# Presentation: 074247

## Roadmap for Soil Community Metagenomics of DOE’s FACE & OTC Sites

**Scientific Opportunity**
- Comprehensive, field-scale understanding of the responses of soil microbial communities and their processes to long-term (10 yr) elevated CO2, and comparison of responses across terrestrial ecosystems.

## Goal

- Develop a science & implementation plan based on metagenomic technology, to assess responses of the belowground communities to CO2 in DOE terrestrial ecosystem FACE and OTC sites.

## August 2007 Workshop

**Facilities:**
- 	FACE sites
- 	JGI sequencing resources
- 	other DOE facilities
**People & Experience**:
- 	FACE site scientists
- 	JGI personnel
- 	National lab & University scientists
- 	DOE program management

**Objective**
- Establish a roadmap for implementing metagenomic analysis of soil communities across several FACE sites

## August Workshop Accomplishments

- Identified major ecological questions that should be addressed across the FACE & OTC sites.
- Determined optimal sequencing technologies to answer those questions.
- Determined the best strategies for implementing sequencing technology in these field-scale experiments.
- Discussed how best to coordinate sample, sequencing, & data management

_**Ecological Research Questions**__ _
- Within each site, has 10 yr elevated CO2 affected the soil microbial community (abundance, composition) and ecosystem processes conducted by this community?
- Have the soil communities in different ecosystems responded similarly or in different patterns to elevated CO2?

## Why Soil Metagenomics ?

_**Benefits & Potential Impacts**_
- Greatest source of biodiversity on Earth.
- Soil communities are central to multiple DOE mission areas & potential for multiple impacts is high.
- Abundant biomass - large amounts of DNA for multiple genomic studies.

_**Technical Challenges**_
- Diversity is high.
- Biofilms and physical attachment affect DNA. extraction efficiency therefore standard protocols are critical.
- Non-uniformity of substrates & patchiness so field sample replication is essential.
- Fungal biomass is present & important, but fungal genomes are complex.

## Metagenomics ~ Community Genomics

***any gene-based comparison ***
***that spans across the collective microbial genome in an environment****** ***

**TARGETED METAGENOMICS**** - focus on specific genes**

_**Pros**_
-  immediate link to function
-  high depth of coverage in complex community can provide strong abundance/composition information

_**Cons**_
- single/few genes studied
- clone/sequence approaches are thorough but time consuming
- only see what you know to look for

_**New Potential w/ 454 pyrosequencing**_
-  larger panels of genes, multiple pathways
-  resolution of sequencing without cloning
-  larger number of sequences for each gene
-  single pass sequencing w/ lower quality

## Metagenomics ~ Community Genomics

**	****	****	****       ****- clone & sequencing random fragments**

_**Characteristics**_
-  broad brush, ‘unbiased’ survey
-  community complexity dictates level of resolution & ability to link to function
-  functional interpretation is limited due to our inability to ID genes in the sequences obtained
-  eukaryotic genomes have lower probability of gene detection by random sequencing

**Bacterial cell**

**2-10 mb genome**

**30-100+ mb genome**

_**Current Applications**_
-  search for new enzymes
-  describe microbial communities
-  describe community dynamics
-  compare community composition

## Challenges & Novelty of Application of Metagenomics to FACE/OTC Sites

- Comparative metagenomics, not just descriptive
- Strong need to tie to ecosystem function
- Similar parent material within a site
- Experimental framework w/ replication
- Link soil microbial results to field-scale ecology
- Will enhance & stretch our current sequencing technology

## Hierarchical Research & Sequencing Strategy

***Tiered datasets cross-inform to improve our ability to interpret ecological relevance***

**100’s**

**10’s**

**# samples**

## Hierarchical Research & Sequencing Strategy

_***Tier 1 Measurements:***_** Ability to relate metagenome data back to field-scale soil response at FACE sites**
*In response to elevated CO**2**:*
-  has the microbial biomass changed?
-  have functional properties or process measures changed?
-  what is the variability across field replicates?

_***Tier 2 Sequencing:***__** phylogenetic species surveys**_
*In response to elevated CO**2**:*
-  has the relative abundance or composition of bacteria/fungi/archaea changed?
-  has the relative abundance of the major phyla/species within a domain changed?

_***Tier 3 Sequencing: ***_**Targeted metagenomics using 454 pyrosequencing**
*In response to elevated CO**2**:*
-  has the functional diversity or composition been altered?
-  how have key microbial species/processes been changed?

## Hierarchical Research & Sequencing Strategy

_**Tier 4 Sequencing**_**: Transcriptome analysis of soil fungi**
*In response to elevated CO**2**:*
-  has activity of abundant soil fungi changed?
-  especially with regard to carbon utilization

_***Tier 5 Sequencing:***__** **_**Seasonal, year-to-year patterns & ozone effects (Rhinelander site)**
*In response to elevated CO**2** and/or ozone:*
- how do changes relate to seasonal responses and year-to-year variability?
- how did soil communities respond to elevated ozone?
- were there any combined effects of elevated CO2 and ozone?

_**Tier 6 Sequencing: Shot-gun metagenomics**_
*In response to elevated CO2:*
-  has the overall community been altered?
-  what new properties are present?
-  how do communities compare across sites?

## Current Status

- Roadmap document circulated to workshop participants and DOE management.
- Two of the six FACE/OTC sites have been deconstructed. Soil samples are archived with the primary site scientist and at LANL.
- Pilot studies are in progress for Tier 2 (rRNA surveys), Tier 3 (454 targeted metagenomics), and Tier 4 (fungal transcriptome) sequencing.

***Cheryl Kuske, kuske@lanl.gov***