Metagenomics Rob Edwards

What is metagenomics Cloning genes from the environment, screening for function 16S sequencing Random community genomics Eukaryotic metagenomics

Screening from the environment Random fragments of DNA Clone into a vector Low copy vectors BACs YACs

BACs Science Creative Quarterly

Screening from the environment Random fragments of DNA Clone into a vector Low copy vectors BACs YACs Screen for a phenotype e.g. Diversa patents > 1,000 amylase genes Why did Diversa sequence whale-falls?

Screening from the environment Expression host? Pathway or single gene? Get what you select But remember … A selection is worth a thousand screens

16S sequencing Catalogs the bacteria that are present PCR amplify the 16S gene with standard primers Sequence the primers Compare to known databases

Ribosomes Ribosomes are made of proteins and RNA Prokaryotic ribosome: Large subunit: 50S 5S and 23S rRNA Small subunit: 30S 16S rRNA

30S Thermus aquaticus subunit Blue: protein Orange: rRNA

16S rRNA secondary structure E. coli 16S rRNA secondary structure Highly conserved Base pairs = stems No pairing = loops

16S rRNA secondary structure E. coli 16S rRNA secondary structure V7 V6 V5 V8 V4 V9 V3 V1 Variable regions in the 16S rRNA. Vn – 9 regions forward/rev primers V2

16S Primers 27F – 1492R full length 1,465 base pairs 967F – 1046R V6 region 1380F – 1510R V9 region 1,465 base pairs 79 base pairs 130 base pairs

Variable regions = Variable results! V1-V3 V3-V5 V6-V9

16S databases Greengenes http://greengenes.lbl.gov/ Gary Andersen, Lawrence Berkeley National Laboratory SILVA – ARB http://www.arb-silva.de/ Frank Oliver Glöckner, MPI, Bremen, Germany VAMPS http://vamps.mbl.edu/ Mitch Sogin, Woods Hole, USA Ribosomal Database Project (RDP) http://rdp.cme.msu.edu/ James Cole, Michigan State University, USA

16S sequencing Cheap Easy Portable PCR bias Variable regions give variable answers Only tells you which organisms are present & abundance Does not explain much of the variance of the data What does 16S sequencing actually tell you?

What does 16S sequencing tell you?

What does 16S sequencing tell you?

What does 16S sequencing tell you?

How do you sequence the environment? Extract DNA Soil extraction kit Water extraction kit Create library LASLs fosmids Sequence fragments

Linker amplified shotgun libraries (LASLs) Soil Extraction Kit This method produces high coverage libraries of over 1 million clones from as little as 1 ng DNA Breitbart (2002) PNAS

Random community genomics

Bioinformatics Bribery Blackmail Coercion Give up!!

Bioinformatics MG-RAST CAMERA IMG/M ebi.ac.uk/metagenomics/ Real time metagenomics

All pipelines work the same way! Raw reads Filter FragGeneScan InterProScan BLAST HMMER3 http://www.ebi.ac.uk/metagenomics/

Eukaryotic metagenomics ITS sequences Internal transcribed spacer regions Individual genes Cox1 Exome sequencing Pull out ESTs and sequence