# Presentation: 344030

## JGI Timeline

**        ****Joint Genome Institute  ****......................****(JGI)****                                  **

**Non Traditional User Facility**

**Notes:**
What to do with the big machine now that the initial reason for creating it create is gone.  During the course of the human Genome program the role of large scale sequencing in nearly all fields of biological sciences has become obvious.  Accordingly the Dept of Energy has made the  decision to instead of putting it in moth balls to  recommission  the JGI a resource where scientist with questions that require  large amount of DNA sequence to answer could come to have the necessary sequence generated to answer them  

**                          **_**The JGI Post Human Genome Project**_
**	****	****	****	**** ****Community Sequencing Program**
**	****	****	****	****	****	****(CSP)**
**	****	****	****	****Microbial Community Genomics**
**	****	****	****	**

**Notes:**
What to do with the  moving from man to microbes may cause a bit of whip lash but I will warn you when 

## Slide 3

## The Community Sequencing Program (CSP) 

- Overview

| 
 |  |
| --- | --- |
|      |                                                     |
|  |  |

**The Community Sequencing Program (CSP) **
**To provide the scientific community through a peer reviewed process access to high throughput sequencing at the JGI. **

**Notes:**
Program goal. Through this we hope to serve as infrastructure of AmericanScience enabling them 

## What types of projects will the JGI/CSP accept?

| 
 |  |
| --- | --- |
|      |                                                     |
|  |  |

**What types of projects will the JGI/CSP accept?**
**A wide range of projects. Ultimately, the most important factor in determining if a project will be accepted is its scientific merit.**

- User Guide > How to Propose a Project

**Notes:**
By making this purily science based we are hoping to also fascilitate revolutionary science .  In many ways the human genome caused much of production sequencing to be a cataloging eercise of the part list .  That’s good but there are also fundemental questons in biology that a billion bases of sequence may be able to answer and we are hoping that these of questions will come tot heJGI

## Sequence

**Users**

**Proposal**
**Study**
**Panel**

**Scientific**
**Advisory**
**Committee**

**Sequence**
**Allocation**

**Designated Lab**
**Director**

**Proposals & Peer Review Process**
**General Scientific Users Proposals**

**Notes:**
A series of structures have been in place to 

## What can researchers get from the CSP program?

| 
 |  |
| --- | --- |
|      |                                                     |
|  |  |

| FAQ |
| --- |
|  |

**What can researchers get from the CSP program?**
**The deliverables can range from raw sequence traces to well-annotated assembled genomes depending on the request in the proposal. **

**Notes:**
Though we have modeled this after light source facilities such as the ALS what we are delivering is more complicated than just delivering a beam .
We will sophisticated users who will just require flat files of sequence while others users it will be ery much a custom job where annotaion how to assemble will not be just offf the shelf and will need to be developed for the project.  We alos wan tnieve users who we will  exploit the informatic capabilities at the JGI to carry out their projects

## Scientific Support for Approved Projects

**Users**

- Scientific
- Support
- Group
- SSG

- Production
- Sequencing

- Informatic
- Analysis
- Of
- Sequence

**Interactions of the JGI and Scientific Users with Approved Sequencing Proposals**

**Notes:**
A crucial component of the JGI for the CSP is is the SSG.  This is the group that will interface with users.  They will make sure that it is  clear what the JGI will deliver and what are the users responsibilities.  It will also serve as the buffer between the JHI informatics and production sequencing and the User.

## Scientific Support for Approved Projects

**Gov Agencies**

- Scientific
- Support
- Group
- SSG

- Production
- Sequencing

- Informatic
- Analysis
- Of
- Sequence

**Interactions of the JGI and Scientific Users with Approved Sequencing Proposals**

- (EPA,USDA, NSF)

- GTL, Microbe

**CSP**

**Notes:**
A crucial component of the JGI for the CSP is is the SSG.  This is the group that will interface with users.  They will make sure that it is  clear what the JGI will deliver and what are the users responsibilities.  It will also serve as the buffer between the JHI informatics and production sequencing and the User.

