Targeted metagenome exploration by solution hybridization-selection capture coupled to next-generation sequencing

The shift from traditional methods to next-generation platforms with high throughput and reduced sequencing costs provided faster acquisition of metagenomic data from various ecosystems. But these next-generation methods still remained difficult to apply to microbial ecology because of the extraordinary diversity of microbial communities within the ecosystems. It would be beneficial, therefore, to reduce the sequence complexity of the sample by focusing on individual genomic subsets of interest. We present an innovative method for targeting large sequences from complex metagenomic DNA, enriched in-solution with biotinylated RNA probes and coupled to next generation sequencing. Initially, the method was evaluated in-solution by targeting the methyl Coenzyme M reductase subunit A gene (mcrA) using a genomic DNA library from the methanogen strain Methanosarcina acetivorans C2A, and metagenomic DNA library from a freshwater lake sample. Our method yielded an enrichment performance of at least 175,365-fold with two cycles of capture, and showed the ability to retrieve large DNA sequence that covering and exploring mcrA flanking regions. For high-throughput mcrA gene survey within the freshwater lake metagenome, we also compared our capture method coupled to 454 pyrosequencing with traditional metagenomic random shotgun and PCR-based sequencing approaches. All methods were effective but highest enrichment performance of 49.3 % and 99.9 % were obtained respectively with our capture method and PCR. In contrast, the capture method allowed the efficient recovering of larger DNA sequence with deeper investigation on minority methanogenic phylotypes related to mcrA gene sequence analysis.

从传统方法向高通量、低测序成本的新一代测序平台的更迭，极大加快了从各类生态系统中获取宏基因组数据的进程。但由于生态系统内微生物群落的极高多样性，这类新一代测序方法仍难以直接应用于微生物生态学研究。因此，通过靶向关注目标基因组亚群以降低样本的序列复杂度，将具有显著应用价值。本研究提出了一种创新方法，可从复杂宏基因组DNA中靶向捕获大片段序列，该方法通过生物素化RNA探针在溶液中完成富集，并结合新一代测序技术开展后续分析。本研究首先以产甲烷菌菌株Methanosarcina acetivorans C2A的基因组DNA文库，以及淡水湖泊样本的宏基因组DNA文库为材料，靶向甲基辅酶M还原酶A亚基基因（methyl Coenzyme M reductase subunit A gene，mcrA），在溶液体系中对该方法进行了评估。经两轮捕获循环后，本方法的富集倍数至少可达175365倍，且能够获取覆盖并解析mcrA侧翼区域的大片段DNA序列。针对淡水湖泊宏基因组的高通量mcrA基因调研，本研究还将结合454焦磷酸测序的捕获方法，与传统宏基因组随机鸟枪法测序以及基于PCR的测序方法进行了对比。所有方法均具备有效性，但本研究的捕获方法与PCR方法分别实现了49.3%与99.9%的最高富集效率。相较之下，捕获方法能够高效获取大片段DNA序列，并可针对与mcrA基因序列分析相关的稀有产甲烷菌系统发育型开展更深入的研究。