Methods and systems for nucleic acid sequencing validation, calibration and normalization

• US 9,169,515 B2
• Filed: 02/18/2011
• Issued: 10/27/2015
• Est. Priority Date: 02/19/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Shen, Min-Yi, Greiner, Douglas
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US13/030,818
• Publication Number:
  US 20110207624A1
• Time in Patent Office: 1,712 Days
• Field of Search: 702/20
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/005 Beads
• 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 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 2201/1242 Validating, e.g. range invalidation, suspending operation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

A system for performing quality control for nucleic acid sample sequencing is disclosed. The system comprises a set of solid supports, each solid support having attached thereto a plurality of nucleic acid sequences, wherein the set comprises plural groups of solid supports and each group contains solid supports having the same nucleic acid sequences attached thereto. The nucleic acid sequences of each group differ from each other. The nucleic acid sequences are synthetically derived, and the nucleic acids sequences are designed such that the nucleic acid sequences produce a predefined pattern of detectable signals during a sequencing run. A method of preparing a quality control for performing nucleic acid sample sequencing, a method of validating a nucleic acid sequencing instrument during a nucleic acid sequencing experiment, and a method of processing nucleic acid sequencing data during a nucleic acid sequencing experiment are also disclosed.

Claims

1. A method of processing nucleic acid sequencing data during a nucleic acid sequencing experiment, comprising:

placing a set of control solid supports each having a plurality of synthetic nucleic acid sequences attached thereto in a detection area of a nucleic acid sequencing instrument, wherein the set of control solid supports comprises plural groups of control solid supports that each have the same synthetic nucleic acid sequences attached thereto and wherein the synthetic nucleic acid sequences are designed such that the synthetic nucleic acid sequences produce a predefined pattern of observable signals during the nucleic acid sequencing experiment;

placing a template solid support having a nucleic acid sample to be sequenced attached thereto in a detection area of the nucleic acid sequencing instrument along with the set of control solid supports;

performing a first ligation cycle to attach dye-labeled probe sequences to the synthetic nucleic acid sequences attached to the control solid supports and to the nucleic acid sample attached to the template solid support;

detecting the dye-labeled probes attached to each of the synthetic nucleic acid sequences and the nucleic acid sample after the first ligation cycle;

performing a second ligation cycle to attach a dye-labeled probe sequences to the synthetic nucleic acid sequences attached to the control solid supports and to the nucleic acid sample attached to the template solid support;

detecting the dye-labeled probes attached to each of the synthetic nucleic acid sequences and the nucleic acid sample after the second ligation cycle;

comparing an intensity of the detected dye-labeled probes attached to the synthetic nucleic acid sequences after the first ligation cycle with an intensity of the detected dye-labeled probes attached to the synthetic nucleic acid sequences after the second ligation cycle; and

adjusting an intensity of the detected dye-labeled probes attached to the nucleic acid sample after the second ligation cycle based on the compared intensities,

wherein the predefined pattern of observable signals comprises transitions based on 12 of the 16 color transitions available when using 4 colors between consecutive ligation cycles while prohibiting transitions of the same color.

2. The method of claim 1, further comprising repeating performing a ligation cycle, detecting the-dye-labeled probe, comparing the intensities, and adjusting the intensity of the detected dye-labeled probe attached to the nucleic acid sample based on the compared intensities.

3. The method of claim 1, further comprising identifying the synthetic nucleic acid sequences based on a presence of a control identification sequence.

4. The method of claim 3, further comprising excluding the synthetic nucleic acid sequences from data corresponding to the nucleic acid sample.

5. The method of claim 1, wherein the synthetic nucleic acid sequence comprised in any one of the plural groups is different than the synthetic nucleic acid sequences comprised in the other ones of the plural groups.

6. The method of claim 1, wherein the plurality of synthetic nucleic acid sequences in the set of control solid supports comprises every possible variation of a set-length sequence.

