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

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 TaxonomyAlignment

31. Assembly TaxonomyAlignment
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