Browsing by Subject "NGS"

Preserving viral nucleic acids is of outmost importance to capture the viral diversity in metagenomic studies. In my master’s thesis, I compare viromes of genus Culex mosquitos stored in two different virus storage media and empty tubes. The mosquitos were collected from Kalajärvi in Espoo, Finland in the summer of 2020 as larvae and were grown to adults in laboratory conditions. Eight pools of five female mosquitos each were stored in each of the two media as well as empty tubes and the samples were homogenized The homogenates were filtered, and the RNA was extracted from them with TRIzol reagent. RNA was then reverse-transcribed to cDNA and amplified with a whole transcriptome amplification kit. The PCR product was prepared with a library preparation kit for sequencing with Illumina Next Generation Sequencer. The resulting reads were processed with a bioinformatic pipeline for identifying viruses from metagenomic sequence data. The results show a clear difference in virus species distribution by storage media. We identified 34 virus species from at least 13 families. Samples stored in ∑-Virocult had the highest yield of viral reads (70.40% of all reads from the pools) as well as the widest variety of mosquito species (n=26). Samples stored in empty tubes had the second most mosquito species (n=10) but the lowest viral read yield (1.25%). RNAlater stored samples had the least virus species (n=7) but a higher viral read percentage than those stored in empty tubes (3.26%). The results indicate the importance of choice of storage media. Since ∑-Virocult had the highest amount of reads and the widest variety, it might be the most useful storage media for our purposes. However, some viruses were found in other samples but not in ∑-Virocult stored samples, indicating a need of different storages conditions for different viruses. It is also important to be consistent in the use of media as it may affect virome results. More work needs to be done to assess if these results are true for other mosquito species as well.

The Oxford Nanopore MinION is a third generation sequencer utilizing nanopore sequencing technology. The nanopore sequencing method allows sequencing of either DNA or RNA strands as they pass through the membrane-embedded nanopores. By measuring the corresponding fluctuations in the ion flow passing through the nanopore the passing strands can be sequenced directly without additional second-hand reactions or measurements. The MinION sequencing has very distinctly different characteristics compared to the market leaders of the sequencing field. The small form factor of the device further helps it to separate itself from the other alternatives. However, the technology has only been on the market for a very short time and thus very little golden standards regarding its capabilities or usage have been established. This thesis describes our experiences testing the capabilities of the MinION sequencer both before its commercial release as a part of a special early access program, as well as our continued experiments with the device following its commercial launch. The main results of this study include successfully sequencing and aligning E.coli and human gDNA samples to their respective reference genomes. Using our sequencing and analysis pipeline specifically tuned to the MinION we were able to sequence the entire E.coli genome on a single MinION flow cell with an average depth of around 180. Over the course of the thesis project the MinION sequencing protocol was evaluated and optimized in order to determine whether it has the potential to achieve our ultimate goal of reliably sequencing the previously inaccessible genomic regions of the human genome. The possibility of augmenting the sequencing protocol by adding the pre-sequencing target enrichment was also explored. Ultimately we were able to confirm that the MinION sequencer can be used to sequence long DNA fragments from a multitude of sample types. The majority of the produced reads could successfully be aligned against a reference genome. However, the limited yield and sequencing quality of a single experiment does limit the applicability of the method for more complicated genomic studies. These issues can be addressed with various techniques, chiefly target enrichment, but adapting such methods into the sequencing pipeline has its own challenges.

The next-generation sequencing (NGS) platforms create a large amount of sequence in short amount of time, when compared to first generation sequencers. An overview of the NGS platforms is provided with more in-depth look into Illumina Genome Analyzer II as that is used to create the data for the thesis. There were two main aims in this thesis. First, to create a pipeline which can be used to analyse genomic sequencing. Second, to use the pipeline to compare whole human exome capture methods from two manufacturers, Roche Nimblegen and Agilent. The pipeline is describe in detail in material and methods. All the inputs for the pipeline are described and examples shown. In the pipeline the given sequences are first aligned against the reference genome. Then various separate analysis is performed to retrieve variants and coverage of the sequencing. Supplementary results include paired-end anomalies, larger insertion and deletion polymorphisms and assembly of non-aligned sequences. The two capture methods are also described and changes to the manufacturers' recommended protocols are listed. Finally, the section has the options and various inputs used in the pipeline runs of the exome data. The results of the pipeline is a basic level of analysis of the sequencing as well as various graphs showing the quality of the run. All the output files intended for user are described. By using the results of the pipeline, the user can do more in-depth analysis as required by the project. When comparing the two exome capture methods, the Nimblegen capture was shown to be more efficient in capturing the CCDS exome. While the Agilent capture kit provided better one fold coverage over the exome, higher fold coverage (over 10 fold), which is required for reliable variant calling in nextgeneration sequencing, was better reached using the Nimblegen capture kit. Also, significantly fewer false positive paired-end anomalies were observed in the library created by using the Nimblegen capture.

