Computational protocol: Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage

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[…] To screen nif-containing fosmids, metagenome microarray was constructed using metagenomic library mentioned above. Each clone was incubated in a shaking incubator at 37°C and 170 r.p.m in the presence of chloramphenicol and an inducer (Epicentre). After overnight incubations, cells were harvested and the fosmid DNA was extracted using QIAprep spin miniprep kit (Qiagen, Germany) according to the manufacturer's protocol. The fosmid DNAs were stored in final concentration of 40 ng·µl−1. 10 µl fosmid DNA of each clone was transferred to a 384-well microplate, and diluted 1∶1 (V/V) in 40% dimethyl sulfoxide (Sigma, USA). Then, the fosmid DNA of each clone was arrayed on microarray with two replicates using Genemachines OmniGrid Accent microarrayer (Genomic Solutions, USA). In addition, the following controls were spotted to check by hybridization, printing, and data analysis: (i) environmental DNA as positive controls, (ii) negative controls with E. coli genomic DNA, and (iii) blanks. The microarrays were post-treated as described previously, and stored in room temperature.Here, we chose PCR products of nifK genes as probe for microarray hybridization, since NifK is indispensable in all diazotrophs. They were labeled with the Bioprime DNA Labeling kit (Invitrogen, Carlsbad, CA) according to the manufacture's protocol. Then, the labeled DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany), concentrated into crystallization in a SpeedVac, and finally resuspended in 20 µl MilliQ water. And labeled DNA was mixed with hybridization solution. The hybridization solution contained 20 µl of labeled DNA, 65 µl of formamide (50%,v/v), 19.5 µl of 20×SSC (1×SSC containing 150 mM NaCl and 15 mM trisodium citrate), 9.1 µl of Herring Sperm DNA(10 mg/mL)(Promega, Madison, WI), 3.9 µl of 10% sodium dodecyl sulfate (SDS), 1.1 µl of DTT(0.1 M) in a total volume of 130 µl. Finally, the mixed solution was incubated at 98°C for 3 min, and then kept at 65°C. Microarray hybridization was performed at 54°C using the HS 4800Pro Hybridization station (TECAN, Switzerland). After hybridization, the microarray was visualized using a GenePix 4100A Microarray Scanner (Axon, USA). The normalized intensity of each spot was calculated as described previously, and positive clones in metagenomic library can be obtained based on the positive spots in metagenome microarray. Each positive clone was tested using PCR amplification with universal nifK primers mentioned above.The verified positive fosmids were individually isolated using QIAprep spin miniprep kit (Qiagen), and pyrosequenced using Roche 454 GS FLX system (Majorbio, China). A total of 5 µg of fosmid DNA from each clone was tagged individually using a multiplex identifier adaptor containing a unique 10 base pair sequence that is recognized by the sequencing analysis software. Finally, each fosmid had ∼1.2 Mb of sequencing data for assembly, equivalent to the >30X clone coverage. And the average length of reads was 410 bp. Each of the fosmids was assembled into one single contig with the program Newbler . [...] Metagenomic sequencing yielded a total of 640,892 reads and 300 Mb of raw sequence. The individual metagenomic sequences >400 bp (approximately 90% sequences) were annotated using the non-redundant (NR) database, KEGG database, COG database. The sequences annotated as nif genes were selected to further analyze (). Protein-coding genes of fosmids were predicted using GLIMMER and the RAST server , and further curated (). Identified ORFs were compared to known proteins in the non-redundant (NR) database, KEGG database and COG database using BLASTX , and all hits with e-value >1e-5 were considered nonsignificant. Other unassigned ORFs were annotated using the hmmpfam program of the HMMER package . The hidden Markov models for the protein domains were obtained from the Pfam database 26.0 (http://pfam.sanger.ac.uk/). For comparative analysis, BLASTN and BLASTX searches among fosmids and different bacterial genomes were performed, leading to the identification of regions of similarity. To allow for the interactive visualization of genomic fragment comparisons, we used Artemis Comparison Tool . Phylogenetic trees were constructed by the use of Molecular Evolutionary Genetics Analysis 4.0 (MEGA 4.0) software. […]

Pipeline specifications

Software tools Newbler, Glimmer, RAST, BLASTX, HMMER, BLASTN, ACT, MEGA
Databases COGs Pfam KEGG
Applications Phylogenetics, Metagenomic sequencing analysis
Organisms Herbaspirillum seropedicae
Chemicals Nitrogen