## Production Sequencing

**DOE**

**Informatics**

**JGI Science Programs**

**Production Sequencing**

## Production Sequencing

**DOE+CSP+Gov A**

**Informatics**

**JGI Science Programs**

**Scientific Support Group**

**Production Sequencing**

## Slide 12

## < 1% of microbes are culturable
Many unculturables live in interdependent consortia of considerable diversity

Aim: to recover genome-scale sequences and reveal metabolic capabilities

What is the structure of natural microbial populations?  What is a microbial species? Can we harness their metabolic capabilities

**< 1% of microbes are culturable**
**Many unculturables live in interdependent consortia of considerable diversity**
**Aim: to recover genome-scale sequences and reveal metabolic capabilities**
**What is the structure of natural microbial populations?  What is a microbial species? Can we harness their metabolic capabilities**

## What Enviroments to Study?

**Ones with minimal microbial complexity**** **

**Notes:**
We have had a particular focus in the beginning of this program to explore communities that we suspect may have limited divirsity.  Assembling communities from sequence alone is a challenge .The thought is that communiies with limited diversity may presented a somewhat simpler version of this problem than more complex communities.

## Iron Mountain

**Jill Banfield et al. **
**UC Berkeley**

**Notes:**
Active for over a century starting right after the US civil war, 1860’s

## Iron Mountain

**  ****Superfund site**
**Discharging >1 ton of toxic metals/day**
**(pH <1)**
**FeS****2**

**Notes:**
Active for over a century starting right after the US civil war, 1860’s Metabolic energy derived from the from iron oxidation  

## “whole metagenome shotgun” dataset

**Purify High Molecular Weight DNA**

**Shotgun Library**
**Construction**

**DNA**
**Sequencing**

**Fosmid Library**
**Construction**

**Fosmid Insert**
**End Sequencing**

**Assembly**
**Annotation**

**=====**
**===**
**==**
**=**

**=====**
**===**
**==**
**=**

- =

**=====**
**===**
**==**
**=**

**Enviromental Sample**

**Purify High Molecular Weight DNA**

**DNA**
**Sequencing**

**Fosmid Library**
**Construction**

**Fosmid Insert**
**End Sequencing**

**Assembly**
**Annotation**

**=====**
**===**
**==**
**=**

**When possible culture isolates**

**=**

**?**

**Enviromental Sample**

## Iron Mtn “whole metagenome shotgun” GC content separates into two components

**Iron Mtn “whole metagenome shotgun” ****GC content separates into two components**

**Forward read average G+C**

**Reverse read average G+C**

**archaea**

**bacteria**

## Iron Mountain “whole metagenome shotgun” GC and depth distributions

**Iron Mountain “whole metagenome shotgun” ****GC and depth distributions**

**Read depth **

**3**

**10**

**Bacterial**

**Lepto II**

**Lepto III**

**Notes:**
Does the stoichiometry tell us about the biofilm structure/function?

**Archaeal**

**Fer 2**
** **

**Fer 1 ****(cultured     and sequenced )**
**G-plasma**

**3**

**10**

**Bacterial**

**Lepto II**

**Lepto III**

**3**

**10**

**Read depth **

**Notes:**
Does the stoichiometry tell us about the biofilm structure/function?

**Archaeal**

**Fer 2 (****3X****)**
** **

**Fer 1 (****1X****)**
**G-plasma (****1X****)**

**3**

**10**

**Bacterial**

**Lepto II (****3X****)**

**Lepto III (****1X****)**

**3**

**10**

**Stoichiometry**** **

**Read depth **

**Notes:**
Does the stoichiometry tell us about the biofilm structure/function?

**Archaeal**

**Fer 2**
** **

**Fer 1**
**G-plasma**

**3**

**10**

**Bacterial**

**Lepto II**

**Lepto III**

**3**

**10**

**Other sampled genomes at low depth (including eukaryotes) 15% of reads**

**Notes:**
Does the stoichiometry tell us about the biofilm structure/function?

## Similarity to Fer1 (isolate) to Sequence in Community

** ****%id to cultivated *****Fer1 *****isolate**

**Number of reads**

**98-100%**

**.50**

**.60**

**.70**

**.80**

**1.**

**.90**

*Fer2*

**    ***G plasma*

*Fer1*

**Mixed Community Reads**

## Conclusions So Far

**The stochiometry of organisms encouraging for the assembly of individual genomes**
**Assemblies support 16S studies suggesting limited diversity**
**Isolated Fer1 genome sequences matches genome in environmental sample **

