Assessment of Molecular Diversity of Soil Bacteria in Rice fields at Itokin, Lagos State.

This study aims to identify the composition of soil bacteria in rice farms at ItokinSampling LocationsThe soil samples were collected at different locations on the rice fields at Itokin in Epe, Lagos State, the stations were marked and Coordinates taken with the aid of a Global Geo-positioning System (GPS)Location 1:Location A: 133SE 6.39'55" N 3.48'16" ELocation B: 37NE 6.39'55" N 3.48'16" ELocation C: 280SW 6.39'55" N 3.48'16" ELocation 2:Location A: 47NE 6.39'55" N 3.48'17" ELocation B: 123SE 6.40'57" N 3.47'50" ELocation C: 290W 6.39'67" N 3.48'18" ELocation 3:Location A: 355N 6.40'29" N 3.49'6" ELocation B: 62SW 6.40'29" N 3.49'6" ELocation C: 140SE 6.40'29" N 3.49'6" ELocation 4:Location A: 236SW 6.39'56" N 3.48'17" ELocation B: 126SE 6.39'56" N 3.48'17" ELocation C: 58NE 6.39'56" N 3.48'17" ELocation 6:Location A: 61SW 6.40'55" N 3.49'17" ELocation B: 45NE 6.39'55" N 3.48'17" ELocation C: 117SE 6.39'56" N 3.48'17" ESample CollectionSoil samples were collected at different locations on the rice fields at Itokin in Epe, Lagos State, during the rice-growing season. Soil samples were randomly collected from a depth of 5 cm with the aid of shovel. Soil samples for bacterial community analysis were stored in sterile ziplock bags and were immediately placed on ice and frozen (-20C) before DNA extractions.Genomic DNA Extraction and Sequencing ServiceThe total bacterial DNA was extracted from the soil samples using Omega Soil DNA kit. Genomic DNA was subjected to polymerase chain reaction (PCR) amplification using the universal bacterial 16S rRNA gene primer pair, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). These primers target the V1-V9 hypervariable regions of the bacterial 16S rRNA gene, enabling near full-length 16S profiling. To facilitate downstream multiplexing and ensure compatibility with PacBio sequencing, the primers were custom-tagged at both 5'- and 3'-ends with PacBio Kinnex adaptors and unique sample-specific index sequences, following the PacBio-recommended barcoding strategy.Amplicon Pooling and ConcatenationFollowing PCR amplification, all tagged amplicons were quantified, normalized to equimolar concentrations, and pooled. The pooled amplicons were then concatenated to generate long DNA fragments (~18 kb in size), in accordance with PacBio's Kinnex concatenation protocol for 16S amplicons. This step increases sequencing throughput by allowing multiple amplicons to be sequenced per Zero-Mode Waveguide (ZMW)Library Preparation and SMRTbell Adapter LigationThe concatenated amplicons were purified using the AMPure PB magnetic bead-based purification method, which selectively binds DNA fragments and removes unwanted by-products such as primers and nucleotides. The purified DNA was then processed to construct a SMRTbell library using the PacBio Kinnex Library Preparation Kit, which involves ligation of hairpin SMRTbell adapters to the concatenated DNA. This step was carried out strictly following the manufacturer's protocol to ensure compatibility with the Revio sequencing platformSequencing Setup and ExecutionThe SMRTbell library was subjected to primer annealing and polymerase binding using protocols executed via SMRT Link software, which provides optimized conditions for sequencing on the PacBio Revio system. The final library was then loaded onto the PacBio Revio System, a high-throughput long-read sequencing platform capable of delivering accurate and high-resolution 16S rRNA sequence dataReferencesBaer M, Hoppe L, Seel W, Lipski A. Impact of DNA extraction, PCR amplification, sequencing, and bioinformatic analysis on food-associated mock communities using PacBio long-read amplicon sequencing. BMC Microbiol. 2024 Dec 6;24(1):521. doi: 10.1186/s12866-024-03677-8. PMID: 39643893; PMCID: PMC11622462Pac BioScience (2025) https://www.pacb.com/wp-content/uploads/Technical-note-Concatenating-amplicons-using-Kinnex-kits.pdf