The Library Construction And Gap Filling In The Whole Genome Sequencing Of Lactobacillus Delbrueckii Ssp.bulgaricus 2038

Lactobacillus delbrueckii ssp.bulgaricus, which inhabits in the fermental food and beverage, is the most commonly Gram-positive bacteria. This bacterium play an important role in the fermentation of the yogurt, Whole genome sequence of this bacterium will provide more extensive information of Lactobacillus delbrueckii ssp.bulgaricus and ultimately would help us improve this bacterium.In our project, we use a "whole-genome-shotgun" method to sequence Lactobacillus delbrueckii ssp.bulgaricus 2038. What an enormous work our project is, nobody can finish it alone. So I just participate in a part of library construction and gap filling. In the sequencing process, two random genomic libraries with small and large insert fragments are necessary. First of all 1.6-4kb fragment are selected and cloned into pUC18/SmaI/BAP vector, then 2-3kb fragment are selected and cloned into Low-copy vector pSMART-LC, and then the fragments range from 6-8kb are selected and cloned into pSMART-LC vector.After other members of our group finishing the large-scale sequencing, assembly by Phrap, there are 120 gaps between every two Contigs, The sequence gap could be filled by direct sequencing with the extending primer on the span clone, whereas physical gap should be first amplified by PCR before sequencing. Finally, one intact circular chromosome sequence which is l,872,907bp are completed, the first nucleotide on the chromosome was located on the first base group beginning sequencing.