Učinkovitost sljedeće generacije sekvenciranja u dijagnostici bakterijskih zoonoza

Brucella, an extremely diverse but yet genetically highly homogenous genus of bacteria, has been a puzzle for scientists for many decades. These bacteria remain a prominent public health issue, particularly in the Balkan region. Correctly identifying and understanding the pathogen is a vital step in the epidemiology and epizootiology of any bacteria. Identification can be challenging, especially in the case of zoonotic species. This study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis strains kept in our archive and to compare this method to the classical and non-sequencing based molecular methods used to date. Classical biotyping is highly subjective and gave inconclusive results for 3 strains. Molecular methods used were multiplex PCR and RFLP methods since no one method can identify both species and biovar which is vital in the case of Brucella suis infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results. Oxford Nanopore long-read sequencing was used on a MinION device for next-generation sequencing (NGS). Various algorithms were implemented for genome assembly and BioNumerics 8.0 software was used for MLST identification and analysis. MLST 21 was used for biovar identification and epidemiological comparison of tested strains. The assembled genomes were 3,2 Mb in size and assembled into two chromosomes. MLST 21 analysis placed our strains into species and biovar clusters in concordance with other molecular tests used. To the extent of our knowledge, this is the first documented use of long-read sequencing in Brucella suis identification in this region. We conclude that bacteriological biotyping is outdated and host-specific identification in this genus is incorrect and that molecular characterisation is always the safer, faster and more suitable option. MinION sequencing proved to be a strong, accessible solution for species determination. Future study is required to determine how detailed genome information it can give, considering the error rate.Rod Brucella biološki je iznimno raznolik, ali genetski vrlo homogen rod bakterija te je već desetljećima znanstvenicima nepoznanica. Ove bakterije su veliki javno-zdravstveni problem, a osobito na Balkanu. Pravilno prepoznavanje i razumijevanje patogena ključan je korak u epidemiologiji i epizootiologiji bilo koje bakterijske vrste, čija identifikacija može biti izazovna, osobito u slučaju zoonotskih vrsta. Cilj je ovog rada bio implementirati sekvenciranje četvrte generacije u tipizaciji 11 sojeva Brucella suis koje se čuvaju u našoj arhivi te ovu metodu usporediti s klasičnim i molekularnim metodama koje se trenutačno primjenjuju, a ne zasnivaju se na sekvenciranju. Klasično je biotipiziranje vrlo subjektivno i dalo je podvojene rezultate za 3 soja. Od molekularnih metoda koristili smo višestruku lančanu reakciju polimerazom (engl. Polymerase Chain Reaction, PCR) i polimorfizam duljine restrikcijskih fragmenata (engl. Restriction Fragment Lenght Polymorphism, RFLP) budući da niti jedna od metoda ne može zasebno identificirati i vrstu i biovar, a što je važno u slučaju Brucella suis infekcije. Vrsta i biovar svih sojeva uspješno su potvrđene i u skladu s rezultatima biotipizacije. Sekvenciranje sljedeće generacije (engl. Next Generation Sequencing, NGS) provodili smo na Oxford Nanopore MinION uređaju koji sekvencira duge lance DNK. Za sastavljanje genoma rabljeni su različiti algoritmi, a za identifikaciju i analizu rezultata MLST-a korišten je softver BioNumerics 8.0. MLST 21 je korišten za identifikaciju biovara i epidemiološku usporedbu ispitivanih sojeva. Genomi su bili veličine 3,2 Mb i sastavljeni u dva kromosoma. Analiza MLST 21 smjestila je naše sojeve u vrsne i biovarne skupine u skladu s drugim korištenim molekularnim testovima. Koliko je nama poznato, ovo je prva dokumentirana uporaba sekvenciranja dugih lanaca DNK u identifikaciji Brucella suis u jugoistočnoj Europi. Zaključujemo da je bakteriološka biotipizacija zastarjela i da je identifikacija biovara u ovom rodu, ovisno o domaćinu, netočna te da je molekularna karakterizacija uvijek sigurnija, brža i prikladnija opcija. MinION sekvenciranje pokazalo se kao vrlo pristupačno rješenje za određivanje vrste i biovara Brucella suis. Daljnja su istraživanja potrebna da bi se ustvrdilo koliko detaljne informacije o genomu može dati, imajući u vidu značajniji postotak pogreške prilikom sekvenciranja

Compendium of 4,941 rumen metagenome-assembled genomes for rumen microbiome biology and enzyme discovery

The Rowett Institute and SRUC are core funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The Roslin Institute forms part of the Royal (Dick) School of Veterinary Studies, University of Edinburgh. This project was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/N016742/1, BB/N01720X/1), including institute strategic programme and national capability awards to The Roslin Institute (BBSRC: BB/P013759/1, BB/P013732/1, BB/J004235/1, BB/J004243/1); and by the Scottish Government as part of the 2016–2021 commission.Peer reviewedPublisher PD

Algoritmi za de novo sastavljanje velikih genoma.

