenković, Lana
AU  - Velimirov, Luka
AU  - Radovanović, Nemanja
AU  - Ninković, Anastasija
AU  - Garai, Nemanja
AU  - Brkušanin, Miloš
AU  - Panić, Marko
AU  - Pešović, Jovan
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2246
AB  - Sekvenciranje dugih fragmenata ili treća generacija sekvenciranja u realnom vremenu produkuje očitavanja
pojedinačnih molekula DNK dužine od 1 kb do nekoliko Mb sa 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.
AB  - 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.
PB  - Beograd : Institut za molekularnu genetiku i genetičko inženjerstvo
T2  - Trendovi u molekularnoj Biologiji
T1  - Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja
T1  - Long read sequencing – the next level in genomic research
EP  - 56
IS  - 3
SP  - 38
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2246
ER  - 

@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 = "Sekvenciranje dugih fragmenata ili treća generacija sekvenciranja u realnom vremenu produkuje očitavanja
pojedinačnih molekula DNK dužine od 1 kb do nekoliko Mb sa 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., 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.",
publisher = "Beograd : Institut za molekularnu genetiku i genetičko inženjerstvo",
journal = "Trendovi u molekularnoj Biologiji",
booktitle = "Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja, Long read sequencing – the next level in genomic research",
pages = "56-38",
number = "3",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2246"
}

Savić-Pavićević, D., Radenković, L., Velimirov, L., Radovanović, N., Ninković, A., Garai, N., Brkušanin, M., Panić, M.,& Pešović, J.. (2023). Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja. in Trendovi u molekularnoj Biologiji
Beograd : Institut za molekularnu genetiku i genetičko inženjerstvo.(3), 38-56.
https://hdl.handle.net/21.15107/rcub_imagine_2246

Savić-Pavićević D, Radenković L, Velimirov L, Radovanović N, Ninković A, Garai N, Brkušanin M, Panić M, Pešović J. Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja. in Trendovi u molekularnoj Biologiji. 2023;(3):38-56.
https://hdl.handle.net/21.15107/rcub_imagine_2246 .

Savić-Pavićević, Dušanka, Radenković, Lana, Velimirov, Luka, Radovanović, Nemanja, Ninković, Anastasija, Garai, Nemanja, Brkušanin, Miloš, Panić, Marko, Pešović, Jovan, "Sekvenciranje dugih fragmenata – sledeći nivo genomskih istraživanja" in Trendovi u molekularnoj Biologiji, no. 3 (2023):38-56,
https://hdl.handle.net/21.15107/rcub_imagine_2246 .

TY  - CONF
AU  - Velimirov, Luka
AU  - Babić, Tamara
AU  - Dragičević, Sandra
AU  - Nikolić, Aleksandra
PY  - 2022
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2084
AB  - Gene LDLRAD4 plays a role in cell proliferation, apoptosis, immunosuppression and cancer
progression. Transcription of LDLRAD4 is regulated by several alternative promoters, two of which
were indicated by in silico analyses to be differentially active in rectal cancer. Promoter A encodes for a
truncated protein-coding transcript and is down-regulated in rectal cancer. Promoter B encodes for a
non-coding transcript up-regulated in rectal cancer identified as lnc-RNMT-2:5. The aim of this study
was to characterize the two alternative promoters in silico in order to explain their differential activity
and to investigate the profile of LDLRAD4 transcripts in colon cell lines. Nucleotide sequences used in
the analyses were downloaded from the Ensemble genome database (reference GRCh37). Three
bioinformatics tools were used for core promoter element prediction: GPMiner, YAPP and
CNNPromoter. Four bioinformatics tools were used for transcription factor binding site prediction:
PROMO, TFBIND, CiiiDER and Tfsitescan. Only the predictions made by two or more tools were
considered. Primer extension followed by fragment analysis was used to characterize LDLRAD4
transcripts present in colon cell lines. The promoter element predictions showed that the promoter A is
typical, while promoter B has most typical elements and lacks GC boxes. The transcription binding site
predictions indicate that three different transcription factors bind only to the promoter A (NF-kB,
EGR1 and IRF-7), while four different transcription factors bind only to the promoter B (HNF1,
POU2F1, POU2F2 and PTF1). The predicted transcription factors are mostly involved in regulation of
cell differentiation and proliferation. The primer extension experiment performed with primer specific
for exon 2-exon 3 junction produced multiple signals of relatively low intensity, indicating the presence
of multiple LDLRAD4 transcripts in colon cell lines. The results obtained by in silico analysis may
explain promoter B activation in rectal cancer. However, based on the results of primer extension, neither
of the LDLRAD4 transcripts is dominant in colon cell lines. Considering that promoter B generates
long non-coding RNA that can exert its function even at low expression level, it can serve as potential
colorectal cancer biomarker and its potential role in carcinogenesis should be investigated.
PB  - Croatian Association for Cancer Research, Zagreb, Croatia
C3  - “HDIR-6: Targeting Cancer” The 6th Meeting of the Croatian Association for Cancer Research with International Participation – Book of Abstracts
T1  - Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer
EP  - 35
SP  - 35
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2084
ER  - 

