title: Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium.
abstract: BACKGROUND
  Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence.
  
  
  RESULTS
  We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions.
  
  
  CONCLUSIONS
  We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. C.Humphreys@nottingham.ac.uk.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Humphreys
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Samantha.McLean@nottingham.ac.uk.
authors.forename: Samantha
authors.initials: S
authors.lastname: McLean
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, UK. Sarah.Schatschneider@nottingham.ac.uk.
authors.forename: Sarah
authors.initials: S
authors.lastname: Schatschneider
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Thomas.Millat@nottingham.ac.uk.
authors.forename: Thomas
authors.initials: T
authors.lastname: Millat
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Anne.Henstra@nottingham.ac.uk.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Henstra
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. mrxfja@exmail.nottingham.ac.uk.
authors.forename: Florence J
authors.initials: FJ
authors.lastname: Annan
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. mrxrb14@exmail.nottingham.ac.uk.
authors.forename: Ronja
authors.initials: R
authors.lastname: Breitkopf
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. mrxbp@exmail.nottingham.ac.uk.
authors.forename: Bart
authors.initials: B
authors.lastname: Pander
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. mrxpp2@exmail.nottingham.ac.uk.
authors.forename: Pawel
authors.initials: P
authors.lastname: Piatek
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. msxpr@exmail.nottingham.ac.uk.
authors.forename: Peter
authors.initials: P
authors.lastname: Rowe
authors.affiliation: School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, UK. pcxatw@exmail.nottingham.ac.uk.
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Wichlacz
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. mrxcw4@exmail.nottingham.ac.uk.
authors.forename: Craig
authors.initials: C
authors.lastname: Woods
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. stxron@exmail.nottingham.ac.uk.
authors.forename: Rupert
authors.initials: R
authors.lastname: Norman
authors.affiliation: Bioinformatics and Systems Biology, Justus-Liebig-University Giessen, 35392, Giessen, Germany. Jochen.Blom@computational.bio.uni-giessen.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Blom
authors.affiliation: Bioinformatics and Systems Biology, Justus-Liebig-University Giessen, 35392, Giessen, Germany. Alexander.Goesmann@computational.bio.uni-giessen.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Goesman
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Charlie.Hodgman@nottingham.ac.uk.
authors.forename: Charlie
authors.initials: C
authors.lastname: Hodgman
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, UK. David.Barrett@nottingham.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Barrett
authors.affiliation: School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, UK. Neil.Thomas@nottingham.ac.uk.
authors.forename: Neil R
authors.initials: NR
authors.lastname: Thomas
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Klaus.Winzer@nottingham.ac.uk.
authors.forename: Klaus
authors.initials: K
authors.lastname: Winzer
authors.affiliation: BBSRC/EPSRC Synthetic Biology Research Centre, School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. Nigel.Minton@nottingham.ac.uk.
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D003013:Clostridium / Q000235:genetics*; D066289:Data Curation / Q000379:methods; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D058977:Molecular Sequence Annotation; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*