Modifications to polynucleotides for sequencing

• US 10,689,684 B2
• Filed: 02/14/2017
• Issued: 06/23/2020
• Est. Priority Date: 02/14/2017
• Status: Active Grant

• Current Assignee: Microsoft Technology Licensing LLC (Microsoft Corporation), University of Washington
• Original Assignee: Microsoft Technology Licensing LLC (Microsoft Corporation)
• Inventors: Chen, Yuan-Jyue, Strauss, Karin, Ceze, Luis H., Organick, Lee, Lopez, Randolph, Seelig, Georg
• Primary Examiner(s): Brusca, John S
• Application Number:
  US15/431,897
• Publication Number:
  US 20180230509A1
• Time in Patent Office: 1,225 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

This disclosure describes techniques to improve the sequencing of polynucleotides by decreasing the likelihood of errors occurring during a sequencing calibration process. In implementations, regions of polynucleotides that are used for the calibration process can be modified to reduce a number of polynucleotides that have a same nucleotide at one or more positions of the calibration regions. In some cases, the calibration regions can be modified by adding a sequence to the polynucleotides that replaces the original calibration regions. Also, the calibration regions can be modified by rearranging the nucleotides at the different positions of the calibration regions. Additionally, the calibration regions can be modified by adding sequences of varying length to the polynucleotides being sequenced to produce polynucleotides having varying length with different calibration regions.

Claims

1. A method comprising:

receiving a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences includes a calibration region, the calibration region read by a sequencing machine during a calibration process;

performing an analysis of sequences of the plurality of polynucleotide sequences;

determining, based at least partly on the analysis, a number of individual polynucleotide sequences of the plurality of polynucleotide sequences having a same nucleotide located in a same position of the calibration region of each of the plurality of polynucleotide sequences;

determining that the number is greater than a threshold number;

modifying a nucleotide sequence of the calibration region of at least one of the plurality of polynucleotide sequences to produce a modified polynucleotide sequence, wherein, after the modifying, the number is less than the threshold number; and

providing the modified polynucleotide sequence to a polynucleotide synthesizer.

2. The method of claim 1, wherein determining the number of polynucleotides of the plurality of polynucleotide sequences having the same nucleotide located in the same position of the calibration region includes:

comparing a first nucleotide at the position of a first calibration region of a first polynucleotide sequence of the plurality of polynucleotide sequences with a second nucleotide at the position of a second calibration region of a second polynucleotide sequence of the plurality of polynucleotide sequences; and

determining that the first nucleotide and the second nucleotide are the same.

3. The method of claim 1, wherein modifying the nucleotide sequence of the calibration regions of the number of polynucleotides to produce the modified polynucleotide sequence is based at least partly on:

determining that an additional number of polynucleotides of the plurality are polynucleotide sequences that have a same nucleotide located in at least a threshold number of positions of the calibration region of each of the additional number of polynucleotides; and

determining that the additional number of polynucleotides is at least the threshold number.

4. The method of claim 1, further comprising:

synthesizing, by the polynucleotide synthesizer, the plurality of polynucleotide sequences to produce a synthesized group of polynucleotides;

wherein modifying the calibration region of the at least one of the plurality of polynucleotide sequences is performed with respect to nucleotide sequences of the synthesized group of polynucleotides.

5. The method of claim 4, further comprising:

adding a first sequencing primer target to a first calibration region of a first synthesized polynucleotide of the synthesized group of polynucleotides to produce a modified first synthesized polynucleotide, the first sequencing primer target having a first extender sequence with a first number of nucleotides; and

adding a second sequencing primer target to a second calibration region of a second synthesized polynucleotide of the synthesized group of polynucleotides to produce a modified second synthesized polynucleotide, the second sequencing primer target having a second extender sequence with a second number of nucleotides different from the first number of nucleotides.

6. The method of claim 5, wherein:

a first modified calibration region of the modified first synthesized polynucleotide includes the first extender sequence and a number of nucleotides of the first calibration region; and

a second modified calibration region of the modified second synthesized polynucleotide includes the second extender sequence and a number of nucleotides of the second calibration region.

7. The method of claim 1, further comprising:

synthesizing, by the polynucleotide synthesizer, a modified polynucleotide based at least partly on the modified polynucleotide sequence.

8. The method of claim 1, wherein the group of polynucleotides includes deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

9. The method of claim 1, wherein:

individual polynucleotide sequences of the plurality of polynucleotide sequences include first regions of nucleotides that encode individual portions of digital data and second regions of polynucleotide sequences that include a file identifier corresponding to the digital data; and

the calibration region of each of the plurality of polynucleotide sequences comprises at least a portion of the nucleotides included in the second regions.

10. The method of claim 9, wherein determining the number of individual polynucleotide sequences of the plurality of polynucleotide sequences having the same nucleotide located in the one or more positions of the calibration regions of the number of polynucleotides includes:

determining that a first file identifier of a first polynucleotide sequence of a one of the plurality of polynucleotide sequences and a second file identifier of a second polynucleotide sequence of a one of the plurality of polynucleotides sequences is the same.

