Fragmentation-based methods and systems for de novo sequencing
First Claim
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1. A method of obtaining sequence information from a target biomolecule, comprising:
- fragmenting the target biomolecule into a plurality of fragments by partial cleavage;
performing mass spectrometry on the plurality of fragments to produce mass spectra of the fragments;
extracting peak information from the produced mass spectra;
constructing sequencing graphs using the extracted peak information; and
traversing the sequencing graphs to reconstruct the sequence information of the target biomolecule.
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Abstract
Methods and systems, particularly mass spectrometric methods and systems, for the analysis and sequencing of biomolecules, particularly nucleic acids, by fragmentation are provided.
156 Citations
84 Claims
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1. A method of obtaining sequence information from a target biomolecule, comprising:
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fragmenting the target biomolecule into a plurality of fragments by partial cleavage;
performing mass spectrometry on the plurality of fragments to produce mass spectra of the fragments;
extracting peak information from the produced mass spectra;
constructing sequencing graphs using the extracted peak information; and
traversing the sequencing graphs to reconstruct the sequence information of the target biomolecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for producing a candidate sequence of a biomolecule, comprising:
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receiving a plurality of sequencing graphs, each sequencing graph having a plurality of vertices and edges, where each vertex represents a compomer of the biomolecule, and each edge represents a cut base of the sequencing graph; and
generating the candidate sequence by traversing the plurality of sequencing graphs. - View Dependent Claims (18, 19, 20, 21, 22, 23)
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24. A program product for use in a computer that executes program instructions recorded in a computer-readable media to produce a candidate sequence of a biomolecule, the program product comprising:
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a recordable medium; and
a plurality of computer-readable program instructions on the recordable media that are executable by the computer to perform a method comprising;
receiving a plurality of sequencing graphs, each sequencing graph having a plurality of vertices and edges, where each vertex represents a compomer of the biomolecule, and each edge represents a cut base of the sequencing graph; and
generating the candidate sequence by traversing the plurality of sequencing graphs. - View Dependent Claims (25, 26, 27, 28, 29, 30, 83, 84)
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31. A sequencing system for obtaining sequence information from a target biomolecule, comprising:
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a biomolecule workstation configured to process the target biomolecule into a plurality fragments and to produce mass spectra; and
an analysis computer configured to construct sequencing graphs using the mass spectra of the target biomelcule. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)
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44. A method of obtaining sequence information from a target biomolecule, comprising:
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fragmenting the target biomolecule into at least two fragments by partial cleavage at specific cleavage sites;
determining the molecular weights of the at least two fragments;
determining the possible compositions of the at least two fragments;
ordering the possible compositions of the at least two fragments according to the number of specific cleavage sites that are not cleaved in each fragment;
constructing at least one sequencing graph that is a graph theoretical representation of the ordered compositions for the at least two fragments; and
traversing the at least one sequencing graph to reconstruct one or more underlying sequence candidates of the target biomolecule. - View Dependent Claims (45, 46, 47, 48, 49, 50, 51, 52, 53, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71)
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54. A method of obtaining nucleic acid sequence information from a target nucleic acid molecule, comprising:
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subjecting the nucleic acid molecule to partial cleavage reactions with one or more specific cleavage reagents, thereby generating two or more fragments that are specific cleavage products;
determining the molecular weights of the two or more fragments;
determining the possible base compositions of the two or more fragments;
ordering the possible base compositions of the two or more fragments according to the number of specific cleavage sites that are not cleaved in each fragment;
constructing one or more sequencing graphs that are graph theoretical representations of the ordered base compositions for the two or more fragments; and
traversing the one or more sequencing graphs to reconstruct one or more underlying sequence candidates, wherein each sequencing graph corresponds to the ordered base compositions derived from a partial cleavage reaction with one base-specific cleavage reagent. - View Dependent Claims (55, 56, 57, 58, 59)
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72. A program product for use in a computer that executes program instructions recorded in a computer-readable media to obtain sequence information in a target biomolecule, the program product comprising:
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a recordable medium; and
a plurality of computer-readable program instructions on the recordable media that are executable by the computer to perform a method comprising;
a) determining mass signals of target biomolecule fragments produced from partially cleaving a target biomolecule into fragments by contacting the target biomolecule with one or more base-specific cleavage reagents;
b) determining the possible compositions of the at least two fragments;
c) ordering the possible compositions of the at least two fragments according to the number of specific cleavage sites that are not cleaved in each fragment;
d) constructing at least one sequencing graph that is a graph theoretical representation of the ordered compositions for the at least two fragments; and
e) traversing the at least one sequencing graph to reconstruct one or more underlying sequence candidates of the target biomolecule. - View Dependent Claims (73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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Specification