Genome sequencing by random priming methods for viral identification Rosseel Toon Dissertation submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Sciences, Faculty of Veterinary Medicine, Ghent University, 2015 Promotors: Dr. Steven Van Borm Prof. Dr. Hans Nauwynck “The real voyage of discovery consist not in seeking new landscapes, but in having new eyes” Marcel Proust, French writer, 1923 Table of contents Table of contents ....................................................................................................................... 1 List of abbreviations ................................................................................................................. 3 Chapter 1 General introduction ................................................................................................ 5 1. Viral diagnostics and genomics ....................................................................................... 7 2. The DNA sequencing revolution ................................................................................... 12 2.1. Classical Sanger sequencing .................................................................................. 12 2.2. Next-generation sequencing ................................................................................... 16 3. The viral metagenomic workflow ................................................................................. 24 3.1. Sample preparation ................................................................................................ 25 3.2. Sequence independent amplification ..................................................................... 27 3.3. DNA sequencing .................................................................................................... 51 3.4. Bioinformatic data analysis .................................................................................... 52 3.5. Follow-up ............................................................................................................... 55 4. The challenges ............................................................................................................... 56 Chapter 2 Aims of the thesis ................................................................................................... 69 Chapter 3 Implementation and Case studies ........................................................................... 73 Chapter 3.1 Implementation of a viral metagenomic workflow ......................................... 75 Chapter 3.2 Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies .................................................................................... 81 Chapter 3.3 Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls .................. 105 Chapter 3.4 What's in a strain? Viral metagenomics identifies genetic variation and contaminating circoviruses in laboratory isolates of pigeon paramyxovirus type 1 ......... 127 Chapter 3.5 DNase SISPA-next generation sequencing confirms Schmallenberg virus in Belgian field samples and identifies genetic variation in Europe ..................................... 147 1 Chapter 3.6 False-positive results in metagenomic virus discovery, a strong case for follow-up diagnosis ........................................................................................................... 161 Chapter 4 Fine-tuning of methodology ................................................................................. 175 Chapter 4.1 The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing ........................ 177 Chapter 4.2 Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples ............................................................................................... 203 Chapter 5 General discussion ............................................................................................... 229 Summary ............................................................................................................................... 261 Samenvatting ........................................................................................................................ 267 Bibliography ......................................................................................................................... 275 Curriculum Vitae ................................................................................................................. 281 Dankwoord ............................................................................................................................ 285 2 List of abbreviations A Adenine aa Amino acids AFLP Amplified fragment length polymorphism AI(V) Avian influenza A (virus) APMV Avian paramyxovirus BLAST Basic Local Alignment Search Tool bp Bases or base pairs BVDV Bovine viral diarrhea virus C Cytosine CCD Charge-coupled device cDNA Complementary DNA CDS Coding sequence Contigs Contiguous overlapping sets of sequences Cp Crossing point value CPE Cytopathic effect Ct Threshold cycle value Da Dalton (molecular weight unit) ddNTP 2’,3’-dideoxynucleotides (ddATP, ddGTP, ddCTP, ddTTP) DNA Deoxyribonucleic acid DNase Deoxyribonuclease dNTP Deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) DOP-PCR Degenerate-oligonucleotide primer PCR dsDNA Double stranded DNA dsRNA Double stranded RNA EDTA Ethylenediaminetetraacetic acid EID50 50 percent embryo infectious dose ELISA Enzyme-linked immunosorbent assay emPCR Emulsion PCR G Guanine HA Hemagglutination or hemagglutinin gene HI Hemagglutination inhibition HIV Human immunodeficiency virus HPAI High-pathogenic avian influenza virus ICPI Intracerebral pathogenicity index IQR Interquartile range LASL Linker Amplified Shotgun Library LPAI Low-pathogenic avian influenza virus MDA Multiple displacement amplification 3 MID Multiplex identifier N Random nucleotide (A, C, G or T) NA Neuraminidase ND(V) Newcastle disease (virus) NGS Next-generation DNA sequencing nt Nucleotides ORF Open reading frame PBS Phosphate buffered saline PCR Polymerase chain reaction PEG Polyethylene glycol PGM Personal Genome Machine PHV Parvovirus-like hybrid virus PiCV Pigeon circovirus PPMV1 Pigeon type 1 paramyxoviruses (q)[RT]-PCR (quantitative real-time) [reverse transcription] PCR reaction pWGA Primase-based whole genome amplification QV Quality score RACE Rapid Amplification of cDNA Ends RCA Rolling circle amplification RNA Ribonucleic acid RNase Ribonuclease rPCR Random PCR rRNA Ribosomal RNA RT Reverse transcription SARS Severe Acute Respiratory Syndrome SBV Schmallenberg virus SE southeast SISPA Sequence independent single primer amplification SMRT Single molecule real time sequencing SPF Specific-pathogen-free SPIA Single primer isothermal amplification ssDNA Single stranded DNA ssRNA Single stranded RNA T Thymine TCID Tissue culture infective dose U Units of polymerase v/v Volume concentration (% volume/volume) VIDISCA Virus-Discovery-cDNA-Amplified fragment length polymorphism VIDISCA-454 VIDISCA combined with 454 pyrosequencing WGA Whole genome amplification WTA Whole transcriptome amplification 4 CHAPTER 1 General introduction Chapter 1 General introduction 6 Chapter 1 General introduction 1. Viral diagnostics and genomics We live in a globalized society where people move increasingly between countries and continents, and livestock animals are farmed and traded extensively. Emerging and reemerging infectious diseases are a constant threat to livestock and the human population. A recent example is the current Ebola virus disease epidemic in West Africa which started in March 2014. This virus has already affected more than 22,000 people, and killed over 9,000 (according to data from World Health Organization on 8 February 2015). Furthermore, recent reviews indicate that the majority of emerging human infections are zoonotic. Therefore, contact with livestock or wildlife increases the probability of infections threatening human populations and individuals lacking immunity. About 15 million (>25 %) of 57 million annual human deaths worldwide are estimated to be linked to infectious diseases [1]. Moreover, livestock diseases may result in significant economic losses and socio-economic consequences. Viral diseases are often spread and transmitted by vectors such as bloodsucking mosquitoes, ticks and wildlife animals. Sometimes, an intermediate host like a domestic animal is the link between viral circulations in wildlife and humans. For instance, some human infections originating from bats, such as Nipah, Hendra, SARS and Ebola viral infections, may involve intermediate amplification in pigs, horses, civets and primates respectively [2]. Complex pathogen lifecycles complicate the control of diseases. Rapid diagnosis of infectious diseases is essential in order to take appropriate action to control livestock and human diseases. Characterization of new pathogens will help to understand diseases and is an important step in the development of vaccines and better diagnostic tests. Viruses are the most abundant infectious agents on the planet. In an apparently sterile environment like sea water, the number of virus particles is estimated at 106 to 109 particles per milliliter [3].