Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
WHOLE GENOME APPROACH TO MICROBIAL
FORENSICS OF PLANT PATHOGENS
Wednesday, 1st June 18:30 La Fonda Mezzanine (2nd Floor) Poster (PS‐2a.09)
Jacqueline Fletcher 1 , Trenna Blagden 1 , Ulrich Melcher 1 , Brittany Campos 2 , David Lin 3 , Kumar
Hari 3 , Richard Winegar 2
1 National Institute for Microbial Forensics & Food and Agricultural Biosecurity,
Oklahoma State University, 2 MRIGlobal, 3 cBio, Inc
A number of bacteria, viruses, and fungi pose serious health concerns to humans, threaten the
U.S. economy, food and energy supplies, and/or the environment. The potential for pathogen use
as biological weapons has been demonstrated, and the U.S. needs strong capabilities to respond
to incidents involving such activity. Use of next‐generation sequencing (NGS) for applications in
forensic investigation enhances microbial detection, strain matching and discrimination capabilities.
The technology also allows for detection of all microbes in complex samples, detection of previously
unknown organisms, and identification of specific signatures or genes.
A multidisciplinary and multi‐institutional team, led by MRIGlobal.,Inc. in cooperation with the
Department of Homeland Security, assessed the genome sequencing status and then performed whole
genome sequencing and assembly for a collection of high threat human, animal and plant pathogens,
including U.S. select agents,
Genome sequence assessment surveys and surveillance plans were completed for the plant pathogenic
bacteria Ralstonia solanacearum, Rathayibacter toxicus, Xylella fastidiosa, and Xan‐ thomonas oryzae,
fungi Coniothyrium glycines, Puccinia graminis, Puccinia striiformis, Puccinia triticina, and Sybchytrium
endobioticum, and oomycetes Peronosclerospora philippinensis and Sclerophthora rayssiae. Information
and data gaps in existing genetic databases were used to set priorities for project collections and genome
sequencing efforts.
Nucleic acid purified from the plant pathogens Ralstonia solanacearum (four strains), Rathayibacter
toxicus (two strains), Xylella fastidiosa subsp. pauca (six strains), Xanthomonas oryzae (pv. USA)
and its near neighbor X. campestris pv. leersiae, and Coniothyrium glycines were obtained from
expert collaborators.
Illumina sequence libraries were prepared using Nextera XT. Whole genome 2×300 paired‐end sequencing
was performed using the Illumina MiSeq instrument. Reads were filtered and trimmed
using Trimmomatic. The iMetAMOS pipeline was used to optimize de novo assembly and perform
quality checks. Elements of the pipeline include FastQC, SPAdes, IDBA, KmerGenie, and QUAST.
Resulting assemblies were polished using Pilon; with Samtools and BLAST used to remove low coverage
and contaminating contigs. Final assemblies were annotated through the NCBI Prokaryotic
Genome Automatic Annotation Pipeline.
An important outcome of this work was the creation of a NCBI‐hosted genomic database for selected
microbial biothreat plant pathogens of priority interest to the United States. The project generated
provided genomic information necessary to assist forensic investigators in genotyping, phylogenetic
analysis, and sample matching of evidentiary materials.
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