METHODS AND SYSTEMS FOR NUCLEIC ACID SEQUENCING VALIDATION, CALIBRATION AND NORMALIZATION

• US 20110207624A1
• Filed: 02/18/2011
• Published: 08/25/2011
• 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
• Granted Patent: US 9,169,515 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 system for performing quality control for nucleic acid sample sequencing, the system comprising:

a set of solid supports, each solid support having attached thereto a plurality of nucleic acid molecules,

wherein the set comprises plural groups of solid supports and each group contains solid supports having the same nucleic acid sequences attached thereto,

wherein the nucleic acid sequences of each group differ from each other,

wherein the nucleic acid sequences are synthetically derived, and

wherein the nucleic acid sequences produce a predefined pattern of detectable signals during a sequencing run.

2. The system of claim 1, wherein the solid supports are beads.

3. The system of claim 1, wherein each nucleic acid sequence is designed such that consecutive ligation cycles during a sequencing-by-ligation process yield a predefined color transition.

4. The system of claim 3, wherein each nucleic acid sequence is designed such that all possible color transitions are observed for consecutive ligation cycles for each subset of the plural groups of solid supports, wherein a number of subsets is equal to a total number of unique detectable signals.

5. The system of claim 1, wherein the set comprises at least 1024 groups of solid supports.

6. The system of claim 1, wherein each nucleic acid sequence comprises a plurality of tag sequences, wherein the plurality of tag sequences comprise the same sequence or different sequences.

7. The system of claim 5, wherein an internal adapter sequence is disposed between each of the plurality of tag sequences.

8. The system of claim 1, wherein the nucleic acid sequences are designed such that a folding free energy of each sequence is minimized.

9. The system of claim 1, wherein the plural groups of solid supports are divided into a plurality of subsets, wherein a number of subsets is equal to a number of unique detectable signals.

10. The system of claim 1, wherein each nucleic acid is designed such that for a given range of bases within each nucleic acid, every possible combination of bases is represented.

11. The system of claim 1, wherein each nucleic acid sequence comprises a control identification sequence.

12. A method of preparing a quality control for performing nucleic acid sample sequencing, comprising:

generating a plurality of synthetic nucleic acid sequences, wherein each synthetic nucleic acid sequence differs from another nucleic acid sequence and wherein the plurality of synthetic nucleic acid sequences produce a predefined pattern of detectable signals during sequencing;

attaching each of the synthetic nucleic acid sequences to solid supports in plural groups of solid supports, wherein the solid supports in each group have the same synthetic nucleic acid sequence attached thereto; and

combining each group of solid supports with the synthetic nucleic acid sequences attached to create a control set of solid supports for performing nucleic acid sample sequencing.

13. The method of claim 12, wherein attaching each of the synthetic nucleic acids to a solid supports comprises attaching each of the synthetic nucleic acids to beads.

14. The method of claim 12, wherein generating a plurality of synthetic nucleic acid sequences comprises designing each synthetic nucleic acid sequence such that consecutive ligation cycles during a sequencing-by-ligation process yield a predefined color transition.

15. The method of claim 12, wherein generating a plurality of synthetic nucleic acid sequences comprises designing each synthetic nucleic acid sequence such that all possible color transitions are observed for consecutive ligation cycles for each subset of the plural groups of solid supports, wherein a number of subsets is equal to a total number of unique detectable signals.

16. The method of claim 12, wherein combining each group of solid supports comprises combining at least 1024 groups of solid supports.

17. The method of claim 12, wherein generating a plurality of synthetic nucleic acid sequences comprises generating each synthetic nucleic acid sequence with a plurality of tag sequences, wherein the plurality of tag sequences comprise the same sequences or different sequences.

18. The method of claim 17, wherein generating a plurality of synthetic nucleic acid sequences further comprises disposing an internal adapter sequence between each of the plurality of tag sequences.

19. The method of claim 12, wherein generating a plurality of synthetic nucleic acid sequences comprising designing the synthetic nucleic acid sequences such that a folding free energy of each sequence is minimized.

20. The method of claim 12, wherein generating a plurality of synthetic nucleic acid sequences comprising designing each nucleic acid such that for a given range of bases within each nucleic acid, every possible combination of bases is represented.

21. The system of claim 12, wherein each nucleic acid sequence comprises a control identification sequence.

22. A method of validating a nucleic acid sequencing instrument during a nucleic acid sequencing experiment, comprising:

placing a set of 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 solid supports comprises plural groups of 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 nucleic acid sequences produce a predefined pattern of observable signals during the nucleic acid sequencing experiment;

placing a nucleic acid sample to be sequenced in a detection area of the nucleic acid sequencing instrument;

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

detecting the dye-labeled probes attached to each of the nucleic acid sequences and the nucleic acid sample; and

comparing the detected dye-labeled probe attached to the nucleic acid sequences with the predefined pattern of detectable signals to determine if the instrument is functioning within predefined parameters.

23. The method of claim 22, further comprising repeating the steps of performing a ligation cycle, detecting the dye-labeled probes, and comparing the detected dye-labeled probe.

24. The method of claim 22, further comprising identifying the synthetic nucleic acid sequences based on a presence of a control identification sequence.

25. The method of claim 24, further comprising excluding the synthetic nucleic acid sequences from data corresponding to the nucleic acid sample.

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

placing a set of 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 solid supports comprises plural groups of 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 nucleic acid sequences produce a predefined pattern of observable signals during the nucleic acid sequencing experiment;

placing a nucleic acid sample to be sequenced in a detection area of the nucleic acid sequencing instrument;

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

detecting the dye-labeled probes attached to each of the 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 nucleic acid sequences attached to the solid supports and to the nucleic acid sample;

detecting the dye-labeled probes attached to each of the 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 nucleic acid sequences after the first ligation cycle with an intensity of the detected dye-labeled probes attached to the 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.

27. The method of claim 26, 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.

28. The method of claim 26, further comprising identifying the synthetic nucleic acid sequences based on a presence of a control identification sequence.

29. The method of claim 28, further comprising excluding the synthetic nucleic acid sequences from data corresponding to the nucleic acid sample.

30. A system for performing quality control for nucleic acid sample sequencing, the system comprising:

a solid support having attached thereto a plurality of nucleic acid sequences,

wherein the plurality of nucleic acid sequences are arranged in a set of distinct locations,

wherein the set of distinct locations comprises plural groups of locations and each group contains locations having the same nucleic acid sequences attached thereto,

wherein the nucleic acid sequences of each group differ from each other,

wherein the nucleic acid sequences are synthetically derived, and

wherein the nucleic acids sequences are designed such that the nucleic acid sequences produce a predefined pattern of detectable signals during a sequencing run.

31. A method comprising:

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

performing a sequencing cycle to attach dye-labeled probes to the nucleic acid sequences;

detecting the dye-labeled probes attached to each of the nucleic acid sequences; and

comparing the detected dye-labeled probe attached to the nucleic acid sequences with the predefined pattern of detectable signals to determine if the instrument is functioning within predefined parameters.

32. The method of claim 31, further comprising modifying the performance of the nucleic acid sequencing instrument in response to comparing.

33. The method of claim 32, wherein modifying the performance includes adjusting data to account for decreased signal-to-noise ratio.

34. The method of claim 31, further comprising placing a nucleic acid sample to be sequenced in a detection area of the nucleic acid sequencing instrument.

35. The method of claim 34, further comprising identifying the synthetic nucleic acid sequences based on a presence of a control identification sequence.

36. The method of claim 35, further comprising excluding the synthetic nucleic acid sequences from data corresponding to the nucleic acid sample.