Unique identifiers for indicating properties associated with entities to which they are attached, and methods for using

• US 20060263789A1
• Filed: 05/19/2005
• Published: 11/23/2006
• Est. Priority Date: 05/19/2005
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Kincaid, Robert
• Application Number:
  US11/133,120
• Publication Number:
  US 20060263789A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

Methods, systems and computer readable media for sequencing a biopolymer specimen and tracking a source from which the specimen was derived. Methods, systems and computer readable media for multiplex sequencing biopolymer samples. Methods, systems and computer readable media for efficiently sequencing biopolymeric specimens through a high-throughput sequencer. Methods, systems and computer readable media for performing ratio-based analysis with a high throughput sequencer.

Claims

1. A method of sequencing a biopolymer specimen and tracking a source from which the specimen was derived, said method comprising the steps of:

processing the biopolymer specimen to provide a unique identifier with the biopolymer specimen as processed, wherein said unique identifier represents metadata identifying a source sample from which the biological specimen was taken, and said unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

passing the biopolymer specimen including the unique identifier through the high-throughput sequencer and identifying a sequence of the biopolymer specimen, as well as identifying the unique identifier as each passes through the high-throughput sequencer; and

correlating the identified sequence of the biopolymer specimen with the source sample from which the identified sequence was derived, based upon the identifier metadata derived from said identification of the unique identifier for that respective sequence.

2. The method of claim 1, wherein said high-throughput sequencer comprises a nanopore device.

3. The method of claim 1, wherein said unique identifier comprises a barcode including a unique sequence of nucleic acid bases, and wherein said processing comprises appending said barcode to the biopolymer specimen.

4. The method of claim 1, wherein the biopolymer specimen comprises a DNA strand.

5. The method of claim 1, wherein the biopolymer specimen comprises an RNA strand.

6. The method of claim 3, wherein said unique sequence of nucleic acid bases comprises SNA.

7. The method of claim 1, wherein said processing comprises digesting said biopolymer specimen with a specific restriction enzyme, and wherein said unique identifier comprises nucleic acid bases resulting from the specific restriction enzyme digest.

8. The method of claim 1, wherein said unique identifier comprises a sequence of nucleic acid bases and said unique identifier is delimited by a unique sequence that is non-homologous to said biopolymer specimen.

9. A method for multiplex sequencing biopolymer samples, said method comprising the steps of:

processing biopolymer strands in a first biopolymer sample to provide a first unique identifier with each said biopolymer strand so processed, wherein said first unique identifier includes metadata identifying said first biopolymer sample, and said first unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

processing biopolymer strands in a second biopolymer sample to provide a second unique identifier with each said to biopolymer strand in said second biopolymer sample so processed, wherein said second unique identifier includes metadata identifying said second biopolymer sample, and said second unique identifier is configured to form a unique, repeatable, characteristic signature different from the signature formed by said first unique identifier, when read by the high-throughput sequencer;

mixing together processed strands of said first biopolymer sample with processed strands of said second biopolymer sample;

randomly passing at least one processed strand through at least one high-throughput sequencer and identifying the strand sequence, as well as identifying the unique identifier as each processed strand passes through the high-throughput sequencer; and

correlating the identified sequences of the biopolymers with the samples from which they were derived, based upon the identifier metadata derived from said identification of the unique identifier for that respective biopolymer strand.

10. The method of claim 9, further comprising processing biopolymer strands in at least one additional biopolymer sample to provide an additional unique identifier for each additional biopolymer sample, respectively, wherein each unique identifier associated with each additional biopolymer sample is unique from all other unique identifiers associated with all other additional biopolymer samples and from said first and second unique identifiers, and wherein processed strands from each additional biopolymer sample are mixed, randomly passed, identified and correlated along with said processed strands from said first and second biopolymer samples.

11. The method of claim 9, wherein at least one of said first and second biopolymer samples comprises DNA strands.

12. The method of claim 9, wherein at least one of said first and second biopolymer samples comprises RNA strands.

13. The method of claim 9, wherein at least one of said first and second unique identifiers comprises a barcode including a unique sequence of nucleic acid bases, and wherein said processing comprises appending said barcode to the biopolymer strand from said respective biopolymer sample.

14. The method of claim 13, wherein said unique sequence of nucleic acid bases comprises SNA.

15. The method of claim 9, wherein at least one of said processing biopolymer strands in said first sample and processing biopolymer strand in said second sample comprises digesting said biopolymer strands in said respective sample, with a specific restriction enzyme, and wherein said unique identifier comprises nucleic acid bases resulting from the specific restriction enzyme digest.

16. The method of claim 9, wherein at least one of said first and second unique identifiers each comprise a sequence of nucleic acid bases that is unique from the other and each said unique identifier is delimited by a unique sequence that is non-homologous to said biopolymer specimen.

