Bacterial Diversity Profiling of Salt marsh halophyte Sesuvium portulacastrum - rhizosphere soil employing metagenomic approach

The study aimed to identify the bacterial metagenomic profiling of Salt marsh halophyte Sesuvium Portulacastrum rhizosphere soil sample by targeting the V3-V4 regions. The metagenomic bacterial DNA of Sesuvium Portulacastrum rhizosphere soil was isolated by DNA Power Soil Kit. Isolated DNA was quantified using Nanodrop 2000. DNA quality and integrity was checked by 1% agarose gel electrophoresis. PCR amplification of targeted regions was performed by using specific primers connecting with barcodes. The PCR products with proper size were selected by 2% agarose gel electrophoresis. Same amount of PCR products from each sample was pooled, end-repaired, A-tailed and further ligated with Illumina adapters. Libraries were sequenced on a paired-end Illumina platform to generate 250bp paired-end raw reads. The experimental procedures of DNA library preparation are PCR Amplification i) PCR Amplification, ii) Size selection, iii) End repair & A-tailing, iv) Adapter ligation and v) Purification. Sequenced reads (raw reads) containing low quality reads and primes/adapters, affecting the analysis quality were filtered to get clean reads by removing i) reads whose length less than 60bp after trimming the primers and adapters from end of reads ii) reads containing N > 10% (N represents the base cannot be determined) and iii) reads containing low quality (Q score <= 5) base which is over 50% of the total base. Among the metagenomes, Streptomyces griseocarneus was the most abundant of about 21% of total bacteria.

本研究旨在通过靶向V3-V4区域，解析盐沼盐生植物海马齿（Sesuvium Portulacastrum）根际土壤的细菌宏基因组谱学特征。实验采用DNA Power Soil试剂盒提取该根际土壤的细菌宏基因组DNA，使用Nanodrop 2000对提取的DNA进行定量，并通过1%琼脂糖凝胶电泳（agarose gel electrophoresis）检测DNA的质量与完整性。随后使用带有条形码（barcodes）的特异性引物对目标区域开展PCR扩增，通过2%琼脂糖凝胶电泳筛选出片段大小符合要求的PCR产物。将各样本的PCR产物按等量混合后，进行末端修复、加A尾，并进一步与Illumina接头（Illumina adapters）连接。最终在双端Illumina测序平台（Illumina platform）上对构建好的文库进行测序，生成250bp的双端原始读段。DNA文库制备的实验流程包括：i) PCR扩增，ii) 片段筛选，iii) 末端修复与加A尾，iv) 接头连接，v) 纯化。针对原始测序读段（raw reads）中影响分析质量的低质量读段及引物/接头序列，我们通过以下规则过滤得到洁净读段（clean reads）：1) 修剪读段两端的引物与接头后长度小于60bp的读段；2) 含不确定碱基（N代表无法确定的碱基）比例超过10%的读段；3) 低质量碱基（质量得分Q≤5）占总碱基比例超过50%的读段。本宏基因组样本中，灰肉链霉菌（Streptomyces griseocarneus）为丰度最高的菌种，约占总细菌群落的21%。