Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction

1. A method for validating detection of an analyte of interest, the method comprising:

detecting the presence of two or more identifier sequences that are uniquely associated with an analyte of interest, wherein the two or more identifier sequences associated with each analyte of interest have four or more nucleotides and are the same; and

excluding templates containing only one identifier or an incorrect pair from analysis.

2. The method of claim 1 , wherein the identifier sequences are nucleic acid sequence tags.

3. The method of claim 1 , wherein the identifiers are first and second barcode sequences.

4. The method of claim 1 , wherein detecting comprises sequencing the analyte.

5. The method of claim 1 , further comprising incorporating the two or more identifier sequences into a template to generate an analyte.

6. The method of claim 5 , wherein a first identifier sequence is incorporated into a 5′ portion of the template, and a second identifier sequence is incorporated into a 3′ portion of the template.

7. The method of claim 6 , wherein after incorporation of the identifier sequences, the template has the following configuration from 5′ to 3′: a first known sequence; a first barcode sequence; a second known sequence; a target polynucleotide sequence; a third known sequence; a second barcode sequence; and a fourth known sequence.

8. The method of claim 5 , wherein a first identifier sequence is incorporated into a 5′ end of the template, and a second identifier sequence is incorporated into a 3′ end of the template.

9. The method of claim 4 , wherein prior to sequencing, the analyte is attached to a substrate.

10. The method of claim 9 , wherein the analyte is directly attached to the substrate.