key: cord-0781097-9iailgv0 authors: Ducatez, Mariette F.; Guérin, Jean-Luc title: Identification of a Novel Coronavirus from Guinea Fowl Using Metagenomics date: 2014-12-18 journal: Coronaviruses DOI: 10.1007/978-1-4939-2438-7_2 sha: 27fbaea4fd3a233f1acfd04b420ac798d206b183 doc_id: 781097 cord_uid: 9iailgv0 While classical virology techniques such as virus culture, electron microscopy, or classical PCR had been unsuccessful in identifying the causative agent responsible for the fulminating disease of guinea fowl, we identified a novel avian gammacoronavirus associated with the disease using metagenomics. Next-generation sequencing is an unbiased approach that allows the sequencing of virtually all the genetic material present in a given sample. The fi eld of pathogen discovery gained a completely new dimension when new genomics tools, next-generation sequencing (NGS), became available. Isolation and identifi cation by electronic microscopy used to be the gold standard techniques to identify a new pathogen. However, some pathogens are diffi cult to culture, and/ or to separate from co-infecting agents. PCR has improved pathogen discovery, but even when pan-species/Genus/family PCRs may be developed (which is not always possible, especially for highly variable RNA viruses), the primers chosen do condition the nucleic acids that will be amplifi ed: it is still a "biased" technique with which only known (or closely related to known) pathogens whose presence was suspected may be detected [ 1 , 2 ] . NGS allows for (1) massive sequencing of genetic material and (2) unbiased sequencing, to a much lower cost per sequenced base than Sanger sequencing technology. Here we describe an NGS technique that can be used to identify novel pathogens, which we recently used to identify the pathogen responsible for the guinea fowl fulminating disease [ 3 ] . Figure 1 summarizes the methodology used for the identifi cation of a novel guinea fowl gammacoronavirus. 1. Pool intestinal contents of experimentally infected guinea fowl poults and resuspend in 500 μl PBS with penicillin and streptomycin. Vortex. 2. Filter (0.45 μm fi lter) the solution to eliminate eukaryoticand bacterial-cell-sized particles. 3. Centrifuge the digestive content at 10,000 × g for 30 min twice to clarify the solution and collect the supernatant in a new tube. 1. Pellet the concentrated material by ultracentrifugation at 100,000 × g for 2 h. 2. Treat with RNAse and DNAse to remove non-particleprotected nucleic acids: make a mix of 500 μl of sample, 10 μl DNAse (100 U), 12 μl RNAse (20 μg/μl), 60 μl 10× DNAse buffer, 16 μl PBS. Incubate the mix for 20 min at 37 °C and then 10 min at 75 °C to stop the reaction. 1. Add 750 μl TRIzol to 250 μl sample from Subheading 3.2 , step 2 and incubate 5 min at room temperature. 2. Add 200 μl chloroform, vortex vigorously, and incubate 10 min at room temperature. 3. Centrifuge for 15 min at 11,000 × g at 4 °C. 13. Analyze the PCR products on a 1 % agarose gel, migrate for 1 h at 60 V. 14. Excise the 300 bp bands and perform a gel purifi cation with a commercial kit. 15. Quality assessment of the prepared library: quantify the DNA generated by a fl uorescence-based method (PicoGreen ® quantitation assay) and aim at 1 μg DNA as input; check the DNA quality and aim for a 260:280 ratio >1.8. 1. Hybridize sample to fl ow cell. 2. Amplify sample (bridge amplifi cation). 3. Linearize fragments. 4. Block fragments. 5. Hybridize sequencing primer. 1. The library DNA fragment act as a template, from which a complementary strand is synthesized. 2. ddNTPs are added one by one (one cycle = one ddNTP added, a picture taken and defl uoration of the ddNTP to be able to add a new ddNTP the next cycle) by a DNA polymerase. The addition of ddNTP is digitally recorded as sequence data cycle after cycle. 1. Preprocess the data to remove adapter sequences and demultiplexing using splitbc (several samples can be multiplexed and run together on the MiSeq Illumina sequencer to reduce cost). 2. Preprocess the data to remove low-quality reads and compiling paired sequences using illuminapairedend. 3. Map the data to a reference genome or de novo align the sequence reads (alignment with bwa [ 4 ] , consensus computed with the SAMtools [ 5 ] software package. Display the results with the IGV [ 6 ] browser). 4. Analyze the compiled sequence with the GAAS software ( http://gaas.sourceforge.net/ ) with an expected value of 10 −3 . 1. Victoria et al. [ 7 ] described a panel of tagged primers (named with alphabet letter), and while we selected "454-A" for the present study, any of the tagged primers described in [ 7 ] could be used instead (454-B, 454-C, etc). 2. To identify pathogens with DNA genomes, a DNA extraction would be performed, followed by a Klenow step before the random PCR: (a) DNA extraction: High Pure template preparation kit (Roche) can be used, following the manufacturer's instructions. (b) Klenow step on DNA: mix 2.5 μl tagged random primer, 3 μl 10× buffer, 1 μl 0.5 mM dNTP, 10 μl DNA, 1 μl 0.5 U/μl DNA pol1 and 12.5 μl water. Incubate at room temperature for 1 h. (c) Proceed from Subheading 3.3 , step 10 . U/μl) 10× DNase buffer: 200 mM Tris-HCl (pH 8.4), 20 mM MgCl 2 , 500 mM KCl TRIzol reagent or similar Nucleospin RNA virus kit (Macherey-Nagel) or similar RevertAid kit (Thermofi sher) or similar Phusion (NEB) or similar Genomics and metagenomics in medical microbiology Metagenomics for pathogen detection in public health Novel avian coronavirus and fulminating disease in guinea fowl Fast and accurate short read alignment with Burrows-Wheeler transform The Sequence alignment/map format and SAMtools Integrative genomics viewer Metagenomic analyses of viruses in stool samples from children with acute fl accid paralysis