title: Ecological patterns of nifH genes in four terrestrial climatic zones explored with targeted metagenomics using FrameBot, a new informatics tool.
abstract: Biological nitrogen fixation is an important component of sustainable soil fertility and a key component of the nitrogen cycle. We used targeted metagenomics to study the nitrogen fixation-capable terrestrial bacterial community by targeting the gene for nitrogenase reductase (nifH). We obtained 1.1 million nifH 454 amplicon sequences from 222 soil samples collected from 4 National Ecological Observatory Network (NEON) sites in Alaska, Hawaii, Utah, and Florida. To accurately detect and correct frameshifts caused by indel sequencing errors, we developed FrameBot, a tool for frameshift correction and nearest-neighbor classification, and compared its accuracy to that of two other rapid frameshift correction tools. We found FrameBot was, in general, more accurate as long as a reference protein sequence with 80% or greater identity to a query was available, as was the case for virtually all nifH reads for the 4 NEON sites. Frameshifts were present in 12.7% of the reads. Those nifH sequences related to the Proteobacteria phylum were most abundant, followed by those for Cyanobacteria in the Alaska and Utah sites. Predominant genera with nifH sequences similar to reads included Azospirillum, Bradyrhizobium, and Rhizobium, the latter two without obvious plant hosts at the sites. Surprisingly, 80% of the sequences had greater than 95% amino acid identity to known nifH gene sequences. These samples were grouped by site and correlated with soil environmental factors, especially drainage, light intensity, mean annual temperature, and mean annual precipitation. FrameBot was tested successfully on three ecofunctional genes but should be applicable to any.
  
  
  OBJECTIVE
  High-throughput phylogenetic analysis of microbial communities using rRNA-targeted sequencing is now commonplace; however, such data often allow little inference with respect to either the presence or the diversity of genes involved in most important ecological processes. To study the gene pool for these processes, it is more straightforward to assess the genes directly responsible for the ecological function (ecofunctional genes). However, analyzing these genes involves technical challenges beyond those seen for rRNA. In particular, frameshift errors cause garbled downstream protein translations. Our FrameBot tool described here both corrects frameshift errors in query reads and determines their closest matching protein sequences in a set of reference sequences. We validated this new tool with sequences from defined communities and demonstrated the tool's utility on nifH gene fragments sequenced from soils in well-characterized and major terrestrial ecosystem types.
authors.affiliation: Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.forename: John F
authors.initials: JF
authors.lastname: Quensen
authors.forename: Jordan A
authors.initials: JA
authors.lastname: Fish
authors.forename: Tae Kwon
authors.initials: TK
authors.lastname: Lee
authors.forename: Yanni
authors.initials: Y
authors.lastname: Sun
authors.forename: James M
authors.initials: JM
authors.lastname: Tiedje
authors.forename: James R
authors.initials: JR
authors.lastname: Cole
mesh_terms: D000413:Alaska; D000465:Algorithms; D000595:Amino Acid Sequence; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics*; D017753:Ecosystem; D005431:Florida; D016368:Frameshift Mutation; D006254:Hawaii; D056186:Metagenomics / Q000295:instrumentation* / Q000379:methods; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010802:Phylogeny; D012988:Soil Microbiology; D014589:Utah
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases; C029130:nitrogenase reductase
references.citation: Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9
references.pmid: 1438297
references.citation: BMC Bioinformatics. 2011 May 24;12:198
references.pmid: 21609463
references.citation: FEMS Microbiol Ecol. 2007 Apr;60(1):85-97
references.pmid: 17381526
references.citation: Comput Appl Biosci. 1996 Feb;12(1):31-40
references.pmid: 8670617
references.citation: J Bacteriol. 2005 Sep;187(18):6258-64
references.pmid: 16159757
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10039-44
references.pmid: 18632554
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):4651-5
references.pmid: 23487761
references.citation: Appl Environ Microbiol. 1999 Jul;65(7):3084-94
references.pmid: 10388707
references.citation: BMC Bioinformatics. 2011 Jan 28;12:38
references.pmid: 21276213
references.citation: ISME J. 2010 Feb;4(2):279-85
references.pmid: 19776767
references.citation: PLoS Comput Biol. 2011 Oct;7(10):e1002195
references.pmid: 22039361
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D290-301
references.pmid: 22127870
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D141-5
references.pmid: 19004872
references.citation: Environ Microbiol. 2007 Jun;9(6):1448-55
references.pmid: 17504482
references.citation: Curr Opin Genet Dev. 2005 Dec;15(6):589-94
references.pmid: 16185861
references.citation: Microbiome. 2013 Mar 04;1(1):8
references.pmid: 24451334
references.citation: Environ Microbiol. 2010 Jul;12(7):1889-98
references.pmid: 20236171
references.citation: Environ Microbiol. 2003 Jul;5(7):539-54
references.pmid: 12823187
references.citation: J Mol Biol. 1970 Mar;48(3):443-53
references.pmid: 5420325
references.citation: Front Microbiol. 2013 Jun 17;4:150
references.pmid: 23785359
references.citation: Mol Biol Evol. 2004 Mar;21(3):541-54
references.pmid: 14694078
references.citation: Nucleic Acids Res. 2010 Nov;38(20):e191
references.pmid: 20805240
references.citation: Science. 2007 Jun 1;316(5829):1307-12
references.pmid: 17540897
references.citation: PLoS One. 2012;7(7):e42149
references.pmid: 22848735
references.citation: Res Microbiol. 2001 Jan-Feb;152(1):95-103
references.pmid: 11281330
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: Nat Methods. 2009 Sep;6(9):636-7
references.pmid: 19718016