The inability of DNA sequencing technologies to interpret entire molecules led to the development of methods that connect the obtained short fragments back together in a puzzle-like process. They are called assemblers and their design is guided with the notion that similar fragments originate from the same genomic region. That is often annulled due to sequencing errors and repetitive nature of the genome. The advent of new sequencing approaches, namely Pacific Biosciences and Oxford Nanopore Technologies, pushed the limit on the fragment lengths at a cost of higher error rates, but still facilitated the assembly problem considerably. First assembly attempts used various types of error correction approaches prior the assembly with existing tools at that time. Although, several long read based assemblers have been proposed in the past years, they demand significant amounts of computational resources. The focus of this research is development of memory efficient and scalable algorithms for de novo assembly of large genomes using raw third generation of sequencing data. In the scope of the thesis we implemented three novel tools for genome assembly: a memory friendly layout module called Rala, which builds the assembly graph from preprocessed sequences and resolves junctions in graph with the help of force directed placement; a fast and accurate consensus module called Racon based on vectorized partial order alignment; and the complete de novo assembler called Raven, which competes with state-of-the-art assemblers both in quality and resource management.Tehnologije za sekvenciranje genoma nisu u mogućnosti interpretirati DNA molekule u cijelosti te je to dovelo do razvoja računalnih metoda koje spajaju kratke fragmente u procesu koji podsjeća na rješavanje slagalica. Alati namijenjeni za ovaj problem nazivaju se asembleri čiji je dizajn baziran na pretpostavci da slični fragmenti potječu iz iste regije genoma. Ta pretpostavka je često poništena zbog pogrešaka prilikom sekvenciranja te repetitivne prirode genoma. Pojava novih tehnologija za sekvenciranje tvrtki Pacific Biosciences i Oxford Nanopore Technologies olakšala je problem sastavljanja genoma zahvaljujući znatno većoj duljini dobivenih fragmenata, ali uz nedostatak veće pogreške. Iako nekoliko asemblera za dugačke fragmente već postoji, oni zahtijevaju pozamašne količine računalnih resursa. Fokus ovog istraživanja je razvoj memorijskih efikasnih i skalabilnih algoritama za de novo sastavljanje velikih genoma, pritom koristeći podatke treće generacije sekvenciranja. U sklopu ove disertacije implementirana su tri nova alata za sastavljanje genoma: memorijski učinkovita faza razmještaja zvana Rala, koja gradi graf sastavljanja iz skupa filtriranih fragmenata te ga pojednostavljuje koristeći algoritam razmještaja simulacijom djelovanja silama; brza i točna faza konsenzusa zvana Racon, koja je bazirana na vektoriziranom poravnanju parcijalnog uređaja; te cjelokupan de novo asembler nazvan Raven, koji konkurira s trenutno najefikasnijim poznatim metodama u kvaliteti i upravljanju resursima

Algoritmi za de novo sastavljanje velikih genoma.

