Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX titanium system in a comparison of multiple genomics core facilities

Vincenzo Forgetta, Gary Leveque, Joana Dias, Deborah Grove, Robert Lyons, Suzanne Genik, Chris Wright, Sushmita Singh, Nichole Peterson, Michael Zianni, Jan Kieleczawa, Robert Steen, Anoja Perera, Doug Bintzler, Scottie Adams, Will Hintz, Volker Jacobi, Louis Bernier, Roger Levesque, Ken Dewar

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30-50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.

Original languageEnglish (US)
Pages (from-to)39-49
Number of pages11
JournalJournal of Biomolecular Techniques
Volume24
Issue number1
DOIs
StatePublished - Mar 2013

Keywords

  • Fungal genomics
  • Massively parallel DNA sequencing
  • Ophiostoma novo-ulmi

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