Soil Microbiome Analysis via 16S rRNA Sequencing

Total genomic DNA was extracted from 0.5 g of each soil sample using the MP FastDNA^® SPIN Kit for soil (MP Biochemicals, Solon, OH, USA) following the manufacturer’s instructions. The V4 and V5 hypervariable regions of the bacterial 16S rRNA gene were PCR amplified using the primer pair 515F and 907R^{7 (link)}. The purified PCR amplicons were sequenced using an Illumina Miseq (300-bp paired-end reads) platform (Illumina Inc., San Diego, CA, USA). The acquired sequences were filtered by quality according to Caporaso, et al.^{51 (link)}. Chimeric sequences were removed using the UCHIME algorithm^{52 (link)} in the USEARCH package v. 6.1544^{53 (link)}. Sequences were grouped by taxonomy and assigned to OTUs at the 97% sequence identity level using the UPARSE package (http://drive5.com/uparse/)^{54 (link)}. After singleton removal, representative sequences were taxonomically assigned using the Ribosomal Database Project naïve Bayesian rRNA classifier within the SILVA database (release 128) at an 80% confidence threshold^{51 (link)}. All sequences were deposited in the NCBI Sequence Read Archive database (Biosample number: SAMN06105854-SAMN06105874).

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Tan W., Wang J., Bai W., Qi J, & Chen W. (2020). Soil bacterial diversity correlates with precipitation and soil pH in long-term maize cropping systems. Scientific Reports, 10, 6012.

Publication 2020

MP FastDNA® SPIN Kit for soil Miseq (300-bp paired-end reads) platform

16s rrna Bacterial gene Chimeric Genomic Primer Ribosomal Rrna Solon

Corresponding Organization :

Other organizations : Northwest A&F University

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Variable analysis

independent variables

Soil sample

dependent variables

Bacterial 16S rRNA gene sequences

control variables

MP FastDNA® SPIN Kit for soil used for DNA extraction
Primer pair 515F and 907R used for PCR amplification
Illumina Miseq (300-bp paired-end reads) platform used for sequencing
Caporaso, et al. method used for sequence quality filtering
UCHIME algorithm used for chimeric sequence removal
UPARSE package used for OTU clustering and taxonomy assignment
SILVA database (release 128) used for taxonomic assignment

controls

No positive or negative controls were explicitly mentioned in the input.

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