18. The method of claim 11, wherein at least one of said first and second biopolymer samples comprises DNA strands.

19. The method of claim 11, wherein at least one of said first and second biopolymer samples comprises RNA strands.

17. A method of sequencing biopolymeric specimens through a high-throughput sequencer, said method comprising the steps of:

processing sequences in a first biopolymeric sample to provide a first unique identifier with each processed sequence, wherein said first unique identifier represents metadata identifying said first biopolymeric sample, and said first unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

passing the sequences having first unique identifiers associated therewith through the high-throughput sequencer and identifying each sequence of the first biopolymeric sample as well as identifying the first unique identifier associated therewith, as each passes through the high-throughput sequencer;

correlating the identified sequences with the first biopolymeric sample from which the identified sequences were derived, based upon the identifier metadata derived from said identification of the first unique identifier for each respective sequence;

processing sequences in a second biopolymeric sample to provide a second unique identifier with each processed sequence from said second biopolymeric sample, wherein said second unique identifier represents metadata identifying said second biopolymeric sample, and said second unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

passing the sequences having second unique identifiers associated therewith through the high-throughput sequencer and identifying each sequence, as well as identifying the second unique identifier associated therewith, as each passes through the high-throughput sequencer; and

correlating the identified sequences associated with the second unique identifiers with the second biopolymeric sample from which the identified sequences were derived, based upon the identifier metadata derived from reading said second unique identifier for each respective sequence, but ignoring the identified sequences when the associated unique identifier read is not the second unique identifier, or there is no unique identifier associated with the sequence.

20. A method of sequencing biopolymeric specimens through a high-throughput sequencer, said method comprising the steps of:

processing sequences in at least one biopolymeric sample to provide a unique identifier with each said sequence so processed, wherein said unique identifiers with respect to each sample are unique from unique identifiers with respect to all other samples and each said unique identifier represents metadata identifying said biopolymeric sample from which each sequence associated with each said unique identifier was taken, and each said unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

passing the sequences having associated unique identifiers through the high-throughput sequencer and identifying each sequence, as well as identifying any unique identifier associated therewith, as each passes through the high-throughput sequencer; and

correlating the identified sequences with the respective biopolymeric samples from which the identified sequences were derived, based upon the identifier metadata derived from said identification of the associated unique identifier for each respective sequence, but ignoring the identified sequences when the associated unique identifier read is not a unique identifier associated by said processing step, or there is no unique identifier associated with the sequence.

21. A method of performing ratio-based analysis with a high throughput sequencer, said method comprising the steps of:

processing sequences in a test biopolymeric sample to associate a first unique identifier with each sequence so processed, wherein said first unique identifier represents metadata identifying said test biopolymeric sample, and said first unique identifier is configured to form a unique, repeatable, characteristic signature when read by a high-throughput sequencer;

processing sequences in a control biopolymeric sample to associate a second unique identifier with each sequence from the control sample so processed, wherein said second unique identifier represents metadata identifying said control biopolymeric sample, and said second unique identifier is configured to form a unique, repeatable, characteristic signature different from the signature formed by said first unique identifier, when read by the high-throughput sequencer;

mixing together processed sequences of said test biopolymeric sample associated with said first unique identifiers, with processed sequences of said control biopolymeric sample associated with said second unique identifiers;

randomly passing processed sequences through at least one high-throughput sequencer and identifying the sequences, as well as identifying the unique identifiers associated therewith as the processed sequences pass through a high-throughput sequencer, respectively;

correlating the identified sequences with the samples from which they were derived, based upon the identifier metadata derived from said identification of the unique identifier associated with that respective sequence;

counting the number of times that a particular sequence is read with regard to said first and second unique identifiers; and

calculating a ratio comparing the number of times that the particular ratio was identified as associated with said first and second identifiers, respectively.

22. The method of claim 21, further comprising processing biopolymer sequences in at least one additional biopolymeric sample to associate a unique identifier with each said sequence so processed from each said additional biopolymeric sample, wherein each unique identifier associated with each additional biopolymeric sample is unique from all other unique identifiers associated with all other additional biopolymeric samples and from said first and second unique identifiers, and wherein processed sequences from each additional biopolymeric sample are mixed, randomly passed, correlated, counted and ratio-calculated against at least one other biopolymeric sample along with said processed sequences from said first and second biopolymeric samples.

23. The method of claim 22, wherein said counting and calculating steps are carried out with regard to at least one additional particular sequence different from said particular sequence.

24. The method of claim 21, wherein said biopolymeric samples are DNA samples.

25. The method of claim 21, wherein said biopolymeric samples are RNA samples.

26. The method of claim 21, wherein said ratio-based abundance analysis comprises CGH analysis.

27. The method of claim 21, wherein said ratio-based abundance analysis comprises gene expression analysis.

28. The method of claim 21, wherein said ratio-based abundance analysis comprises SNP analysis.