High-throughput Sequencing of Picea crassifolia: Rhizosphere Soil Microbiome from Helan Mountain, Inner Mongolia
Design Description:This study aims to analyze the rhizosphere soil microbiome of different plant species in the Helan Mountain National Nature Reserve, Inner Mongolia, China. The soil samples were collected from the rhizosphere of Pinus tabuliformis, Picea crassifolia, and Betula platyphylla. High-throughput sequencing was performed to investigate both bacterial and fungal communities.    Sample Collection:        Samples were collected from the rhizosphere soil of three plant species (Pinus tabuliformis, Picea crassifolia, and Betula platyphylla) in the Helan Mountain National Nature Reserve.        The soil is well-drained and fertile, located at coordinates 10525'-10528'E, 3836'~3840'N.    Total DNA Extraction:        The total DNA of the microbiome was extracted from various sources using the most appropriate method based on past project experience.        DNA quality was checked by 0.8% agarose gel electrophoresis and quantified using a UV spectrophotometer.    Target Fragment PCR Amplification:        Primers were designed targeting conserved regions of microbial ribosomal RNA to amplify rRNA gene variable regions or specific gene fragments.        PCR amplification was performed using NEB Q5 High-Fidelity DNA Polymerase with controlled cycle numbers to ensure consistency.        Specific Barcode sequences were added to identify samples.    Amplification Product Recovery and Purification:        PCR products were checked by 2% agarose gel electrophoresis, and target fragments were gel-purified using the AXYGEN gel recovery kit.    Fluorescent Quantification of Amplification Products:        Fluorescent quantification was performed using the Quant-iT PicoGreen dsDNA Assay Kit and the BioTek FLx800 Microplate Reader.        PCR products were mixed according to the sequencing volume required for each sample.    Library Preparation:        Libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit.        End repair, A-tailing, adapter ligation, and purification steps were performed.        Libraries were purified using BECKMAN AMPure XP Beads and checked by 2% agarose gel electrophoresis.    High-throughput Sequencing:        Libraries were quality checked on the Agilent Bioanalyzer using the Agilent High Sensitivity DNA Kit, and quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor system.        Libraries were sequenced on the Illumina MiSeq platform with 2300 bp paired-end reads using the MiSeq Reagent Kit V3 (600 cycles).        Target fragment length for sequencing was optimized between 200-450 bp.    Analysis Workflow:        Initial quality filtering of raw sequencing data was performed based on sequence quality.        Sequences were assigned to corresponding samples based on primer and Barcode information, and chimeric sequences were removed.        Operational Taxonomic Units (OTUs) were clustered, and representative sequences were used for taxonomic classification and phylogenetic analysis.        Diversity levels were evaluated based on OTU abundance distributions, with rarefaction curves indicating sequencing depth sufficiency.        Compositional analysis of samples/groups at different taxonomic levels, including statistical testing for differences between groups.        Multivariate statistical analysis tools were used to assess microbial community structure differences and species associated with these differences.        Interaction networks were constructed based on species composition in samples.        Predictive functional profiling of microbial communities was conducted based on 16S rRNA gene sequencing results.