A novel retrieval system for nearly complete microbial genomic fragments from soil samples.
The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1-1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of alpha-Proteobacteria and Actinobacteria, which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.
Yu
Wen-Han
WH
Graduate Institute of Agricultural Chemistry, National Taiwan University, No. 1 Section 4 Roosevelt Road, Taipei, Taiwan.
Su
Shiun-Cheng
SC
Lee
Chia-Yin
CY
D001419:Bacteria / Q000145:classification / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D020459:Genes, rRNA; D005821:Genetic Techniques; D016680:Genome, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S