7. The method of claim 1, wherein the plurality of synthetic nucleic acid sequences in the set of control solid supports comprises a set of 64 synthetic nucleic acid sequences comprising each possible combination of a 3 base sequence.

8. The method of claim 1, wherein the plurality of synthetic nucleic acid sequences in the set of control solid supports comprises a set of 256 synthetic nucleic acid sequences comprising each possible combination of a 4 base sequence.

10. The method of claim 1, wherein the groups of control solid supports comprise beads having a plurality of synthetic nucleic acid sequences attached thereto.

11. The method of claim 1, wherein the groups of control solid supports comprise one or more of (i) nucleic acid sequences provided as a fragment library, (ii) nucleic acid sequences provided as a mate-pair library, and (iii) nucleic acid sequences provided as analogous synthetic libraries.

12. The method of claim 1, wherein the groups of control solid supports comprise one or more of (i) nucleic acid sequences provided as a template comprising multiple inserts and multiple internal adapters, and (ii) nucleic acid sequences including concatenates.

13. The method of claim 1, wherein the predefined pattern of observable signals comprises predefined color transitions between consecutive ligation cycles.

17. The method of claim 1, further comprising identifying an error when a detected intensity in the comparison does not match with the predefined pattern of observable signals, and pausing operation of the nucleic acid sequencing instrument to determine a cause of the identified error.

18. The method of claim 1, further comprising comparing a signal to noise ratio for the synthetic nucleic acid sequences to a prior sequencing cycle.

19. The method of claim 18, comprising adjusting intensities based on the signal to noise ratio comparison to account for an increased noise and/or decreased signal during the sequencing experiment.

9. A method of processing nucleic acid sequencing data during a nucleic acid sequencing experiment, comprising:

placing a set of control solid supports each having a plurality of synthetic nucleic acid sequences attached thereto in a detection area of a nucleic acid sequencing instrument, wherein the set of control solid supports comprises plural groups of control solid supports that each have the same synthetic nucleic acid sequences attached thereto and wherein the synthetic nucleic acid sequences are designed such that the synthetic nucleic acid sequences produce a predefined pattern of observable signals during the nucleic acid sequencing experiment;

placing a template solid support having a nucleic acid sample to be sequenced attached thereto in a detection area of the nucleic acid sequencing instrument along with the set of control solid supports;

performing a first ligation cycle to attach dye-labeled probe sequences to the synthetic nucleic acid sequences attached to the control solid supports and to the nucleic acid sample attached to the template solid support;

detecting the dye-labeled probes attached to each of the synthetic nucleic acid sequences and the nucleic acid sample after the first ligation cycle;

performing a second ligation cycle to attach a dye-labeled probe sequences to the synthetic nucleic acid sequences attached to the control solid supports and to the nucleic acid sample attached to the template solid support;

detecting the dye-labeled probes attached to each of the synthetic nucleic acid sequences and the nucleic acid sample after the second ligation cycle;

comparing an intensity of the detected dye-labeled probes attached to the synthetic nucleic acid sequences after the first ligation cycle with an intensity of the detected dye-labeled probes attached to the synthetic nucleic acid sequences after the second ligation cycle; and

adjusting an intensity of the detected dye-labeled probes attached to the nucleic acid sample after the second ligation cycle based on the compared intensities,

wherein the plurality of synthetic nucleic acid sequences in the set of control solid supports comprises a set of 1024 synthetic nucleic acid sequences comprising each possible combination of a 5 base sequence.

14. The method of claim 9, wherein the predefined pattern of observable signals comprises color transitions from a first color to a second color, from the second color to a fourth color, from the fourth color to the fourth color, and from the fourth color to a third color.

15. The method of claim 9, wherein the predefined pattern of observable signals comprises color transitions from a second color to a first color, from the first color to a third color, from the third color to the third color, and from the third color to a second color.

16. The method of claim 9, wherein the predefined pattern of observable signals comprises color transitions from a third color to a fourth color, from the fourth color to a second color, from the second color to the second color, and from the second color to a first color.