@conference{
author = "Velimirov, Luka and Babić, Tamara and Dragičević, Sandra and Nikolić, Aleksandra",
year = "2022",
abstract = "Gene LDLRAD4 plays a role in cell proliferation, apoptosis, immunosuppression and cancer
progression. Transcription of LDLRAD4 is regulated by several alternative promoters, two of which
were indicated by in silico analyses to be differentially active in rectal cancer. Promoter A encodes for a
truncated protein-coding transcript and is down-regulated in rectal cancer. Promoter B encodes for a
non-coding transcript up-regulated in rectal cancer identified as lnc-RNMT-2:5. The aim of this study
was to characterize the two alternative promoters in silico in order to explain their differential activity
and to investigate the profile of LDLRAD4 transcripts in colon cell lines. Nucleotide sequences used in
the analyses were downloaded from the Ensemble genome database (reference GRCh37). Three
bioinformatics tools were used for core promoter element prediction: GPMiner, YAPP and
CNNPromoter. Four bioinformatics tools were used for transcription factor binding site prediction:
PROMO, TFBIND, CiiiDER and Tfsitescan. Only the predictions made by two or more tools were
considered. Primer extension followed by fragment analysis was used to characterize LDLRAD4
transcripts present in colon cell lines. The promoter element predictions showed that the promoter A is
typical, while promoter B has most typical elements and lacks GC boxes. The transcription binding site
predictions indicate that three different transcription factors bind only to the promoter A (NF-kB,
EGR1 and IRF-7), while four different transcription factors bind only to the promoter B (HNF1,
POU2F1, POU2F2 and PTF1). The predicted transcription factors are mostly involved in regulation of
cell differentiation and proliferation. The primer extension experiment performed with primer specific
for exon 2-exon 3 junction produced multiple signals of relatively low intensity, indicating the presence
of multiple LDLRAD4 transcripts in colon cell lines. The results obtained by in silico analysis may
explain promoter B activation in rectal cancer. However, based on the results of primer extension, neither
of the LDLRAD4 transcripts is dominant in colon cell lines. Considering that promoter B generates
long non-coding RNA that can exert its function even at low expression level, it can serve as potential
colorectal cancer biomarker and its potential role in carcinogenesis should be investigated.",
publisher = "Croatian Association for Cancer Research, Zagreb, Croatia",
journal = "“HDIR-6: Targeting Cancer” The 6th Meeting of the Croatian Association for Cancer Research with International Participation – Book of Abstracts",
title = "Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer",
pages = "35-35",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2084"
}

Velimirov, L., Babić, T., Dragičević, S.,& Nikolić, A.. (2022). Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer. in “HDIR-6: Targeting Cancer” The 6th Meeting of the Croatian Association for Cancer Research with International Participation – Book of Abstracts
Croatian Association for Cancer Research, Zagreb, Croatia., 35-35.
https://hdl.handle.net/21.15107/rcub_imagine_2084

Velimirov L, Babić T, Dragičević S, Nikolić A. Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer. in “HDIR-6: Targeting Cancer” The 6th Meeting of the Croatian Association for Cancer Research with International Participation – Book of Abstracts. 2022;:35-35.
https://hdl.handle.net/21.15107/rcub_imagine_2084 .

Velimirov, Luka, Babić, Tamara, Dragičević, Sandra, Nikolić, Aleksandra, "Analysis of Alternative LDLRAD4 Gene Promoters and Transcripts in Colorectal Cancer" in “HDIR-6: Targeting Cancer” The 6th Meeting of the Croatian Association for Cancer Research with International Participation – Book of Abstracts (2022):35-35,
https://hdl.handle.net/21.15107/rcub_imagine_2084 .