Alternative nucleic acid sequencing methods

• US 9,404,155 B2
• Filed: 04/11/2013
• Issued: 08/02/2016
• Est. Priority Date: 08/29/2007
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Bortner, Scott
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US13/860,769
• Publication Number:
  US 20130303381A1
• Time in Patent Office: 1,209 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C40B 20/08 Direct analysis of the library members per se by physical methods, e.g. spectroscopy
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

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.

Claims

1. A method for sequencing a polynucleotide in a preparation by a sequencing instrument, wherein the sequencing instrument comprises a processor, said method comprising:

obtaining a polynucleotide preparation comprising the polynucleotide, comprising amplifying the polynucleotide in a semisolid support,

determining, by the sequencing instrument, a first apparent sequence of a first region of a polynucleotide by subjecting a sample of the polynucleotide preparation to a first type of sequencing technique;

simultaneously determining, by the sequencing instrument, a second apparent sequence of a second region of the polynucleotide that overlaps with the first region, by subjecting a sample of the same polynucleotide preparation to a second different type of sequencing technique,

wherein the first and second sequencing techniques are selected from the group consisting of Maxam-Gilbert sequencing, chain termination methods, dye termination methods, sequencing using reversible terminators, sequencing of nucleic acid by pyrophosphate detection, sequencing by ligation, closed complex single molecule sequencing, nanoscale fluidic sequencing, and force spectroscopy platform sequencing;

identifying, by the processor, any discrepancies between the first apparent sequence and the second apparent sequence; and

selecting, by the processor, the least error prone sequence from the first apparent sequence and the second apparent sequence based upon the likelihood of an error or error rate in one sequencing technique compared to the other sequencing technique.

2. The method of claim 1, wherein the polynucleotide is immobilized on a solid support.

3. The method of claim 2, wherein the solid support is a bead.

4. The method of claim 1, wherein the polynucleotide is a single molecule.

5. The method of claim 1, wherein the polynucleotide is an amplified clone.

6. The method of claim 5, wherein the amplified clone is produced by PCR.

7. The method of claim 6, wherein the PCR is emulsion PCR.

8. The method of claim 5, wherein the amplification takes place on a solid support.

9. The method of claim 5, wherein the amplified clone is present on an array.