Methods and Compositions for Using Error-Detecting and/or Error-Correcting Barcodes in Nucleic Acid Amplification Process

• US 20100323348A1
• Filed: 01/26/2010
• Published: 12/23/2010
• Est. Priority Date: 01/31/2009
• Status: Abandoned Application

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Knight, Robin D., Hamady, Micah L.
• Application Number:
  US12/693,612
• Publication Number:
  US 20100323348A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The present invention provides methods and compositions for detecting and correcting errors in nucleic acid amplification processes, and methods for using the same. In particular, barcode amplification errors are detected and corrected such that integrity in sample assignment is maintained. The methods are compatible with high throughput sequencing techniques as some of the barcodes are based upon Hamming codes, thereby allowing self-correction for single bit errors. Some methods and compositions of the invention allow characterization (e.g., sequencing) of a plurality of nucleic acid samples simultaneously within a single sequencing reaction.

Claims

1. A pyrosequencing compatible primer comprising a first region containing a unique error-detecting/correcting hamming barcode.

2. The pyrosequencing compatible primer of claim 1, wherein the primer further comprises a second region complementary to a bacterial 16S rRNA gene.

3. A method of assigning sequence data to individual samples from a mixture of samples, comprising:

a) providing;

i) a pyrosequencing compatible primer comprising a first region containing a unique error-detecting/correcting barcode and a second region complementary to a target nucleic acid molecule, and

ii) a target nucleic acid molecule,

b) amplifying said target nucleic acid molecule with said primer,

c) pooling a plurality of said amplification product, and

d) pyrosequencing said pooled amplification products to determine their respective nucleotide sequences.

4. The method of claim 3, wherein said plurality of amplification products are pooled in equimolar ratios.

5. The method of claim 3, wherein said unique error-detecting/correcting barcode is a Hamming code.

6. The method of claim 3, wherein said target nucleic acid molecule comprises a portion of the 16S rRNA gene.

7. The method of claim 3, further comprising identifying amplification products with unique barcode sequence errors.

8. The method of claim 3, further comprising correcting the unique barcode sequence of amplification products containing correctable unique barcode sequence errors.

9. The method of claim 3, further comprising discarding the nucleotide sequence of amplification products containing non-correctable unique barcode sequence errors.

10. The method of claim 3, further comprising step e) aligning the nucleotide sequences of said amplification products to generate a phylogenetic tree.

11. A method comprising:

a) providing;

i) a plurality of samples comprising nucleic acid sequences;

ii) a plurality of primers error correcting or error-detecting sequence tags wherein said primers are at least partially complementary to said nucleic acid sequences;

iii) a parallel sequencing technique capable of simultaneously characterizing said nucleic acid sequences from said plurality of samples;

b) amplifying said plurality of nucleic acid samples using said plurality of primers; and

c) analyzing said sequence tags of said amplified nucleic acids.

12. The method of claim 11, wherein said sequence tag identifies a sample assignment thereby identifying one of said samples from which said nucleic acid was derived.

13. The method of claim 12, wherein said sequence tag identifies the presence of an error in said nucleic acid, thereby establishing a probability that said sample assignment is incorrect.

14. The method of claim 12, wherein said sequence tag identifies the absence of any error in said nucleic acid, thereby establishing a probability that said sample assignment is correct.

15. The method of claim 11, wherein said sequence technique comprises pyrosequencing.