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

A method for validating the sequence of a nucleic acid analyte of interest in a multiplex sequencing reaction, the method comprising:

• i) detecting the presence of two or more identifier sequences that are uniquely associated with the nucleic acid analyte of interest,
• wherein at least a first identifier sequence is incorporated into a 5′ portion of the nucleic acid analyte of interest and wherein at least a second identifier sequence is incorporated into a 3′ portion of the nucleic acid analyte of interest, and
• wherein the first and second identifier sequences have four or more nucleotides and are different;
• ii) sequencing the first identifier sequence, the nucleic acid analyte of interest and the second identifier sequence;
• and
• iii) validating the sequence of the nucleic acid analyte of interest by analyzing both identifiers and excluding the sequences of those nucleic acid analytes of interest containing only one identifier or an incorrect pair of identifiers from sequence analysis of the multiplex sequencing reaction.