Download - Virus Hunting in French Guiana

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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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78

1081136

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78

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