Annotation and Visualization

Doreen Ware

Project Challenges

•Rapidly growing sequence data

•Full annotation of all clones

•New high-performance computing cluster

•2,000 nodes

•Scheduling system (SunGrid Engine)

•NFS issues

•EnsEMBL Code Integration

Milestones• www.maizesequence.org released

• Customized entry points of the Ensembl browser for the maize community.

• Adapted modules to the new compute cluster Blue Helix and automated gene predictions, MDR analysis, repeat masker

• Alignments of cereal sequence using Gramene Biopipe (needs to be automated)

• Transitioned from annotating Finished BACs to all BACs as they become available

• Blast Server

• FTP site

• DAS server (displaying Twinscan annotations)

Index Page

Maizesequence.org RSS BAC Notification

• Users can be notified of sequence and annotation updates to a particular region of interest on the FPC map via a RSS (Really Simple Syndication) notification system.

• Data is delivered as XML to the user’s favorite feed reader or is parsed in RSS enabled browsers.

• The URL for any given query is persistent and dynamically retrieves database updates in the user-specified region.

…

www.maizesequence.org/Zea_mays/notification

Maizesequence.org FTP and Blast DB

Ensembl BAC DB

Weekly

Bulk Genome Dump

Maize FTP

BACsBAC Contigs

Ab initio predictionsAb initio translations

Maize Blast

BAC ContigsAb initio predictionsAb initio translations

• BACs, BAC Contigs, FgenesH predictions (TE and non-TE classes), and FgenesH translations are dumped on a weekly basis.

• Sequence dumps are posted to the FTP site. (ftp.maizesequence.org)

• Sequence dumps are also used to update the blast databases. (www.maizesequence.org/Multi/blastview)

MapView

CytoView

ContigView

GeneView

GeneView

ExportView

ExportView

ExportView

Maize Databases and Annotation Pipeline

Classification of Gene Models

• Ab initio gene prediction on non-masked contigs with FGENESH using Monocot parameters.

• Classified gene models by BLASTP to Genbank NRAA.

• TE = Alignment to transposable elements (TE), as specified within curated database.

• NH = No detectable homology.

• WH = Significant alignment to non-TE.

• Corrupted_translation = Ensembl translation inconsistent with FGENESH.

Gene Model Class Minimum Maximum Average Median

Standard Deviation

TE size (bases) 51 23,913 2,739 2,402 1,916

WH size (bases) 73 25,816 2,465 1,829 2,146

NH size (bases) 3 19,465 975 645 944

Corrupted_translation (bases)

8 25,869 2,251 1,845 1,813

10,352 Annotated BACs (309,845 Gene Models)

TENHWHCorrupted_translation

TE, 198580, 64%

NH, 51805, 17%

WH, 45055, 14%

Corrupted_Translation, 14405, 5%

Data generated as of September 2007

Nucleotide Coverage of Mathematically-Defined Repats in 10,352 Annotated BACs

(130,978 Contigs)

MDR Type*Total Nucleotides

Nucleotide Coverage

2 copies 1,325,811,407 79.11%

10 copies 937,789,153 55.96%

100 copies

602,350,024 35.94%

1000 copies

218,650,689 13.05%

*Mathematically defined repeats indicate regions of repetitive DNA. The frequency of each constituent 20-mer along the BAC sequence was determined within the raw reads of the maize whole genome shotgun sequence (DOE Joint Genome Institute). “MDR type 2 copies” indicates regions over which 20-mers occurred two or more times. Thus, “MDR type 10 copies”, “MDR type 100 copies”, and “MDR type 1000 copies” indicate; respectively, regions over which 20-mers occurred, ten or more times, one hundred or more times, and one thousand or more times. The most repetitive regions correspond to regions in the MDR type 1000 copies. The least repetitive regions correspond to areas in the MDR type 2 copies.

Data generated as of September 2007

Nucleotide Coverage of Repeats in 10,352 Annotated BACs (130,978

Contigs)

Repeat Type*Total Nucleotides

Nucleotide Coverage

MIPS/REcat Class I Retroelements 1,503,929,793 75.66%

MIPS/REcat Class II/III Transposable Elements

36,620,646 1.84%

MIPS/Recat Other 16,048,937 0.81%

All Repeats 1,553,118,769 78.13%

*Repetitive sequences were annotated and masked using RepeatMasker and the MIPS-Redat library.

Data generated as of September 2007

Outreach and Collaborations

•MaizeGDB•EBI EnsEMBL•Gramene•Maize Array Working Group

•Maize Optical Map

•Transposon Annotation

•TWINSCAN•Vmatch•Student Annotation (Howard Hughes)

Objectives for Year 3

• Whole Genome Alignments for rice, maize and arabidopsis

• Evidence based gene builds Gramene modified Ensembl pipeline and FGENESH++ in combiner mode BioMart for complex query generation

• Whole Genome Alignments for rice, maize and arabidopsis

• SyntenyView based on whole genome alignment• Transition from Gramene Biopipe -> Ensembl

Exonerate pipeline to automate sequence alignments• Annotation of non-coding RNA using tRNAScan and

microRNA • Gene Ontology using dbxref pipeline• Incorporation in Gramene Compara builds; GeneTree

view• MySQL Database dumps• Tutorials for website using Camptasia• Submit paper on MDR analysis

Shiran Pasternak, Apurva Narechania, Linda McMahan, Joshua Stein