Paired-end sequencing method

A paired-end sequencing method for sequencing a target genome comprising a string of oligonucleotides, the method comprising:

• preparing a template comprising a plurality of wells, each well containing a pair of strands from the target genome, the pair of strands comprising a forward strand and a corresponding reverse strand with an insert size distance from each other along the target genome, the insert size distance being in a range between 100 nucleotide bases and 1000 nucleotide bases;
• generating a reads pairs set comprising a plurality of reads pairs by simultaneously sequencing the pair of strands within each well, each reads pair comprising a read associated with the forward strand of the well and a corresponding read-pair associated with the reverse strand of the well, comprising detecting fluorescence intensity of hybridized nucleotide bases along the forward strand and the corresponding reverse strand at the same time in each well;
• and
• processing the reads pairs set, comprising:
• generating a sequences pairs set comprising a plurality of sequences pair, each sequences pair comprising a read sequence and a read-pair sequence corresponding to a reads pair of the reads pairs set, comprising:
• classifying each pair of nucleotide bases of each reads pair within one of three classes, the three classes comprising:
• a unique class U comprising a set of pairs of nucleotide bases with a unique possibility for each nucleotide base;
• a double class D comprising a set of pairs of nucleotide bases with two possibilities for each nucleotide base;
• and
• a triple class T comprising a set of pairs of nucleotide bases with three possibilities for each nucleotide base;
• and
• detecting each pair of nucleotide bases of the read and the corresponding read-pair of each reads pair within one of the three classes based on the detected fluorescence intensities;
• generating a mapped sequences pairs set by mapping the sequences pairs set along a reference genome, comprising:
• searching for each sequences pair of the sequences pairs set along the reference genome by considering nucleotide bases of the sequences pair classified in the unique class U;
• locating the read sequence and read-pair sequence of the searched sequences pair along the reference genome by retaining nucleotide bases of the searched sequences pair classified in the unique class U and positioning an ambiguous character at nucleotide bases of the searched sequences pair classified in at least one of the double class D or triple class T;
• aligning the located sequences pair along the reference genome responsive to the read sequence and the read-pair sequence being associated with a distance equal to the pre-defined insert size distance by matching nucleotide bases of the located sequences pair to corresponding nucleotide bases of the reference genome;
• and
• accepting the aligned sequences pair along the reference genome responsive to nucleotide bases of the aligned sequences pair classified in one of the double class D or the triple class T being consistent with corresponding nucleotide bases of the reference genome;
• and
• obtaining a sequence of the target genome by iteratively re-sequencing the mapped sequences pairs set, comprising determining not accepted nucleotide bases of each aligned sequences pair of the aligned sequences pair set based on nucleotide bases of other aligned sequences pairs of the aligned sequences pair set.