title: IncI shufflons: Assembly issues in the next-generation sequencing era.
abstract: The shufflon is a site-specific recombination system first identified in the IncI1 plasmid R64. The R64 shufflon consists of four segments, separated by short repeats, which are rearranged and inverted by the recombinase protein Rci, generating diversity in the C-terminal end of the PilV protein. PilV is the tip adhesin of the thin pilus structure involved in bacterial conjugation and may play a role in determining recipient cell specificity during liquid mating. The variable arrangements of the shufflon region would be expected to make plasmid assembly difficult, particularly with short-read sequencing technology, but this is not usually mentioned in recent publications reporting IncI plasmid sequences. Here we discuss the issues we encountered with assembly of IncI1 sequence data obtained from the Roche-454 and Illumina platforms and make some suggestions for assembly of the shufflon region. Comparison of shufflon segments from a collection of IncI1 plasmids from The Netherlands and Australia, together with sequences available in GenBank, suggests that the number of shufflon segments present is conserved among plasmids grouped together by plasmid multi-locus sequencing typing but the different reported arrangements of shufflon segments may not be meaningful. This analysis also indicated that the sequences of the shufflon segments are highly conserved, with very few nucleotide changes.
authors.affiliation: Central Veterinary Institute of Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands. Electronic address: mike.brouwer@wur.nl.
authors.forename: Michael S M
authors.initials: MS
authors.lastname: Brouwer
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia.
authors.forename: Kaitlin A
authors.initials: KA
authors.lastname: Tagg
authors.affiliation: Central Veterinary Institute of Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands.
authors.forename: Dik J
authors.initials: DJ
authors.lastname: Mevius
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Iredell
authors.affiliation: Central Veterinary Institute of Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Bossers
authors.affiliation: Central Veterinary Institute of Wageningen UR, PO Box 65, 8200 AB Lelystad, The Netherlands.
authors.forename: Hilde E
authors.initials: HE
authors.lastname: Smith
authors.affiliation: Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, The University of Sydney and Westmead Hospital, Westmead, NSW 2145, Australia. Electronic address: sally.partridge@health.nsw.gov.au.
authors.forename: Sally R
authors.initials: SR
authors.lastname: Partridge
mesh_terms: D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA
keywords: Assembly; IncI1 plasmids; Next-generation sequencing; Shufflon