Indexing based deep DNA sequencing to identify rare sequences

An assay to identify, from a plurality of polynucleotides, a polynucleotide having a mutation within a target sequence, the assay comprising the steps of:

• a) producing a single-stranded complement of each of the plurality of polynucleotides containing the target sequence by conducting one cycle of PCR using a plurality of first primers, wherein each of the plurality of first primers comprises, from the 5′ end:
• i) an outer PCR primer motif,
• ii) an inner PCR primer motif,
• iii) a tag comprising a sequence unique for each of the first primers, wherein the unique sequence comprises about 4-20 nucleotides, and
• iv) a 3′ target sequence which has a sequence that hybridizes to the sequence at the 3′ end of the target sequence,
• wherein each of the single-stranded complements of each of the plurality of polynucleotides produced in this step comprises, from the 5′ end:
• i) the outer PCR primer motif,
• ii) the inner PCR primer motif,
• iii) the tag comprising a unique sequence of about 4-20 nucleotides, and
• iv) the 3′ target sequence,
• b) optionally, isolating the single-stranded complements produced in step a),
• c) PCR amplifying the single-stranded complements produced in step a) or isolated in step b) using a first primer set comprising an outer PCR primer and a first 5′ target primer to produce multiple double-stranded copies of each of the single-stranded complements produced in step a), wherein the outer PCR primer has a sequence that corresponds to the outer PCR primer motif portion of the first primers and the first 5′ target primer has a sequence that corresponds to the sequence at the 5′ end of the target sequence,
• d) optionally, isolating the double-stranded copies produced in step c),
• e) PCR amplifying the double-stranded copies produced in step c) or isolated in step d) using a second primer set comprising:
• i) a first barcode primer comprising, from the 5′ end:
• a sequence corresponding to a 3′ portion of a first sequencing primer, a first barcode and an inner PCR primer sequence, wherein the inner PCR primer sequence corresponds to the inner PCR primer motif portion of the first primer, and
• ii) a second barcode primer comprising, from the 5′ end:
• a sequence corresponding to a 3′ portion of a second sequencing primer, a second barcode and a second 5′ target sequence, wherein the second 5′ target sequence corresponds to the portion of the target sequence that is at the 3′ end of the sequence corresponding to the first 5′ target primer,
• f) optionally, isolating the amplified double-stranded copies produced in step e),
• g) PCR amplifying the double-stranded copies produced in step e) or isolated in step f) using a third primer set comprising a first sequencing primer and a second sequencing primer, wherein the first sequencing primer has a sequence corresponding to a first paired-end sequencing primer and the second sequencing primer has a sequence corresponding to a second paired-end sequencing primer,
• h) optionally, isolating the amplified double-stranded copies produced in step g), and
• i) subjecting the double-stranded copies produced in step g) or isolated in step h) to paired-end sequencing using the first paired-end sequencing primer and the second paired-end sequencing primer.