11. A method comprising:

generating polynucleotide data indicating a polynucleotide sequence including a payload and a file identifier, the payload encoding a segment of digital data and the file identifier being associated with the digital data;

determining that a position located in a calibration region of the polynucleotide sequence has a same nucleotide as the same position located in additional calibration regions of at least a threshold number of additional polynucleotide sequences, the calibration region and the additional calibration regions including a sequence of nucleotides used by a sequencing machine during a calibration process of the sequencing machine;

modifying a portion of the polynucleotide data corresponding to the calibration region of the polynucleotide to produce a modified polynucleotide sequence having a modified calibration region such that less than the threshold number of the calibration regions include the same nucleotide at the same position; and

providing the modified polynucleotide sequence to a polynucleotide synthesizer.

12. The method of claim 11, further comprising:

generating a plurality of segments of the digital data;

encoding individual segments of the plurality of segments of the digital data as polynucleotide sequence data for the payload; and

generating additional polynucleotide data indicating a plurality of polynucleotide sequences, the plurality of polynucleotide sequences including the polynucleotide and individual polynucleotides of the plurality of polynucleotides including one of the individual sequences of nucleotides and the file identifier.

13. The method of claim 11, wherein modifying the calibration region includes modifying the polynucleotide data to add one or more nucleotides to at least one of a 5′

end of the polynucleotide sequence or a 3′

end of the polynucleotide sequence to produce the modified calibration region.

14. The method of claim 11, wherein the sequencing machine determines a sequence of the polynucleotide by:

attaching the polynucleotide to a surface of a flow cell;

producing a cluster of the polynucleotide on the surface of the flow cell by replicating the polynucleotide at a location of the surface of the flow cell;

attaching a fluorophore-bound nucleotide to complementary nucleotides at a position of individual polynucleotides of the cluster;

applying energy to the fluorophore;

capturing one or more images of the surface of the flow cell after applying energy to the fluorophore;

performing an analysis of the one or more images to determine wavelengths emitted by different locations of the surface of the flow cell; and

determining, based at least partly on the analysis, a type of nucleotide at the position based on a distribution of wavelengths of electromagnetic radiation emitted by the fluorophore.

15. The method of claim 11, wherein modifying the portion of the polynucleotide data corresponding to the calibration region of the polynucleotide includes modifying an order of the nucleotides included in the calibration region.

16. A system comprising:

a processor;

a memory in communication with the processing unit, the memory storing computer-readable instructions that when executed by the processor perform operations comprising;

performing an analysis of nucleotide sequence data of a plurality of polynucleotide sequences, wherein individual ones of the plurality of polynucleotide sequences include calibration regions comprising a sequence of nucleotides used by a sequencing machine during a calibration process of the sequencing machine and payload regions that encode a segment of digital data;

determining, based at least partly on the analysis, that a likelihood of an error occurring during the calibration process of the sequencing machine while sequencing polynucleotides with nucleotide sequences indicated by the plurality of polynucleotide sequences is at least a threshold probability;

modifying the calibration regions of at least a portion of the plurality of polynucleotide sequences to produce a modified plurality of polynucleotide sequences having modified calibration regions such that the likelihood of an error occurring during the calibration process of the sequencing machine while sequencing polynucleotides with nucleotide sequences indicated by the modified plurality of polynucleotide sequences is below the threshold probability; and

providing the modified plurality of polynucleotide sequences to a polynucleotide synthesizer.

17. The system of claim 16, wherein individual sequences of the plurality of polynucleotide sequences include:

a file identifier region that encodes a file identifier associated with the digital data; and

an individual calibration region that includes at least a portion of the file identifier region.

18. The system of claim 16, wherein the operations further comprise:

determining changes between a calibration region of a polynucleotide sequence of the plurality of polynucleotide sequences and a modified calibration region of a modified polynucleotide sequence of the modified plurality of polynucleotide sequencers; and

generating a data structure that stores the changes.

19. The system of claim 18, wherein the operations further comprise:

receiving a request for the digital data;

obtaining sequence data from the sequencing machine, the sequence data including a sequence of nucleotides;

determining that the sequence of nucleotides corresponds to the modified plurality of polynucleotide sequences;

accessing the data structure to determine the changes;

generating, based on the sequence data and the changes stored in the data structure, nucleotide sequence data; and

decoding the nucleotide sequence data to produce a series of bits related to a portion of the digital data.

20. The system of claim 16, wherein determining that the likelihood of an error occurring during the calibration process of the sequencing machine while sequencing polynucleotides with nucleotide sequences indicated by the plurality of polynucleotide sequences is at least the threshold probability and includes at least one of:

determining that at least a threshold number of the plurality of polynucleotide sequences have a same nucleotide located in a same position of the calibration regions of the threshold number of the plurality of polynucleotide sequences;

determining that at least the threshold number of the plurality of polynucleotide sequences have a same nucleotide located in at least a threshold number of positions of the calibration regions of the threshold number of the plurality of polynucleotide sequences;

or

determining that a distribution of nucleotides located in at least one position of the calibration regions of the plurality of polynucleotide sequences is outside of a specified distribution of nucleotides.