Virus Hunting in French Guiana

40
French Guiana Virus Hunting in Nacho Caballero

description

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

Page 2: Virus Hunting in French Guiana

French Guiana

Page 3: Virus Hunting in French Guiana

Rodents

Bats

Page 4: Virus Hunting in French Guiana

Rodents

Bats

Leishmania

Page 5: Virus Hunting in French Guiana

Capture

Page 6: Virus Hunting in French Guiana

Capture Isolate viral particles

Page 7: Virus Hunting in French Guiana

Capture Isolate viral particles

Extract RNA

Page 8: Virus Hunting in French Guiana

Capture Isolate viral particles

Extract RNA

Sequence

Page 9: Virus Hunting in French Guiana

Estimated read coverage

% reads with coverage smaller than x

Rodents

Page 10: Virus Hunting in French Guiana

Estimated read coverage

% reads with coverage smaller than x

Rodents

Page 11: Virus Hunting in French Guiana

Estimated read coverage

% reads with coverage smaller than x

Rodents Bats

Page 12: Virus Hunting in French Guiana

Read

How can we estimate the coverage without a reference genome?

Page 13: Virus Hunting in French Guiana

Read

How can we estimate the coverage without a reference genome?

Page 14: Virus Hunting in French Guiana

K-mers

Read

How can we estimate the coverage without a reference genome?

Page 15: Virus Hunting in French Guiana

How can we estimate the coverage without a reference genome?

Page 16: Virus Hunting in French Guiana

1111111

How can we estimate the coverage without a reference genome?

Page 17: Virus Hunting in French Guiana

78

1081136

Page 18: Virus Hunting in French Guiana

78

1081136

Median k-mer count ≈

Read coverage

Page 19: Virus Hunting in French Guiana
Page 20: Virus Hunting in French Guiana

k-mers make it possible to align without a reference

Page 21: Virus Hunting in French Guiana
Page 22: Virus Hunting in French Guiana

Problem: each sequencing error introduces k erroneous k-mers

Page 23: Virus Hunting in French Guiana

Problem: each sequencing error introduces k erroneous k-mers

Page 24: Virus Hunting in French Guiana

78

1081136

Over a threshold, additional reads are redundant

Page 25: Virus Hunting in French Guiana

5555535

Solution: digital normalization reduces redundancy and errors

Page 26: Virus Hunting in French Guiana

Assembly

Page 27: Virus Hunting in French Guiana

Assembly

SPADes

Page 28: Virus Hunting in French Guiana

Assembly Alignment

Page 29: Virus Hunting in French Guiana

Assembly Alignment

BLAST

Page 30: Virus Hunting in French Guiana

Assembly TaxonomyAlignment

Page 31: Virus Hunting in French Guiana

Assembly TaxonomyAlignment

NCBI

Page 32: Virus Hunting in French Guiana

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

Page 33: Virus Hunting in French Guiana

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

Page 34: Virus Hunting in French Guiana

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

Page 35: Virus Hunting in French Guiana

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)

Page 36: Virus Hunting in French Guiana

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

5 Kb genome

Page 37: Virus Hunting in French Guiana

Lessons

Page 38: Virus Hunting in French Guiana

Assume that 50% of your samples are going to fail

Lessons

Page 39: Virus Hunting in French Guiana

Assume that 50% of your samples are going to fail

Lessons

Design a small experiment, then iterate

Page 40: Virus Hunting in French Guiana

Assume that 50% of your samples are going to fail

Lessons

Design a small experiment, then iterate

Come up with excuses to learn