UNIVERSIDAD MIGUEL HERNÁNDEZMetagenomics and the NGS technology  Francisco Rodriguez-Valera
1: Introduction to Metagenomics (The era of cloning)2: The advent of NGS, is there a role for clone libraries?4 : Screenin...
The pure cultureLouis Pasteur                    Robert Koch Study of large populations of microoganisms that reproduce   ...
Culture is not enough• Most prokaryotes are extremely dificult  to retrieve in pure culture• Even if you get them in pure ...
We need to understand prokaryotes, they are an          essential part of our lives
And our planet!
(P.Hugenholtzadapted)                Genomics and Metagenomics                isolate               community             ...
Applications•   Exploration and conservation•   Metabiogeochemistry•   Systematics•   Population genomics and evolution•  ...
CLASSIC METAGENOMICS:AMPLIFICATION BY CLONING                                               Environmental DNA             ...
Sanger Sequencing                                                            Metagenomic Libraries                      $$...
eDNA can be screend for interesting phenotypes            Function-driven analysis
METAGENOMICS : Sanger Sequencing                                            Microbial                                     ...
AMPLIFICATONemPCR NGS (New GenerationSequencing)   Originally, NextGeneration Sequencing   but actually 2ndgeneration, we ...
Single Molecule Sequencing          Munroe and Harris, Nature Biotechnology, 28: 226 (2010)Pac Bio                     Hel...
METAGENOMICS BY NGS: NO NEED FOR CLONING                               Microbial                                          ...
NGS METAGENOMICS• Straight forward simple and cheap• Large volume of sequence (800 Mbp by 454  FLX plus pyrosequencing), 1...
What about screening for useful genes ? • From sequence to function   – Screen bulk sequences for     tell-tale domains   ...
Outlook for the next 10 years• Human, farm animals and Earth microbiomes  catalogued In-depth exploitation of microbial  ...
Thank you !
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Future of metagenomics

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The future of metagenomics

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Future of metagenomics

  1. 1. UNIVERSIDAD MIGUEL HERNÁNDEZMetagenomics and the NGS technology Francisco Rodriguez-Valera
  2. 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 sequence5: What the future may bring
  3. 3. The pure cultureLouis Pasteur Robert Koch Study of large populations of microoganisms that reproduce clonally from a single cell
  4. 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. 5. We need to understand prokaryotes, they are an essential part of our lives
  6. 6. And our planet!
  7. 7. (P.Hugenholtzadapted) Genomics and Metagenomics isolate community Sequencing analysis Not only Genomics 16S rRNA! Metagenomics
  8. 8. Applications• Exploration and conservation• Metabiogeochemistry• Systematics• Population genomics and evolution• Biotechnology
  9. 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. 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-endedLarge database s like GOS (35-40 Kpb) …. Interesting fosmids can be fully sequenced!! but Interesting genes are at the end incomplete operons sometimes
  11. 11. eDNA can be screend for interesting phenotypes Function-driven analysis
  12. 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. 13. AMPLIFICATONemPCR NGS (New GenerationSequencing) Originally, NextGeneration Sequencing but actually 2ndgeneration, we are nowon the brink of the 3rd Array PCR
  14. 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. 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 cloninggenerate •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 genesgenes (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. 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. 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. 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. 19. Thank you !
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