Method of whole-genome sequencing

A whole-genome sequencing method based on clone DNA mixing pool, the method comprising:

• 1) extracting a whole-genome DNA from cells of a species, using a restriction endonuclease to digest the whole-genome DNA, constructing a bacterial artificial chromosome (BAC) library, and preserving the BAC library in a plurality of multi-well plates;
• 2) constructing BAC clone mixing pools;
• 3) extracting a plurality of DNA clones from the BAC clone mixing pools, inoculating and culturing the plurality of DNA clones in culture plates, mixing the plurality of DNA clones to obtain sequencing samples according to the BAC clone mixing pools of 2), and extracting plasmids from the sequencing samples;
• 4) performing pair-end sequencing for the plasmids of 3) by next-generation sequencing (NGS) sequences to obtain sequences of each of the mixing pools;
• 5) scanning sequences of each of the mixing pools to obtain a feature sequence set and a k-mer set of each mixing pool;
• 6) inputting the feature sequence set and the k-mer set of each mixing pool of 5) into a computer to analyze a corresponding feature sequence set and a corresponding k-mer set of each clone according to the feature sequence set and the k-mer set of each mixing pool;
• 7) activating the computer to construct clone contigs according to feature sequence sets of clones;
• 8) activating the computer to divide the k-mer sets of the clones into small k-mer sets by the clone contigs and to position the small k-mer sets onto the clone contigs;
• 9) activating the computer to assemble the NGS sequences in the mixing pool of 3) to yield sequence contigs;
• 10) activating the computer to position the sequence contigs onto the clone contigs, to use sequenced paired-end information to connect the sequence contigs, and to determine directions thereof, to obtain sequences of the clone contigs;
• and
• 11) using Southern blot hybridization or PCR to determine positions and directions of molecular markers on the plurality of DNA clones, inputting the positions and the directions of the molecular markers into the computer to determine relative positions and directions of the clone contigs and to connect the sequences of clone contigs as whole stripe of chromosome sequences to obtain whole-genome sequences;
• wherein
• algorithms for analyzing the feature sequence sets and the k-mer sets of the clones are as follows:
• a total number of clones of the BAC library of a certain species is defined as a, and a total number of the constructed mixing pools is defined x, then:
• the k-mer set of the mixing pool at a κ-th dimension and having an index of λ is expressed as:
• P(κ,λ);
• the k-mer sets of the mixing pools comprising a given clone are expressed as:
• {P(δ)|δ<m, P(δ)∈P};
• an intersection set of k-mer sets of the mixing pools comprising the given clone, namely an initial k-mer set of the given clone, is expressed as:
• Cτ=∩δ=1xP(δ);
• an excluded union k-mer set (EUKS) of the given clone in the mixing pools is expressed as:
• CU(κ,τ)=∪ν=0aCν (ν ≠τ, 0<ν<a);
• an intersection set of all excluded union k-mer sets (EUKS) of the given clone is expressed as:
• CIτ=∩κ=0xCU(κ,τ);
• and
• a final k-mer set (FKS) of the given clone is:
• CF=Cτ−CIτ.