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

A method of discarding a chimeric sequence read, said method comprising:

• (i) attaching a first pair of identifier sequences to a first template nucleic acid from a first sample and a second pair of identifier sequences to a second template nucleic acid from a second sample, thereby forming a first identified template nucleic acid and a second identified template nucleic acid, wherein
• a. each of said first pair of identifier sequences are different and both of said first pair of identifier sequences are specific for said first sample, and
• b. each of said second pair of identifier sequences are different and both of said second pair of identifier sequences are specific for said second sample;
• (ii) combining said first identified template nucleic acid and said second identified template nucleic acid, thereby forming pooled identified template nucleic acids;
• (iii) attaching said pooled identified template nucleic acids to a surface of a flow cell;
• (iv) amplifying said pooled identified template nucleic acids on the surface of said flow cell, thereby forming first template amplicons, second template amplicons and chimeric amplicons, wherein said first template amplicons comprise said first pair of identifier sequences, said second template amplicons comprise said second pair of identifier sequences and said chimeric amplicons comprise one identifier sequence of said first pair of identifier sequences and one identifier sequence of said second pair of identifier sequences, and wherein said chimeric amplicons are formed during amplification from said first identified template nucleic acid and said second identified template nucleic acid;
• (v) sequencing said first template amplicons, said second template amplicons and said chimeric amplicons, thereby forming a first template sequence read, a second template sequence read and a chimeric sequence read;
• and
• (vi) discarding said chimeric sequence read.

A method of validating a first template sequence read and a second template sequence read, said method comprising:

• (i) attaching a first pair of identifier sequences to a first template nucleic acid from a first sample and a second pair of identifier sequences to a second template nucleic acid from a second sample, thereby forming a first identified template nucleic acid and a second identified template nucleic acid, wherein
• a. each of said first pair of identifier sequences are different and both of said first pair of identifier sequences are specific for said first sample, and
• b. each of said second pair of identifier sequences are different and both of said second pair of identifier sequences are specific for said second sample;
• (ii) combining said first identified template nucleic acid and said second identified template nucleic acid, thereby forming pooled identified template nucleic acids;
• (iii) attaching said pooled identified template nucleic acids to a surface of a flow cell;
• (iv) amplifying said pooled identified template nucleic acids on the surface of said flow cell, thereby forming first template amplicons, second template amplicons and chimeric amplicons, wherein said first template amplicons comprise said first pair of identifier sequences, said second template amplicons comprise said second pair of identifier sequences and said chimeric amplicons comprise one identifier sequence of said first pair of identifier sequences and one identifier sequence of said second pair of identifier sequences, and wherein said chimeric amplicons are formed during amplification from said first identified template nucleic acid and said second identified template nucleic acid;
• (v) sequencing said first template amplicons, said second template amplicons and said chimeric amplicons, thereby forming a first template sequence read, a second template sequence read and a chimeric sequence read;
• and
• (vi) validating said first template sequence read and said second template sequence read.