Provided is a method of gap closing in nucleotide sequence. The nucleic acid sequence comprises a first contig at one end of a gap in an unassembled region, and a second contig at the other end of the gap in the unassembled region. The method comprises: selecting reads having an overlap with one end of the first contig close to the gap as a set of reads for gap closing; selecting reads having a shortest overlap with the first contig in the set of reads for gap closing as a candidate read; determining whether reads having an overlapping length with the first contig shorter than an overlapping length between the candidate read and the first contig present in the set of reads for gap closing, and determining whether reads having no overlapping relationship with the candidate read present in the set of reads for gap closing; obtaining a result of presenting an extension conflict, and determining an unconfident candidate read, if reads having an overlapping length with the first contig shorter than an overlapping length between the candidate read and the first contig present in the set of reads for gap closing, reads having no overlapping relationship with the candidate read present in the set of reads for gap closing, or both reads having an overlapping length with the first contig shorter than an overlapping length between the candidate read and the first contig, and reads having no overlapping relationship with the candidate read present in the set of reads for gap closing; reselecting the candidate read until obtaining a confident candidate read, if the candidate read is unconfident; connecting the confident candidate read to the first contig, to form a new first contig; determining whether one end of the new first contig close to the gap has an overlap with one end of the second contig close to the gap; performing the step of selecting the set of reads for gap closing on the basis of the new first contig, if the one end of the new first contig close to the gap has no overlap with the one end of the second contig close to the gap, wherein the first contig in the step of selecting the set of reads for gap closing is replaced with the new first contig; connecting the new first contig to the second contig to complete gap closing, if one end of the new first contig close to the gap has an overlap with one end of the second contig close to the gap.