Computational protocol: Soil Microbial Functional and Fungal Diversity as Influenced by Municipal Sewage Sludge Accumulation

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[…] The most dominant fungi that were isolated from the sludge were identified using a molecular method. The method is based on rDNA-extraction and the subsequent amplification of the D2 large subunit region of the fungal rDNA [] by using the Fast MicroSeq D2 LSU rDNA fungal sequencing kit (Applied Biosystems, Foster City, CA, USA). The fungal strains were grown on PDA medium (Biocorp). Preparation of the fungal DNA was performed with the PrepMan Ultra reagent (Applied Biosystems). A very small amount of one fungal colony was collected using tweezers and suspended in 200 µL of the PrepMan Ultra reagent. After incubation for 10 min at 100 °C, the suspension was mixed and centrifuged for 10 s allow the cell debris to settle. The supernatant solution was transferred to a new microcentrifuge tube and diluted at a ratio of 1:100 in deionized nuclease-free water []. A fast MicroSeq D2 LSU rDNA fungal PCR kit (Applied Biosystems) was used to amplify the D2 LSU rDNA region. The PCR was performed using the conditions listed in .The ExoSAP-IT® PCR Products Purification Kit for ABI (Affymetrix Inc., Santa Clara, CA, USA) was used to clean the PCR products before sequencing to eliminate any remaining labelled ddNTPs and for desalting. Next, 5 µL of the PCR product was mixed with 2 µL of the ExoSap reagent and incubated in a thermal cycling profile as follows: 37 °C for 15 min and 80 °C for an additional 15 min. The next step after PCR purification was to prepare the cycle sequencing. For this purpose, 13 µL of the forward/reverse mix from the MicroSeq D2 Fungal ID Sequencing Kit was used with 7 µL of the purified product. The cycling program is listed as follows: 96 °C for 1 min; (96 °C for 10 s; 50 °C for 5 s; 60 °C for 1 min 15 s) × 25. Next, the Performa Purification System (EdgeBio-Performa Gel Filtration Cartridges) was used and 20 µL of the reaction samples eluate was mixed with 20 µL of deionized formamide. Electrophoresis was run through an ABI 3130 ×l capillary sequencer (Applied Biosystems) with a 50 cm capillary array and polymer POP6_1. The MicroSEQ® ID software was used to assess the raw sequence files and to perform sequence matching to the MicroSEQ® ID (Applied Biosystems) to validated the reference database and to create Neighbor-Joining trees. [...] The average well-color development, richness and the Shannon-Weaver index were statistically analyzed using an analysis of variance (ANOVA). The significance differences were determined using the Tukey test at p = 0.05. A principle component analysis was conducted on the optical density (OD) data from the 31 carbon sources following 48 h of incubation. Cluster analysis, including grouping of the treatments and features, was performed on the standardized data from the average absorbance values at 120 h. A dendrogram was prepared to present the similarity of the carbon utilization patterns of the substrates that were located on the Biolog EcoPlateTM between the soil samples with scaled bond distances on the axis (Ward's method) and boundaries marked according to Sneath`s criteria (33% and 66%). The data were standardized according to AWCD in each microplate to remove the inoculum density effects []. All statistical analyses were performed using the Statistica 10.0 software (StatSoft Inc., Tulsa, OK, USA). […]

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