**The *Populus* Genome Science Plan :**

# From Draft Sequence to a Catalogue of All Genes through the Advancement of Genomics Tools -- 2004-2009

## **Panel: Informatics, Annotation & Database Development**

[Francis Martin]{.underline} <fmartin@nancy.inra.fr> INRA -- Nancy

Jan Karlsson <jan.karlsson@plantphys.umu.se> Umeå Plant Science Centre

Dan Weems <dcw@ncgr.org> National Center Genome Research

Loren Hauser [hoi@ornl.gov](mailto:larimerfw@ornl.gov) Oak Ridge
National Laboratory

Natalie Pavy <nathalie.pavy@rsvs.ulaval.ca> Université Laval

Pierre Rouzé <Pierre.Rouze@gengenp.rug.ac.be> Ghent University

**1. Delineation of the scope**

Aims of this panel include the formation of publicly accessible, readily
updateable, globally linked intraspecific and interspecific genomics
databases. Three main tasks:

## -- Development of bioinformatic resources

## -- Annotation of the *Populus* genome

## -- Development and curation of databases 

**2. Description of the current state of the science, including a
catalog of the up-to-date status of physical, financial, and human
resources**

**2.1. Physical resources**

*Canada:*

*--Laval University (CRBF)* Gene prediction & genome annotation, gene
profiling databasing (Mac Kay *et al.*).

-- *UBC:* ?

*Belgium*

-   Gene prediction & genome annotation, with special emphasis on plants
    (Ghent, Rouzé et al.)

-   Databasing & search for transcriptional regulatory elements (Leuven,
    Y.Moreau\'s Team; Brussels, J.Van Helden; Ghent, Rouzé et al.)

-   Modeling Pathways & Gene Interactions (Brussels, S.Wodak/J.VH;
    Ghent, M.Kuiper; Leuven, Y. Moreau)

> -- Comparative genomics, genome duplication, gene families (Ghent,
> Rouzé et al./ Y. Van de Peer)

*France:*

-   EST annotation, SAGE, search for transcriptional regulatory
    elements, microarray databasing: INRA-Orléans (Leplé & Pilate et
    al.).

-   The INRA PoplarDB database containing annotations, functional
    classifications for unigenes of root ESTs, and blast services:
    INRA-Nancy (Martin et al.).

-   The LIGNOME EST database & EST clustering: INRA-Bordeaux (Plomion et
    al.)

*Germany & Finlande* ??

#### Sweden: The UPSC PopulusDB database containing annotations, functional classifications for unigenes of \>100.000 ESTs, and blast services. Curation of the database is funded for 2004 (Karlsson et al.).

#### USA: ORNL facilities + Michigan Tech (?)

**2.2.Financial resources:** No specific funding, except at UPSC
(Sweden). The EU \"Network of Excellence\", so-called EVOLTREE, if
agreed will allocate funding to Populus genome annotation.

**2.3. Human resources**

Human resources are requested at three steps of the process:

(1) Development/tuning of software & tools for annotation

(2) Structural (syntactic) annotation : modeling of genes for the whole
    genome

(3) Functional annotation: give functional attributes to every gene
    and/or gene product.

**Step.1** will be done in a very few teams which are already involved
in such developments, like ORNL, The Ghent Team (Rouzé et al.) and
possibly Umea IPGC & UBC.

**Step.2** is straightforward as soon as the tools are developed and
validated in Step.1. It is mostly automated and will need a few
dedicated curation people, well organized, database-minded, but not
necessarily highly qualified, their number and duration of the task
depending on the flow and quality of sequence data produced. Their job
will be finished (as a first version) quite early on after a decently
full sequence will be obtained. This job should better be done inside,
or in close contact with (and feedback from), the teams having performed
step.1 (ORNL & Ghent). The output could be [one]{.underline} initial
gene model for the genome, or (more likely) [several]{.underline}
concurrent gene models, as produced using the different tools available.
Pierre Rouzé suggests to keep these alternative models separate,
documented as completely as possible, and to leave the choice for one or
the other models to the annotators in step.3 here below.

**Step.3** is the most human-demanding step. It can be done in a
processive way, with a first covering done merely through BLAST
homology. This simple pass is time-demanding, since the curator has to
check if this annotation fits with the gene model, and correct it
otherwise. A more elaborate functional annotation will come through
analysis of gene families (potentially through external experts), and
more elaborate analysis of the gene products according to the accepted
Gene Ontology procedure, and using additional criteria. The Ghent team
would only take part in this step if additional financial support would
be allocated specifically for it (see below).

**Identified human resources (May 2003)**

####### Canada:

-   Laval University: 1 post-doctoral fellow dedicated to *Populus*
    genome

-   UBC: ??

*Belgium*

The Ghent bioinformatics team (Rouzé et al.) is a partner in PLANeT, an
EU-founded initiative coordinated by Klaus Mayer (MIPS) which aims at
providing plant scientists access to plant genomics data, knowledge and
resources collected in the different partners countries, and to share
curation tasks and know-how. One post-doc will be funded for *Populus*
genome annotation in addition to the current staff and PhDs currently
involved in such task.

*France:*

*--* Antoine Kramer (INRA-Bordeaux) is coordinating the EU network
EVOLTREE (involving up to 200 scientists from a dozen of european
countries), aiming at investigating tree biodiversity and involving
genomics as the first component. Poplar is one of the three species
chosen in this programme, and helping its annotation one of the
milestone of the proposal. If funded, this proposal will provide support
to hire people to perform this task, up to the functional step if
funding allows.