The inability of DNA sequencing technologies to interpret entire molecules led to the development of methods that connect the obtained short fragments back together in a puzzle-like process. They are called assemblers and their design is guided with the notion that similar fragments originate from the same genomic region. That is often annulled due to sequencing errors and repetitive nature of the genome. The advent of new sequencing approaches, namely Pacific Biosciences and Oxford Nanopore Technologies, pushed the limit on the fragment lengths at a cost of higher error rates, but still facilitated the assembly problem considerably. First assembly attempts used various types of error correction approaches prior the assembly with existing tools at that time. Although, several long read based assemblers have been proposed in the past years, they demand significant amounts of computational resources. The focus of this research is development of memory efficient and scalable algorithms for de novo assembly of large genomes using raw third generation of sequencing data. In the scope of the thesis we implemented three novel tools for genome assembly: a memory friendly layout module called Rala, which builds the assembly graph from preprocessed sequences and resolves junctions in graph with the help of force directed placement; a fast and accurate consensus module called Racon based on vectorized partial order alignment; and the complete de novo assembler called Raven, which competes with state-of-the-art assemblers both in quality and resource management.Tehnologije za sekvenciranje genoma nisu u mogućnosti interpretirati DNA molekule u cijelosti te je to dovelo do razvoja računalnih metoda koje spajaju kratke fragmente u procesu koji podsjeća na rješavanje slagalica. Alati namijenjeni za ovaj problem nazivaju se asembleri čiji je dizajn baziran na pretpostavci da slični fragmenti potječu iz iste regije genoma. Ta pretpostavka je često poništena zbog pogrešaka prilikom sekvenciranja te repetitivne prirode genoma. Pojava novih tehnologija za sekvenciranje tvrtki Pacific Biosciences i Oxford Nanopore Technologies olakšala je problem sastavljanja genoma zahvaljujući znatno većoj duljini dobivenih fragmenata, ali uz nedostatak veće pogreške. Iako nekoliko asemblera za dugačke fragmente već postoji, oni zahtijevaju pozamašne količine računalnih resursa. Fokus ovog istraživanja je razvoj memorijskih efikasnih i skalabilnih algoritama za de novo sastavljanje velikih genoma, pritom koristeći podatke treće generacije sekvenciranja. U sklopu ove disertacije implementirana su tri nova alata za sastavljanje genoma: memorijski učinkovita faza razmještaja zvana Rala, koja gradi graf sastavljanja iz skupa filtriranih fragmenata te ga pojednostavljuje koristeći algoritam razmještaja simulacijom djelovanja silama; brza i točna faza konsenzusa zvana Racon, koja je bazirana na vektoriziranom poravnanju parcijalnog uređaja; te cjelokupan de novo asembler nazvan Raven, koji konkurira s trenutno najefikasnijim poznatim metodama u kvaliteti i upravljanju resursima

De Novo Transcriptome Assembly

U ovom radu, implementiran je de novo asembler transkriptoma koji je baziran na preklapanje-razmještaj-konsenzus paradigmi. Napisan je u programskom jeziku C++ i imenovan je Ra što je skraćeno od RNA asembler. Faza preklapanja bazirana je na poboljšanom sufiksnom polju i reproducira egzaktna preklapanja između ulaznih očitanja. Faza razmještaja koristi nekoliko metoda za pojednostavljenje grafova koji su izgrađeni nad očitanjima i njihovim preklapanjima. Kako faza preklapanja pronalazi samo egzaktne parove, trenutno ne postoji potreba za fazom konsenzusa ali ona je svejedno dio implementacije i bazirana je na partial order alignment algoritmu. Provedeni testovi upućuju da su Ra asembleru potrebna dodatna poboljšanja kako bi mogao konkurirati drugim asemblerima transkriptoma. Izvorni kod dostupan je na https://github.com/rvaser/ra.In this thesis, a de novo transcriptome assembler was implemented based on the overlap-layout-consensus paradigm. It was written in the C++ programming language and was named Ra which is short for RNA assembler. Its overlap phase relies on the enhanced suffix arrays and reproduces exact overlaps between input reads. The layout phase uses several methods for graph simplification which includes trimming and bubble popping. Due to the exact overlap phase there is no need for a consensus phase at this moment but there exists one which is based on the partial order alignment algorithm. Conducted tests have shown that Ra needs improvements to compete with other transcriptome assemblers. Source code is available at https://github.com/rvaser/ra

De Novo Transcriptome Assembly

U ovom radu, implementiran je de novo asembler transkriptoma koji je baziran na preklapanje-razmještaj-konsenzus paradigmi. Napisan je u programskom jeziku C++ i imenovan je Ra što je skraćeno od RNA asembler. Faza preklapanja bazirana je na poboljšanom sufiksnom polju i reproducira egzaktna preklapanja između ulaznih očitanja. Faza razmještaja koristi nekoliko metoda za pojednostavljenje grafova koji su izgrađeni nad očitanjima i njihovim preklapanjima. Kako faza preklapanja pronalazi samo egzaktne parove, trenutno ne postoji potreba za fazom konsenzusa ali ona je svejedno dio implementacije i bazirana je na partial order alignment algoritmu. Provedeni testovi upućuju da su Ra asembleru potrebna dodatna poboljšanja kako bi mogao konkurirati drugim asemblerima transkriptoma. Izvorni kod dostupan je na https://github.com/rvaser/ra.In this thesis, a de novo transcriptome assembler was implemented based on the overlap-layout-consensus paradigm. It was written in the C++ programming language and was named Ra which is short for RNA assembler. Its overlap phase relies on the enhanced suffix arrays and reproduces exact overlaps between input reads. The layout phase uses several methods for graph simplification which includes trimming and bubble popping. Due to the exact overlap phase there is no need for a consensus phase at this moment but there exists one which is based on the partial order alignment algorithm. Conducted tests have shown that Ra needs improvements to compete with other transcriptome assemblers. Source code is available at https://github.com/rvaser/ra

