16S rRNA gene (V4 region) amplicon sequencing of the microbiota in ambient sediment and multiple body compartments of the sea cucumber Holothuria leucospilota
This project presents an amplicon sequencing study targeting the bacterial 16S rRNA V4 region to investigate the microbiota associated with the sea cucumber Holothuria leucospilota and its immediate environment. The study systematically compares the microbial community structure across four distinct sample types: (1) ambient marine sediment, (2) the body wall epidermis, (3) the foregut, and (4) the hindgut. The primary objective is to elucidate the compositional differentiation and potential functional niches of the prokaryotic communities inhabiting these different compartments.Samples were homogenized in liquid nitrogen using a sterile mortar and pestle. Approximately 0.5 mL of each homogenate was suspended in 0.8 mL TE buffer for storage prior to DNA extraction. Total genomic DNA was extracted using the E.Z.N.A. Soil DNA Kit (Omega Biotek). The V4 region of the 16S rRNA gene was amplified with the primers 515F (CCTACGGRRBGCASCAGKVRVGAAT) and 806R (GGACTACNVGGGTWTCTAATCC) in 25 uL PCR reactions. Thermal cycling conditions were: initial denaturation at 94C for 3 min; 24 cycles of 94C for 5 s, 57C for 90 s, and 72C for 10 s; followed by a final extension at 72C for 5 min. The resulting amplicon libraries were quantified, normalized, pooled, and sequenced on an Illumina MiSeq platform. Bioinformatics processing included merging paired-end reads, quality filtering, chimera removal, and clustering into amplicon sequence variants (ASVs) at 97% similarity using VSEARCH. Taxonomic assignment was performed against the SILVA 138 database using the RDP classifier.