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United States Department of Agriculture

Agricultural Research Service

Research Project: Genomics Based Discovery of Novel Traits in Beneficial Plant-associated Pseudomonas Species
2010 Annual Report

1a.Objectives (from AD-416)
1. Generate draft genomic sequences for seven plant-associated strains of Pseudomonas spp. that suppress plant disease. 2. Generate assemblies of the genomes. 3. Perform automatic annotation of the genomes. 4. Provide an instructor and materials for a workshop for 14 collaborators.

1b.Approach (from AD-416)
Prepare random genomic shotgun libraries and sequence both ends of randomly selected clones. Assemble genome from shotgun libraries and sequencing. For initial identification of protein coding sequences, the Glimmer algorithm will be used. All regions of a genome without Glimmer predictions will then be re-evaluated using BLASTP to search against a database of non-redundant proteins (nraa) maintained at JCVI. Documents Grant with J. Craig Venter Institute.

3.Progress Report

To improve the draft genomes of four strains of Pseudomonas fluorescens, JCVI constructed MID barcoded paired-end libraries and applied 454 Titanium sequencing. Bar-coded, paired-end 454 Titanium sequencing represents a state-of-the art new sequencing technology. Application of this technique represented a milestone at JCVI and the Pseudomonas spp. genomes re-sequenced as part of this project were among the first at JCVI or anywhere sequenced in this manner. Because 454 FLX sequencing is a fragment sequencing approach, there were expectations that paired-end (or mate pair) information would significantly enhance the accuracy and quality of assembled sequence data. The new sequence data was merged with the original FLX and Sanger data. The merged data sets assembled with both Newbler 2.3 and Celera Assembler 5.42 represented significantly improved genome sequences. The data was incorporated into fewer overall scaffolds and the contigs within scaffolds were higher quality in terms of accuracy and ordering. Each genome was subsequently evaluated for additional assembly improvement with the CA 5.42 assembly versions providing the starting points. Multiple gaps were closed by merging overlapping contigs and resolving repetitive gaps. PCR end reads produced by both collaborators and JCVI were generated for several additional gaps which were then incorporated to improve contiguity. Gap sequence was provided by collaborators for P. fluorescens SS101 and P. fluorescens A506. For P. fluorescens Q8r1-96 and P. fluorescens Q2-87 PCR sequences were generated at JCVI. After completing the manual improvement, these genomes were released to annotation as Improved High-Quality Drafts with the following contig counts: P. fluorescens SS101 in one contig with one unresolved gap, P. fluorescens A506 in one contig with one unresolved gap, P. fluorescens Q2-87 in two contigs with two unresolved gaps, P. synxanta BG33R in two contigs with two unresolved gaps and P. fluorescens Q8r1-96 is in five contigs with five unresolved gaps. The genomes are currently undergoing annotation mapping from the previous draft assemblies.

Methods of ADODR monitoring included meetings, phone calls, e-mail and site visits.

Last Modified: 1/31/2015
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