De Novo Transcriptome Assembly

U ovom radu, implementiran je de novo asembler transkriptoma koji je baziran na preklapanje-razmještaj-konsenzus paradigmi. Napisan je u programskom jeziku C++ i imenovan je Ra što je skraćeno od RNA asembler. Faza preklapanja bazirana je na poboljšanom sufiksnom polju i reproducira egzaktna preklapanja između ulaznih očitanja. Faza razmještaja koristi nekoliko metoda za pojednostavljenje grafova koji su izgrađeni nad očitanjima i njihovim preklapanjima. Kako faza preklapanja pronalazi samo egzaktne parove, trenutno ne postoji potreba za fazom konsenzusa ali ona je svejedno dio implementacije i bazirana je na partial order alignment algoritmu. Provedeni testovi upućuju da su Ra asembleru potrebna dodatna poboljšanja kako bi mogao konkurirati drugim asemblerima transkriptoma. Izvorni kod dostupan je na https://github.com/rvaser/ra.In this thesis, a de novo transcriptome assembler was implemented based on the overlap-layout-consensus paradigm. It was written in the C++ programming language and was named Ra which is short for RNA assembler. Its overlap phase relies on the enhanced suffix arrays and reproduces exact overlaps between input reads. The layout phase uses several methods for graph simplification which includes trimming and bubble popping. Due to the exact overlap phase there is no need for a consensus phase at this moment but there exists one which is based on the partial order alignment algorithm. Conducted tests have shown that Ra needs improvements to compete with other transcriptome assemblers. Source code is available at https://github.com/rvaser/ra

Algoritmi za de novo sastavljanje velikih genoma.

The inability of DNA sequencing technologies to interpret entire molecules led to the development of methods that connect the obtained short fragments back together in a puzzle-like process. They are called assemblers and their design is guided with the notion that similar fragments originate from the same genomic region. That is often annulled due to sequencing errors and repetitive nature of the genome. The advent of new sequencing approaches, namely Pacific Biosciences and Oxford Nanopore Technologies, pushed the limit on the fragment lengths at a cost of higher error rates, but still facilitated the assembly problem considerably. First assembly attempts used various types of error correction approaches prior the assembly with existing tools at that time. Although, several long read based assemblers have been proposed in the past years, they demand significant amounts of computational resources. The focus of this research is development of memory efficient and scalable algorithms for de novo assembly of large genomes using raw third generation of sequencing data. In the scope of the thesis we implemented three novel tools for genome assembly: a memory friendly layout module called Rala, which builds the assembly graph from preprocessed sequences and resolves junctions in graph with the help of force directed placement; a fast and accurate consensus module called Racon based on vectorized partial order alignment; and the complete de novo assembler called Raven, which competes with state-of-the-art assemblers both in quality and resource management.Tehnologije za sekvenciranje genoma nisu u mogućnosti interpretirati DNA molekule u cijelosti te je to dovelo do razvoja računalnih metoda koje spajaju kratke fragmente u procesu koji podsjeća na rješavanje slagalica. Alati namijenjeni za ovaj problem nazivaju se asembleri čiji je dizajn baziran na pretpostavci da slični fragmenti potječu iz iste regije genoma. Ta pretpostavka je često poništena zbog pogrešaka prilikom sekvenciranja te repetitivne prirode genoma. Pojava novih tehnologija za sekvenciranje tvrtki Pacific Biosciences i Oxford Nanopore Technologies olakšala je problem sastavljanja genoma zahvaljujući znatno većoj duljini dobivenih fragmenata, ali uz nedostatak veće pogreške. Iako nekoliko asemblera za dugačke fragmente već postoji, oni zahtijevaju pozamašne količine računalnih resursa. Fokus ovog istraživanja je razvoj memorijskih efikasnih i skalabilnih algoritama za de novo sastavljanje velikih genoma, pritom koristeći podatke treće generacije sekvenciranja. U sklopu ove disertacije implementirana su tri nova alata za sastavljanje genoma: memorijski učinkovita faza razmještaja zvana Rala, koja gradi graf sastavljanja iz skupa filtriranih fragmenata te ga pojednostavljuje koristeći algoritam razmještaja simulacijom djelovanja silama; brza i točna faza konsenzusa zvana Racon, koja je bazirana na vektoriziranom poravnanju parcijalnog uređaja; te cjelokupan de novo asembler nazvan Raven, koji konkurira s trenutno najefikasnijim poznatim metodama u kvaliteti i upravljanju resursima