Computational protocol: amoA-encoding archaea and thaumarchaeol in the lakes on the northeastern Qinghai-Tibetan Plateau, China

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Protocol publication

[…] Five lakes (Erhai Lake, Gahai Lake 1, Gahai Lake 2, Xiaochaidan Lake, and Lake Chaka) were selected for the AEA amoA gene diversity analysis. Two primer sets of Arch-amoAF/Arch-amoAR () and CrenamoA23f (5′-ATGGTCTGGCTWAGACG-3′)/CrenamoA616r (5′-GCCATCCATCTGTATGTCCA-3′; ) were used to PCR-amplify the archaeal amoA genes from the extracted DNA and synthesized cDNA. Each PCR mixture (25 μL reaction volume) contained the following ingredients: 2.5 μL 10× buffer (Takara), 2 μL deoxynucleoside triphosphate (dNTP; a 2.5 mM dNTP; Takara), 16.2 μL sterilized ultra pure water (Millipore), 1 μL bovine serum albumin (BSA; Takara), 1 μL each primer (10 pmole), and 0.3 μL rTaq DNA polymerase (Takara). PCR conditions were same as those described previously (; ). The resulting PCR products (635 and 629 bp from the primer sets of Arch-amoAF/Arch-amoAR and CrenamoA23f/CrenamoA616r, respectively) were examined on 1% agarose gel and no bands were observed for PCR negative controls (with distilled water as the template). PCRs failed for the DNA samples from Lake Chaka (with 325 g L-1 salinity). Transcription of archaeal amoA gene was shown to occur in Erhai Lake and Gahai Lake 1 in our previous study (). Therefore cDNA synthesis was performed only on the samples from Gahai Lake 2 and Xiaochaidan Lake. However, Xiaochaidan Lake cDNA sample was only successfully amplified with primer Arch-amoAF/Arch-amoAR. The appropriate bands were excised and PCR gels were purified with Agarose Gel DNA purification Kit (Takara). The purified PCR products were ligated into the pGEM-T vector (Promega Inc.) and transformed into Escherichia coli JM109 competent cells (Takara) according to the manufacturer’s instructions. The transformed cells were spread on Luria–Bertani plates containing 100 μg mL-1 of ampicillinsodium, 80 μg mL-1 of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyrano-side), and 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and cultivated overnight at 37°C.Sixteen (eight for each primer set) DNA clone libraries were constructed from the following samples: EHL-1-W, GHL1-32-W, GHL2-84-W, XCDL-160-W, EHL-1-S, GHL1-32-S, GHL2-84-S, and XCDL-160-S. One cDNA clone library (XCDL-160-SR) was constructed for the sediment from Xiaochaidan Lake. Around 30–40 randomly selected clones per sample were analyzed for the insert amoA gene sequences. Positive clones were sequenced using M13F with the BigDye Terminator version 3.1 chemistry (Applied Biosystems, Foster City, CA, USA) on an ABI 3730 automated sequencer.The obtained raw nucleotide sequences were checked and trimmed manually by using the BioEdit program. The sequences of poor quality were removed from further analysis. The operational taxonomic units (OTUs) of the amoA gene clone sequences were determined based on a cutoff value of 98% by using nearest neighbor algorithm in the DOTUR program (). The saturation of the sampled clones from each amoA gene clone library was assessed by calculating the coverage (C) values as follows: C = 1 - (n1/N), where n1 is the number of OTUs that occurred only once in the clone library and N is the total number of analyzed clones (). One representative sequence was selected from each OTU for phylogenetic analysis. Closest references of the amoA gene were retrieved from the GenBank using BLAST. Maximum likelihood trees were constructed from the amoA gene sequences obtained in this study and their references () with the use of the MEGA program version 5.0 (), and were assessed using 1000 bootstrap replications. The nucleotide sequences obtained in this study were deposited in the GenBank database under accession numbers JX488399–JX488453 and KF606897–KF606927. […]

Pipeline specifications

Software tools BioEdit, DOTUR, MEGA
Applications Phylogenetics, 16S rRNA-seq analysis
Chemicals Ammonia