Metagenomics Unbiased Shuttle BAC Libraries
Construction of Unbiased Random Shear shuttle BAC libraries of bacteria, fungi, and metagenomes with average insert 100kb or larger
We have successfully constructed unbiased Random Shear shuttle BAC libraries from bacteria, fungi, soils and other metagenome samples with average insert size at least 100~150kb (Figure 1). To date, besides providing more than 200 unbiased Random Shear BAC libraries with large inserts for researchers around the world, arrayed a total of more than 2,500,000 clones from human, animals, plants, and other species, our group has also constructed at least 56 unbiased Random Shear BAC libraries of bacteria, fungi, algae, soil and other environmental metagenome samples with average insert sizes of at least 100kb (Table 1).
Figure 1 Genomic DNA was isolated from bacteria, fungi or environmental samples, randomly sheared, size-selected to >100 kb, and cloned into either shuttle vector: pIG-BAC4 or pIG-BAC5. DNA from minipreps was digested with NotI to excise inserts. The vector band is visible at about 15kb, and average insert size of this Random Shear shuttle BAC library of soil metagenomes was estimated at 120kb (top panel) and 125kb (bottom panel).
Due to the advancement of next-gen sequencing (NGS) technology, the enormous numbers of reads generated by NGS enabled the sequencing of entire genomes of any species: human individuals, animals, plants, and microorganisms at an unprecedented speed (Van Dijk et al. Trends in Genetics 2014). However, very little of the daunting amount of next-gen sequence data has been translated into functional studies. On the other hand, synthetic biology is at its infancy and complete synthesis of a large DNA with 100 kb is too expensive for routine functional studies. Novel large DNA technology, such as unbiased bacterial artificial chromosomes (unbiased BACs), could bridge this gap to translate sequence information into genome biology and functional genomics, as well as metagenomics. Since traditional BAC libraries, which are built by using restriction partial digestion, are inherently biased, they will inevitably cause missing information and genomic gaps in sequences. Unbiased BAC technology is crucial to bridge these gaps, completely cover target genomic regions of interest, obtain the highest quality of genome sequencing assembly, finish genomes, and for functional applications. We have successfully developed and continuously optimize the novel technologies for unbiased large DNA isolation, purification, manipulation, and Random Shear BAC cloning. Intact Genomics is the worldwide sole provider of high-quality, unbiased Random Shear BAC libraries. We have also demonstrated broad applications of unbiased BAC libraries in genome studies and discoveries in animals, plants, human, as well as bacteria, fungi, metagenomes, and other species.
A single and unbiased Random Shear BAC library provides the optimal resource for efficient genomic studies and discovery applications. We have provided hundreds of unbiased Random Shear BAC library related projects (Tree of Life) worldwide since 2008.