INRA: Full time equivalent for 2004 : 5 mans/year, mainly on functional
genomics and QTLs. One man/year could be involved in gene annotation.

*Germany* ??

#### USA: Currently, no human resources for doing gene modeling and annotation???

#### Sweden: One man/year that could be more or less directly involved in the annotation of the Populus genome and post-genomic activities.

### 3. List of short-term \[1-2 years\], mid-term \[2-5 years\] and long-term \[5+ years\] goals

**3.1. \[1-2 years\]**

-   Generate a data-set of non-redundant full length cDNAs and ESTs to
    build a relevant database and train GRAILEXP or/and EuGene
    components. This will involve ORNL, UPSC, UBC, Laval, and INRA. Up
    to 1000 full-length cDNAs and 150K ESTs are currently available.

-   Check (mostly through automatic routines) all poplar BACs for these
    documented genes, as well as evolutionary conserved genes. Annotate
    automatically & check manually all these annotations. Build the
    relevant training sets from these annotation

-   Validate the existing comparative annotation program for adequation
    to comparison of poplar and Arabidopsis genomes

-   Depending on the results, tune one or several of them, and possibly
    develop additional in-house capabilities

-   Through collaboration between ORNL and Ghent Team search for and
    enter in database specific genome features to be filtered out (or
    annotated separately) in the annotation process (repeats, rRNAs,
    transposons, ..)

-   Build the BLAST databases for the protein and cDNA comparisons
    components of EUGENE_POP and/or GRAILEXP

-   Validate, tune and train the ab-initio components of EUGENE_POP
    and/or GRAILEXP (Markov models for Exon, Introns, UTRs and
    Intergenic, Splice site predictors, Translation Start, ..)

-   Integrate Genome Comparison Algorithm(s) as EUGENE_POP component(s)

-   Train EUGENE_POP and/or GRAILEXP and validate

-   Beta-trial of EUGENE_POP/GRAILEXP on routine with feedbacks

-   Syntactic (structural) annotation of the poplar genome, BAC-wise
    using EUGENE_POP/GRAILEXP

-   Comparative evaluation of syntactic annotation (collaboration
    ORNL-Ghent). Depending on sequencing status, provide a provisional
    complete genome annotation (with or without functional annotation,
    see below)

-   Agree on stategy and share of tasks with ORNL-Ghent and other teams
    on functional annotation. Build routines to collect comparative
    functional annotation.

-   Routine first functional annotation through database matches
    (SwissProt, Gene Ontology and Interpro, ...)

-   Enter Ontology consortium and validate for poplar

**3.2. \[2-5 years\]**

-   Compare GRAILEXP and EuGene gene models (and any other modeler that
    IPGC scientists want to use).  This comparison will be on going for
    a number of years.  To be discussed at the yearly meetings.

-   After an agreed upon time and as more full length cDNAs and
    assembled ESTs become available, the entire genome will be remodeled
    with retrained GRAILEXP and EuGene gene models.

-   Final annotation and distribution of the workload.  To be discussed
    at the yearly meetings

-   Train a number of poplar biologists on how to do annotation since
    they will be the ones who will  primarily use and edit the final
    poplar database.

-   Developpement of the Poplar Genome Anatomy Project (PGAP) aiming to
    determine the gene expression profiles of poplar tissues/cells,
    leading eventually to improved detection and diagnosis for the
    economically-relevant traits. The PGAP will provide comprehensive
    genomic data, including expressed sequence tags (ESTs), gene
    expression patterns, single nucleotide polymorphisms (SNPs), cluster
    assemblies, and cytogenetic information, together with informatics
    tools to query and analyze the data & Information on methods and
    resources for reagents developed by the project.

### 3.3. \[long-term \>5years\]

-   Build a visualization tool to understand architectural structuration
    of gene expression in trees (linking gene annotation, Gene Ontology,
    microarray gene expression profilings).

**4. Discussion on a) strategies for reaching each goal and b) potential
future applications.**

Gene prediction & annotation in poplar will be largely using tools
(GRAILEXP/EuGene) that have been developed for other genomes (e.g.,
Arabidopsis). Nevertheless several points have to be noticed:

-   the coverage of *Populus* sequencing will be quite low (x6) which
    will have a negative influence on the performance of *ab initio*
    gene finding.

-   there are many ESTs (150 K) but not plenty, and quite few entire
    cDNAs: gene finding will gain only marginally from data from the
    expressed genome (contrary to human or even Arabidopsis & Rice).

-   In contrast, there is/will be soon several plant genome entirely
    sequenced, or with large sequence data (Arabidopsis, rice, medicago,
    maize, ..): gene prediction in poplar should use comparative
    genomics to a large extent. This approach has been promoted recently
    for the human genome, but mainly dealing with more closely related
    organisms (human/mouse). There will be a need to tune or re-develop
    the existing tools to cope with our needs. We suggest to balance the
    low coverage concern which will leave many gene models with
    uncertainties by a back-and-forth mechanism between sequencing and
    annotating teams, in order for the second to pinpoint potential
    anomalies (e.g. frameshifts) to be checked on sequence readings and
    spectra by the firsts (Rouzé's team did it on a small scale when
    sequencing Arabidopsis, and corrected several sequencing errors this
    way). Ghent team is planning to build an integrated gene prediction
    platform based on EUGENE, the one they developed for Arabidopsis in
    collaboration with Thomas Schiex (INRA Toulouse) plugging in an
    additional comparative genome component.