Multiple tagging of long dna fragments
AssigneeComplete Genomics, Inc.
InventorsRadoje Drmanac, Brock A. Peters, Andrei Alexeev
Life of this patent
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Abstract
The present invention provides methods and compositions for tagging long fragments of a target nucleic acid for sequencing and analyzing the resulting sequence information in order to reduce errors and perform haplotype phasing, for example.
Claims28
A method for sequence analysis of a target nucleic acid comprising:
- (a) combining a plurality of long DNA fragments of the target nucleic acid with a population of tag-containing sequences, wherein the population comprises at least 1000 different tag sequences;
- (b) producing tagged long fragments, wherein each tagged long fragment comprises target nucleic acid sequence and multiple interspersed tag sequences, wherein the multiple interspersed tag sequences in an individual tagged long fragment may be the same or different;
- (c) producing from each tagged long fragment a plurality of tagged subfragments, wherein the tagged subfragments each comprise one or more tag sequences;
- (d) obtaining sequence of individual tagged subfragments, wherein the obtained sequence includes target nucleic acid sequence and at least one tag sequence;
- (e) combining sequences obtained in (d) to produce assembled sequence(s) of the target nucleic acid, wherein the combining comprises (i) determining that sequences obtained in (d) originated from the same long DNA fragment if said sequences comprise the same tag sequence and/or (ii) identifying pairs of sequences as being adjacent sequences in the target nucleic acid if the pair comprise the same tag sequence.
3-22. (canceled)
25-27. (canceled)
32-44. (canceled)
46-51. (canceled)
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60-70. (canceled)
A method of sequencing a target nucleic acid comprising:
- (a) combining in a single reaction vessel (i) a plurality of long fragments of the target nucleic acid, and (ii) a population of polynucleotides, wherein each polynucleotide comprises a tag and a majority of the polynucleotides comprise a different tag;
- (b) introducing into a majority of the long fragments tag-containing sequences from said population of polynucleotides to produced tagged long fragments, wherein each of the tagged long fragments comprises a plurality of the tag-containing sequences at a selected average spacing, and each tag-containing sequence comprises a tag;
- (c) producing a plurality of subfragments from each tagged long fragment, wherein each subfragment comprises one or more tags;
- (d) sequencing the subfragments to produce a plurality of sequence reads;
- (e) assign a majority of the sequence read to corresponding long fragments;
- and
- (f) assembling the sequence reads to produce an assembled sequence of the target nucleic acid.
72-76. (canceled)
78-89. (canceled)
A kit comprising a library comprising 103 or more distinct bar codes or sources of clonal bar codes:
- i) a library of barcodes associated with transposon ends, and optionally adaptor sequences;
- ii) a library of clonal barcodes, optionally with adaptor sequences, comprising a plurality of 104 or more distinct sources of clonal bar codes;
- iii) a library of concatemers comprising monomers, wherein the monomers comprise bar codes;
- iv) a library of templates suitable for rolling circle amplification, wherein the templates comprise a monomer as described in (iii);
- and/or
- v) a library of hairpin oligonucleotides, each oligonucleotide comprising two copies of a barcode sequence, wherein the library comprises a plurality of at least about 104 barcodes.
91-95. (canceled)
Dependencies are inferred from the claim text.