Kissoilla esiintyy monia ruoansulatuskanavan viruksia. Taudinaiheuttajasta riippuen osa tartunnoista voivat olla täysin oireettomia, mutta myös kuolemaan johtavia tartuntoja esiintyy. Ihmiselle tautiriskin voi aiheuttaa esimerkiksi kissojen rotavirukset. Uuden sukupolven sekvensointimenetelmiä (Next-generation sequencing, NGS) käyttämällä voidaan sekvensoida viruksen koko perimä eli genomi nopeasti yhdellä kerralla. NGS:ää käytettäessä sekvensointi voidaan tehdä ilman ennakkotietoja ja täten löytää myös aiemmin tuntemattomia viruksia. Suomalaisten kissojen virusten esiintyvyydestä ei ole aikaisemmin tehty kattavia tutkimuksia. Työn tavoitteena oli tutkia, mitä viruksia löytyy terveiden ja ripuloivien kissojen ulosteista Suomessa. Tutkimuksessa analysoitiin yhteensä 15 kpl kissojen ulostenäytteitä, joista yhdeksän oli eläinsuojeluyhdistyksen kissaa ja kuusi lemmikkikissaa. Kahdeksalla kissalla oli esiintynyt ruoansulatuskanavan oireita. Työhön kuului nukleiinihappojen eristys kissojen ulosteista. Esikäsittelyn avulla pyrittiin poistamaan ulosteesta bakteerit, jonka jälkeen näytteistä tehtiin kirjasto, joka sekvensoitiin Illumina Miseq-laitteistolla. Data analysoitiin bioinformatiikan avulla, johon kuului muun muassa huonolaatuisten sekvenssien poisto ja yhdistelmäsekvenssien eli contigien koostaminen lyhyistä sekvensseistä. Contigit käännettiin aminohapposekvenssiksi, joille etsittiin vastaavaa sekvenssiä geenipankista. Löydettyjen virusten nimet perustuivat geenipankkisekvenssien nimiin. Näytteistä löydettiin yhteensä kuusi virusta, joista kaksi esiintyi samalla kissalla. Yhdestä näytteestä löydettiin täysi vastaavuus kissan panleukopenia virukseen (FPV). Kissa oli saanut yhden rokotuksen ennen näytteiden keräämistä eikä kissalla ollut minkäänlaisia oireita. Löydetty virus ei ollut täysin identtinen rokotekantojen virusten kanssa, mutta ero ei ollut suuri. Taustalla voi olla luonnollinen tartunta tai esimerkiksi heikentyneen immuunivasteen aiheuttama rokotekannan pitkäaikainen replikoituminen. Yhdestä näytteestä löydettiin circovirukseen vastaavaa sekvenssiä ja lisäksi tunnistamaton virus, jonka sekvenssi vastasi myös circovirusta. Circovirusten tiedetään aiheuttavan ruoansulatuskanavaoireita, mutta kissoilta ei ole aiemmin todettu tartuntoja. Löydetty virus voi olla aiemmin tunnistamaton kissojen oma circovirus, mutta lisätutkimuksia tulisi tehdä asian varmentamiseksi. Viruksen yhteys ripuliin on myös epäselvä. Muut löydetyt virukset olivat todennäköisesti peräisin ruoasta tai esimerkiksi RNA-eristyskolumnista. Ulosteista löytyneiden virusten määrä oli pieni eikä suuria johtopäätöksiä pystytä tekemään näytteiden vähäisen lukumäärän vuoksi. Kuitenkin ulkomailla tehdyissä ulosteen viromia selvittävissä tutkimuksissa vastaavilla kissamäärillä on löydetty huomattavasti enemmän viruksia kuin tässä tutkimuksessa. Tulevaisuudessa suuremmalla kissamäärällä tehty tutkimus vertaillen eroavaisuuksia maantieteellisesti sekä sisä- ja ulkokissojen välillä, antaisi luotettavampaa dataa Suomen tilanteesta. NGS:n käyttäminen ulosteen viromin selvittämiseksi vaikuttaisi olevan käyttökelpoinen tutkimusmenetelmä.