METHOD FOR TARGETED SEQUENCING

摘要

The method of the present invention now provides a technique for generating sequence information from nucleic acid samples based on knowledge from part(s) of the nucleotide sequence. The knowledge of the partial sequence may include knowledge about the presence of restriction sites. The knowledge of the partial sequence can be used to generate adaptor ligated or nucleotide-elongated fragments. From the combination of information on the ligated adaptor and the Known Nucleotide Sequence Section, probes can be designed. The probes can be used in the provision of circularised fragments that can be sequenced. Combining the known and determined sequences adds sequence information to the already existing sequence information and complements the available genomic sequence information.

主权项

1. A method for obtaining sequence information from a nucleic acid sample, the method comprising the steps of:
(a) providing a nucleic acid sample wherein at least part of the nucleotide sequence information for the nucleic acid sample is available in the form of at least one Known Nucleotide Sequence Section; (b) fragmenting the nucleic acid sample to obtain one or more fragments; (c) optionally, blunting the ends of the fragments; (d) optionally, adding one or more 3′ nucleotides to the fragments; (e) ligating one or more adaptors to one or both of the ends of the fragments to obtain adaptor-ligated fragments; (f) providing for at least one circularization probe that comprises at least part of the Known Nucleotide Sequence Section and at least part of the sequence of the adaptor; (g) combining the adaptor-ligated fragments with the circularization probes; (h) denaturing the adaptor-ligated fragments to obtain denatured adaptor-ligated fragments; (i) allowing the circularization probes and the denatured adaptor-ligated fragments to hybridize and form circularized denatured adaptor-ligated fragments; (j) optionally, removing an overhang; (k) optionally, filling in missing nucleotides between the Known Nucleotide Sequence Section and the adaptor; (l) ligating the ends of the circularized adaptor-ligated fragments to obtain ligated circularized adaptor-ligated fragments; and (m) sequencing the ligated circularized adaptor-ligated fragments;
wherein, for each fragment, sequence information of only one single Known Nucleotide Sequence section is required to obtain sequence information of the ligated circularized adaptor-ligated fragment.