Computational protocol: The effects of climate, catchment land use and local factors on the abundance and community structure of sediment ammonia-oxidizing microorganisms in Yangtze lakes

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Protocol publication

[…] The primers Arch-amoAF/Arch-amoAR (Francis et al. ) and amoA-1F/amoA-2R (Rotthauwe et al. ) were used to amplify the AOA and AOB amoA (ammonia monooxygenase subunit A) genes, respectively. The sequences of primers and thermal cycling procedures are shown in Additional file : Table S1. Each reaction was performed in a 25 μL volume consisting of 1 μL of DNA template (10–100 ng/μL), 0.5 μL of each primer (10 mM), 0.2 μL of rTaq polymerase (5U/μL) (TaKaRa, DaLian, China), 0.5 μL of deoxynucleotide triphosphates (10 mM) and 2.5 μL of 10× buffer. The PCR products were inserted into the pMD18-T vector (TaKaRa, DaLian, China) after gel purifying and transformed into Trans-5α competent cells (Transgen Biotech, Beijing, China).Approximately sixty positive clones were screened by PCR and sequenced with an ABI-3730XL (Applied BioSystems, CA, USA) by Sangon Biotech Co., Ltd. After removing sequences of poor-quality or insufficient length by Geneious Pro 8.0.2 software (Biomatters Ltd., Auckland, New Zealand), the remaining sequences were aligned using the MAFFT software (Katoh et al. ). Operational taxonomic units (OTUs) with a more than 95% similarity level were calculated using Mothur software by the furthest neighbour algorithm (Schloss et al. ). Neighbour-joining phylogenetic trees were constructed with sequences from the main OTUs (containing at least two clones) and the most similar sequences retrieved from GenBank by software MEGA version 6. The diversity indices (i.e., Chao 1, Shannon–Weiner and Simpson) were calculated for each sediment sample using the software Mothur version 1.23.0.The archaeal and bacterial amoA sequences obtained in this study have been deposited in GenBank with the following Accession Numbers: KU204801–KU204822, KY244149–KY244154, and KY244243–KY244299 for archaeal amoA and KU168323–KU168339 and KY244155–244242 for bacterial amoA. […]

Pipeline specifications

Software tools Geneious, MAFFT, mothur
Application Phylogenetics
Organisms Hemisus marmoratus
Chemicals Ammonia, Carbon, Chlorophyll