ALTERNATIVE NUCLEIC ACID SEQUENCING METHODS
Embodiments are provided that provide for parallel sequencing of nucleic acid segments. In some embodiments, a single sequence is sequenced by at least two different sequencing techniques and the results compared, allowing for deficiencies or strengths of one technique to be complemented by the second technique.
1 . A method for sequencing a polynucleotide, said method comprising:
determining a sequence of a first region of a polynucleotide using a first set of nucleic acid sequencing reagents; and
determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different from the second set of nucleic acid sequencing reagents.
2 . The method of claim 1 , wherein the first region and the second region overlap.
3 . The method of claim 1 , wherein the first region and the second region are on different strands of the polynucleotide.
4 . The method of claim 3 , wherein the first region and the second region overlap by at least 5 nucleotides.
5 . The method of claim 4 , wherein the first and second region overlap by at least 10 nucleotides.
6 . The method of claim 5 , wherein the first region can be adjacent to the second region.
7 . The method of claim 1 , wherein the polynucleotide is immobilized on a solid support.
8 . The method of claim 7 , wherein the solid support can be a bead.
9 . The method of claim 1 , wherein the polynucleotide can be a single molecule.
10 . The method of claim 1 , wherein the polynucleotide can be an amplified clone.
11 . The method of claim 10 , wherein the amplified clone is produced by PCR.
12 . The method of claim 11 , wherein the PCR is emulsion PCR.
13 . The method of claim 10 , wherein the amplification takes place in a semisolid support.
14 . The method of claim 10 , wherein the amplification takes place on a solid support.
15 . The method of claim 10 , wherein the amplified clone is present on an array.
16 . The method of claim 1 , wherein the first sequencing chemistry or the second sequencing chemistry can be a sequencing by ligation chemistry.
17 . The method of claim 1 , wherein the first sequencing chemistry or the second sequencing chemistry can be a reversible terminator chemistry.
18 . The method of claim 1 , wherein the first sequencing chemistry or the second sequencing chemistry can be a pyrosequencing chemistry
19 . The method of claim 16 , wherein the first sequencing chemistry and the second sequencing chemistry can be are sequencing by ligation chemistry.
20 . The method of claim 17 , wherein the first sequencing chemistry and the second sequencing chemistry are reversible terminator chemistry.
21 . A method of sequencing a polynucleotide of interest, said method comprising:
applying a first nucleic acid sequencing chemistry to a clonal library derived from a polynucleotide of interest;
determining a sequence of a first region of the polynucleotide using a first set of nucleic acid sequencing reagents; and
determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different than the second set of nucleic acid sequencing reagents.
22 . The method of claim 21 , wherein the clonal library comprised the genome of an organism of interest.
23 . The method of claim 22 , wherein the organism of interest is prokaryotic.
24 . The method of claims 22 , wherein the organism of interest is eukaryotic.
25 . The method of claim 22 , wherein the clonal library comprises clones derived from amplicons derived from a genome of interest.
26 . The method of claim 21 , wherein the clonal library is derived from a nucleic acid library
27 . A method of sequencing a polynucleotide, said method comprising:
determining a sequence of a first region of the polynucleotide using a first set of nucleic acid sequencing reagents, whereby a first nucleic acid sequence is produced;
determining a sequence of a second region of the polynucleotide using a second set of nucleic acid sequencing reagents, wherein the first set of nucleic acid sequencing reagents are different than the second set of nucleic acid sequencing reagents, whereby a second nucleic acid sequence is produced, wherein the first region and the second region have at least 1 nucleotide base position in common; and
comparing the first nucleic acid sequence and the second nucleic acid.
28 . A system for determining a base sequence of a polynucleotide of interest, the system comprising:
an array of polynucleotides for analysis;
a flow cell containing the array and having at least one input port;
a reservoir set comprising a first set of nucleic acid sequencing reagents, wherein the reservoir set is connection with an input port;
a reservoir set comprising a second set of nucleic acid sequencing reagents, wherein the reservoir set is in fluid connection with an input port;
a first optical signal collector configured to detect optical signal generated by reactions between the first set of nucleic acid sequencing reagents and the polynucleotides for analysis; and
a second optical signal collector configured to detect optical signal generated by reactions between the second set of nucleic acid sequencing reagents and the polynucleotides for analysis.
27 . The system of claim 26 , wherein the first optical signal collector and the second optical signal collector are the same component.
28 . The system of claim 26 , wherein at least one of the optical signal collectors comprises a CCD.
29 . The system of claim 26 , wherein the system further comprises a laser configured to induce excitation of fluorescent signal present on the array of polynucleotides.
30 . A method for sequencing a polynucleotide, said method comprising:
fragmenting a polynucleotide for analysis into a plurality of polynucleotide fragments;
clonally amplifying at least a part of the polynucleotide fragments, whereby a set of fragment clones is produced;
sequencing a first portion of the set of fragment clones, with a first set of nucleic acid sequencing reagents, whereby a first nucleotide base sequence assembly is produced;
producing error values for at least some of the bases in the second nucleotide base sequence assembly;
sequencing a second portion of the set of fragment clones, with a second set of nucleic acid sequencing reagents, whereby a second nucleotide base sequence assembly is produced;
producing error values for at least some of the bases in the second nucleotide base sequence assembly;
comparing the first nucleotide base sequence assembly with the second nucleotide base sequence assembly; and
selecting at least one base identity between the first and second base sequence assemblies based upon a lower error value for the base identity in the corresponding nucleotide base sequence assembly compared to the base identity of the base in the other base sequence assembly.