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French Guiana Virus Hunting in Nacho Caballero

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French Guiana

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Rodents Bats

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Rodents Bats Leishmania

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Capture

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Capture Isolate viral particles

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Capture Isolate viral particles Extract RNA

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Capture Isolate viral particles Extract RNA Sequence

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Estimated read coverage % reads with coverage smaller than x Rodents

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Estimated read coverage % reads with coverage smaller than x Rodents

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Estimated read coverage % reads with coverage smaller than x Rodents Bats

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Read How can we estimate the coverage without a reference genome?

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Read How can we estimate the coverage without a reference genome?

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K-mers Read How can we estimate the coverage without a reference genome?

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How can we estimate the coverage without a reference genome?

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1 1 1 1 1 1 1 How can we estimate the coverage without a reference genome?

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7 8 10 8 11 3 6

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7 8 10 8 11 3 6 Median k-mer count ≈ Read coverage

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k-mers make it possible to align without a reference

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Problem: each sequencing error introduces k erroneous k-mers

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Problem: each sequencing error introduces k erroneous k-mers

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7 8 10 8 11 3 6 Over a threshold, additional reads are redundant

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5 5 5 5 5 3 5 Solution: digital normalization reduces redundancy and errors

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Assembly

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Assembly SPADes

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Assembly Alignment

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Assembly Alignment BLAST

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Assembly Taxonomy Alignment

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Assembly Taxonomy Alignment NCBI

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Problem: 67% of contigs in rodent dataset (serum) align to human sequences

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Problem: 67% of contigs in rodent dataset (serum) align to human sequences Night-heron coronavirus HKU19 (1 Kb) Simian hemorrhagic fever virus (300 bp) Equine arteritis virus (3.7 Kb) Possum nidovirus Rodent hepacivirus Chipmunk parvovirus Theiler's disease-associated virus Reticuloendotheliosis virus Mosquito VEM Anellovirus SDBVL A Porcine reproductive and respiratory syndrome virus Dragonfly-associated circular virus 1 Gemycircularvirus 3 Rodent pegivirus Cyclovirus PK5510 Hypericum japonicum associated circular DNA virus

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Pig stool associated circular ssDNA virus (1Kb) Avian gyrovirus 2 Torque teno sus virus 1a Mosquito VEM virus SDBVL G Turdivirus 3 Problem: 92% of contigs in bat dataset (droppings) don’t align to anything in NCBI

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Lymphocytic choriomeningitis virus (7kb) Hepatitis C virus Amphotropic murine leukemia virus Murid herpesvirus 1 Mosquito VEM Anellovirus SDBVL A Rat retrovirus SC1 Mason-Pfizer monkey virus (retrovirus) Eidolon helvum parvovirus 2 Periplaneta fuliginosa densovirus (also a parvovirus) Moloney murine sarcoma virus Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 Problem: 95% of contigs in rodent dataset 2 (serum, spleen) align to mouse sequences (2)

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7 out of 10 samples contained more than 1Kb of Leishmania RNA virus (94% ident) 5 Kb genome

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Lessons

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Assume that 50% of your samples are going to fail Lessons

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Assume that 50% of your samples are going to fail Lessons Design a small experiment, then iterate

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Assume that 50% of your samples are going to fail Lessons Design a small experiment, then iterate Come up with excuses to learn