Hunting for viruses in French Guiana

Lab meeting presentation about my work doing viral metagenomic analysis in French Guiana

Transcript

1. French Guiana Virus Hunting in Nacho Caballero
2. French Guiana
3. Rodents Bats
4. Rodents Bats Leishmania
5. Capture
6. Capture Isolate viral particles
7. Capture Isolate viral particles Extract RNA
8. Capture Isolate viral particles Extract RNA Sequence
9. Estimated read coverage % reads with coverage smaller than x Rodents
10. Estimated read coverage % reads with coverage smaller than x Rodents
11. Estimated read coverage % reads with coverage smaller than x Rodents Bats
12. Read How can we estimate the coverage without a reference genome?
13. Read How can we estimate the coverage without a reference genome?
14. K-mers Read How can we estimate the coverage without a reference genome?
15. How can we estimate the coverage without a reference genome?
16. 1 1 1 1 1 1 1 How can we estimate the coverage without a reference genome?
17. 7 8 10 8 11 3 6
18. 7 8 10 8 11 3 6 Median k-mer count ≈ Read coverage
20. k-mers make it possible to align without a reference
22. Problem: each sequencing error introduces k erroneous k-mers
23. Problem: each sequencing error introduces k erroneous k-mers
24. 7 8 10 8 11 3 6 Over a threshold, additional reads are redundant
25. 5 5 5 5 5 3 5 Solution: digital normalization reduces redundancy and errors
26. Assembly
27. Assembly SPADes
28. Assembly Alignment
29. Assembly Alignment BLAST
30. Assembly Taxonomy Alignment
31. Assembly Taxonomy Alignment NCBI
32. Problem: 67% of contigs in rodent dataset (serum) align to human sequences
33. 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
34. 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
35. 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)
36. 7 out of 10 samples contained more than 1Kb of Leishmania RNA virus (94% ident) 5 Kb genome
37. Lessons
38. Assume that 50% of your samples are going to fail Lessons
39. Assume that 50% of your samples are going to fail Lessons Design a small experiment, then iterate
40. Assume that 50% of your samples are going to fail Lessons Design a small experiment, then iterate Come up with excuses to learn