## How do we know that our assembly is correct?

## How do you know you’ve done it right?Check pair ends against scaffold

**At the gross level: check pairs (expect few % due to failing/chimeric clones)**
**Align all reads back against assembled scaffolds**
**scaffolds end where there is no clone coverage in 3kb plasmids**
**Identifies potentially repetitive areas and/or rearrangements**

**How do we know that our assembly is correct?**

## Fer2  vs. fer1 shows local synteny

**Fer1 and **
**Fer2 have avg. nt identity of 78%**

**Fer1 gene on contig**

**Fer2 gene on contig**

## What does it mean to assemble a community genome?

**  ****Sample derived from  millions of genomes.**
**                              ****?**

**What is a “species” in the enviroment?**
**Members of the same species  **
**significantly different (many lineages survive and diverge)**
**highly similar (selective sweeps)**

## What does it mean to assemble a community genome?

**  ****Lepto II : 1 nucleotide variation / 3,000 bp**
**                              **

**Fer II: 2.2 nucleotide variation  / 100 bp**

## CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012
XYG46314.b1 162    A.......C........................A...........A.............. 103
XYG44123.b1 673    A.......C........................A...........A.............. 732
XYG44918.b1 48     A.......C........................A...........                4
XYG13291.g3 2                                                        .......... 11
XYG40116.g1 192    ......G..................................................... 133
XYG3051.b2  396    ......G..................................................... 455

CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072
XYG46314.b1 102    ............................................................ 43
XYG44123.b1 733    ............................................................ 792
XYG13291.g3 12     ............................................................ 71
XYG40116.g1 132    ...A........................................................ 73
XYG3051.b2  456    ...A........................................................ 515

CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132
XYG46314.b1 42     ..........................................                   1
XYG44123.b1 793    ........................                                     816
XYG13291.g3 72     ............................................................ 131
XYG40116.g1 72     T............G....C....................A.................... 13
XYG3051.b2  516    T............G....C....................A.................... 575

**CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012**
**XYG46314.b1 162    A.......C........................A...........A.............. 103**
**XYG44123.b1 673    A.......C........................A...........A.............. 732**
**XYG44918.b1 48     A.......C........................A...........                4**
**XYG13291.g3 2                                                        .......... 11**
**XYG40116.g1 192    ......G..................................................... 133**
**XYG3051.b2  396    ......G..................................................... 455**
**CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072**
**XYG46314.b1 102    ............................................................ 43**
**XYG44123.b1 733    ............................................................ 792**
**XYG13291.g3 12     ............................................................ 71**
**XYG40116.g1 132    ...A........................................................ 73**
**XYG3051.b2  456    ...A........................................................ 515**
**CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132**
**XYG46314.b1 42     ..........................................                   1**
**XYG44123.b1 793    ........................                                     816**
**XYG13291.g3 72     ............................................................ 131**
**XYG40116.g1 72     T............G....C....................A.................... 13**
**XYG3051.b2  516    T............G....C....................A.................... 575**

**5 Reads of the Same Sequence from            ****5  Different Members of the Same Species (FerII)**

*1*

**3**

**3**

## CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012
XYG46314.b1 162    A.......C........................A...........A.............. 103
XYG44123.b1 673    A.......C........................A...........A.............. 732
XYG44918.b1 48     A.......C........................A...........                4
XYG13291.g3 2                                                        .......... 11
XYG40116.g1 192    ......G..................................................... 133
XYG3051.b2  396    ......G..................................................... 455

CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072
XYG46314.b1 102    ............................................................ 43
XYG44123.b1 733    ............................................................ 792
XYG13291.g3 12     ............................................................ 71
XYG40116.g1 132    ...A........................................................ 73
XYG3051.b2  456    ...A........................................................ 515

CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132
XYG46314.b1 42     ..........................................                   1
XYG44123.b1 793    ........................                                     816
XYG13291.g3 72     ............................................................ 131
XYG40116.g1 72     T............G....C....................A.................... 13
XYG3051.b2  516    T............G....C....................A.................... 575

**CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012**
**XYG46314.b1 162    A.......C........................A...........A.............. 103**
**XYG44123.b1 673    A.......C........................A...........A.............. 732**
**XYG44918.b1 48     A.......C........................A...........                4**
**XYG13291.g3 2                                                        .......... 11**
**XYG40116.g1 192    ......G..................................................... 133**
**XYG3051.b2  396    ......G..................................................... 455**
**CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072**
**XYG46314.b1 102    ............................................................ 43**
**XYG44123.b1 733    ............................................................ 792**
**XYG13291.g3 12     ............................................................ 71**
**XYG40116.g1 132    ...A........................................................ 73**
**XYG3051.b2  456    ...A........................................................ 515**
**CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132**
**XYG46314.b1 42     ..........................................                   1**
**XYG44123.b1 793    ........................                                     816**
**XYG13291.g3 72     ............................................................ 131**
**XYG40116.g1 72     T............G....C....................A.................... 13**
**XYG3051.b2  516    T............G....C....................A.................... 575**

**Two Haplotypes Among the                                    ****5  Different Members of the Same Species (FerII)**

*1*

**3**

**3**

## CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012
XYG46314.b1 162    A.......C........................A...........A.............. 103
XYG44123.b1 673    A.......C........................A...........A.............. 732
XYG44918.b1 48     A.......C........................A...........                4
XYG13291.g3 2                                                        .......... 11
XYG40116.g1 192    ......G..................................................... 133
XYG3051.b2  396    ......G..................................................... 455

CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072
XYG46314.b1 102    ............................................................ 43
XYG44123.b1 733    ............................................................ 792
XYG13291.g3 12     ............................................................ 71
XYG40116.g1 132    ...A........................................................ 73
XYG3051.b2  456    ...A........................................................ 515

CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132
XYG46314.b1 42     ..........................................                   1
XYG44123.b1 793    ........................                                     816
XYG13291.g3 72     ............................................................ 131
XYG40116.g1 72     T............G....C....................A.................... 13
XYG3051.b2  516    T............G....C....................A.................... 575

**1**

**CONSENSUS   130953 gtttatattaaatccattgatttctaagcttccggttcttcttccgtataatggagattt 131012**
**XYG46314.b1 162    A.......C........................A...........A.............. 103**
**XYG44123.b1 673    A.......C........................A...........A.............. 732**
**XYG44918.b1 48     A.......C........................A...........                4**
**XYG13291.g3 2                                                        .......... 11**
**XYG40116.g1 192    ......G..................................................... 133**
**XYG3051.b2  396    ......G..................................................... 455**
**CONSENSUS    131013 atagcttaataattcatcctccatcatacttatgcttgaacctgataatattatgtatag 131072**
**XYG46314.b1 102    ............................................................ 43**
**XYG44123.b1 733    ............................................................ 792**
**XYG13291.g3 12     ............................................................ 71**
**XYG40116.g1 132    ...A........................................................ 73**
**XYG3051.b2  456    ...A........................................................ 515**
**CONSENSUS   131073 ccttgtagtatccattaattcatcaaatattttctgcattatagatataataccatggtt 131132**
**XYG46314.b1 42     ..........................................                   1**
**XYG44123.b1 793    ........................                                     816**
**XYG13291.g3 72     ............................................................ 131**
**XYG40116.g1 72     T............G....C....................A.................... 13**
**XYG3051.b2  516    T............G....C....................A.................... 575**

**Two haplotypes Among the                                        ****5  Different Members of the Same Species (Fer II)**

*1*

**3**

**3**

## Polymorphisms occur in blocks

**Polymorphisms occur in blocks**

**Long quiet regions separate highly variable segments**
**Variation is found in blocks of 5-10 genes**

**Local depth**

**% polymorphic sites**

**ORFs**

## Summary of Iron Mountain Biofilm

** **
**Limited number of predominant  species present in biofilm the majority have never been cultured**
**Several lines of evidence suggest that we can assemble genomes of these organisms**
**Simplicity of community suggests removal of most variants by natural selection**
** **
**Now studying the metabolic capabilities of microbes**

## Slide 37