Virus Hunting in French Guiana

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French Guiana Virus Hunting in Nacho Caballero

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Lab meeting presentation about my work doing viral metagenomics in French Guiana Rat by Francisca Arévalo from The Noun Project Bat by Adam Heller from The Noun Project

Transcript of Virus Hunting in French Guiana

Page 1: Virus Hunting in French Guiana

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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1111111

How can we estimate the coverage without a reference genome?

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1081136

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1081136

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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78

1081136

Over a threshold, additional reads are redundant

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5555535

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 TaxonomyAlignment

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Assembly TaxonomyAlignment

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