The future of metagenomics

1. UNIVERSIDAD MIGUEL HERNÁNDEZ
Metagenomics and the NGS technology
Francisco Rodriguez-Valera

2. 1: Introduction to Metagenomics (The era of cloning)
2: The advent of NGS, is there a role for clone libraries?
4 : Screening for genes: activity vs sequence
5: What the future may bring

3. The pure culture
Louis Pasteur Robert Koch
Study of large populations of
microoganisms that reproduce
clonally from a single cell

4. Culture is not enough
• Most prokaryotes are extremely dificult
to retrieve in pure culture
• Even if you get them in pure culture you might not be able to
perform experiments with them (physiology)
• The genome of one strain may not represent the genetic
repertoir of the “species” in the community PAN-GENOME
• Many microbes require close interaction with others to show
their abilities

5. We need to understand prokaryotes, they are an
essential part of our lives

6. And our planet!

7. (P.Hugenholtz
adapted)
Genomics and Metagenomics
isolate community
Sequencing
analysis
Not only
Genomics 16S rRNA! Metagenomics

8. Applications
• Exploration and conservation
• Metabiogeochemistry
• Systematics
• Population genomics and evolution
• Biotechnology

9. CLASSIC METAGENOMICS:AMPLIFICATION BY CLONING
Environmental DNA Metagenomic Libraries
eDNA eDNA
cloning 3Kbp (35-40 Kpb)
Sequencing
primers
DNA fragmentation plasmid
(3 Kpb / 30-40 Kpb /BAC)
Large insert vectors
Small insert vectors Sequencing
primers
fosmid / cosmid
transduction/
transformation
Metagenomic Libraries
Eschericchia coli
Microtiter plate
Gene “repository”

10. Sanger Sequencing Metagenomic Libraries
$$$
eDNA
(35-40 Kpb)
eDNA $$$$$$
(3 Kpb)
plasmid
$$$
(0.8 Kpb)
(0.8 Kpb) Pair-ended fosmid
(0.8 Kpb)
fosmid-ends Pair-ended
Large database s like GOS (35-40 Kpb)
….
Interesting fosmids can be fully
sequenced!! but Interesting genes are at the end
incomplete operons sometimes

11. eDNA can be screend for interesting phenotypes
Function-driven analysis

12. METAGENOMICS : Sanger Sequencing
Microbial
community
DNA
Cloning: Small insert Cloning: long insert vector
vector (ca 3 Kbp) (e.g. fosmids,ca. 35-40 Kbp eDNA) e.g. HOTs
(Hawaii Ocean Time-Series)
e.g. GOS •Large libraries easy to •Large libraries more difficult
(Global Ocean Sampling) generate •Natural contigs of ca: 35 Kbp
•Natural contigs of ca: 3Kbp (pair ended)
(pair ended) •Complete sequence allows
•.Annotation of single genes annotation of clusters of genes
(unreliable) (very reliable)
•Phenotype can be detected •PCR or fosmid end screening
(very unlikely) •Phenotype can be detected
(unlikely)

13. AMPLIFICATON
emPCR
NGS (New
Generation
Sequencing)
Originally, Next
Generation Sequencing
but actually 2nd
generation, we are now
on the brink of the 3rd
Array PCR

14. Single Molecule Sequencing
Munroe and Harris, Nature Biotechnology, 28: 226 (2010)
Pac Bio Helicos Nanopore Ion Torrent
NO AMPLIFICATION
Gigantic technological drive for the
1000$ human genome

15. METAGENOMICS BY NGS: NO NEED FOR CLONING
Microbial
LOW COST!!
community mRNA
DNA
Cloning: Small insert Cloning: long insert vector Direct NGS
vector (ca 3 Kbp) (e.g. fosmids,ca. 35-40 Kbp eDNA) Sequencing
•Large libraries easy to
•Large libraries more difficult •No need for cloning
generate
•Natural contigs of ca: 35 Kbp
•Natural contigs of ca: 3Kbp •Low cost
(pair ended)
(pair ended) •Natural contigs of ca: 0.4-
•Complete sequence allows
•. Annotation of single 0.8 Kbp 16/18S rRNA
annotation of clusters of genes
genes (unreliable) •. Annotation of fragments
(very reliable)
•Phenotype can be detected of genes (very unreliable)
•PCR screening
(very unlikely) •Phenotype can not be
•Phenotype can be detected
detected
(unlikely)

16. NGS METAGENOMICS
• Straight forward simple and cheap
• Large volume of sequence (800 Mbp by 454
FLX plus pyrosequencing), 10 Gb Solexa
• Thanks to the high coverage ASSEMBLY of
large fragments is feasibleAnnotation
reliable
• Large insert libraries can be sequenced by
NGS
• Sequence driven search for activities

17. What about screening for useful genes ?
• From sequence to function
– Screen bulk sequences for
tell-tale domains
– Synthetic DNA from eDNA seq
– Clone in adequate host

18. Outlook for the next 10 years
• Human, farm animals and Earth microbiomes
catalogued In-depth exploitation of microbial
diversity
• Sequence analysis (assembly and annotation)
limiting step
• A new MicrobiologySystematics, Ecology ,
Evolution
• Sequence driven screening for useful metabolic
pathways (PKs, NRP etc), enzymes, new
antimicrobials, new probiotics Huge
oportunities for biotech Better health

19. Thank you !