Methods and materials for assessing nucleic acids

A method, comprising:

• a) amplifying a plurality of adapter-ligated double-stranded cell-free DNA fragments (ALDScfDFs) with first and second universal primers,
• wherein each of said ALDScfDFs comprise:
• a target Watson strand hybridized to a target Crick strand, a universal 3′ adapter attached to a 3′ end of said target Watson and Crick strands, and a universal 5′ adapter attached to a 5′ end of said target Watson and Crick strands, wherein said universal 3′ adapters each comprise a universal 3′ adapter sequence,
• wherein attachment of said universal adapters provides a double stranded unique identifier (dsUID) sequence on each end of each of said ALDScfDFs, wherein one strand of each dsUID is generated in situ after said universal 3′ adapters are attached to the strands and prior to said amplifying,
• wherein said first universal primer is complementary to said universal 3′ adapter and said second universal primer is complementary to the complement of said universal 5′ adapter,
• wherein said amplifying in step a) generates a duplex sequencing library sample comprising:
• i) a plurality of dsUID-labelled double-stranded templates (dsUID-dsTs);
• b) amplifying a first aliquot from said duplex sequencing library sample with a first set of Watson target-selective primer pairs comprising:
• (i) a first Watson target-selective primer comprising a sequence complementary to the universal 3′ adapter, and (ii) a second Watson target-selective primer comprising a first Watson target-selective sequence, thereby:
• i) generating dsUID labelled double-stranded target Watson amplification products (dsUID-dsTWAPs) and ii) not generating dsUID labelled double-stranded target Crick amplification products (dsUID-dsTCAPs) in said first aliquot;
• c) amplifying said dsUID-dsTWAPs with a second set of Watson target-selective primers comprising:
• (i) a third Watson target-selective primer comprising:
• A) a sequence complementary to said universal 3′ adapter, and B) a Watson sample index barcode, and (ii) a fourth Watson target-selective primer comprising, in 5′ to 3′ direction:
• A) an R1 sequencing primer binding site, and B) a second Watson target-selective sequence that is nested with respect to said first Watson target-selective sequence, thereby creating a first sample comprising a plurality of dsUID-labelled double-stranded target Watson library members (dsUID-dsTWLMs);
• d) amplifying a second aliquot from said duplex sequencing library sample with a first set of Crick target-selective primer pairs comprising:
• (i) a first Crick target-selective primer comprising a sequence complementary to the complement of said universal 5′ adapter, and (ii) a second Crick target-selective primer comprising a first Crick target-selective sequence that comprises the same target-selective sequence as said first Watson target-selective sequence, thereby:
• i) generating dsUID-dsTCAPs and ii) not generating said dsUID-dsTWAPs in said second aliquot;
• e) amplifying said dsUID-dsTCAPs with a second set of Crick target-selective primers comprising:
• (i) a third Crick target-selective primer comprising:
• A) a sequence complementary to the complement of said universal 5′ adapter, and B) a Crick sample index barcode, and (ii) a fourth Crick target-selective primer comprising, in 5′ to 3′ direction:
• A) an R2 sequencing primer binding site, and B) a second Crick target-selective sequence that is nested with respect to said first Crick target-selective sequence and comprises the same target-selective sequence as said second Watson target-selective sequence, thereby creating a second sample comprising a plurality of dsUID-double-stranded target Crick library members (dsUID-dsTCLMs);
• f) pooling said first sample and said second sample to generate a combined sample comprising dsUID-dsTWLMs and dsUID-dsTCLMs;
• g) sequencing said dsUID-dsTWLMs and dsUID-dsTCLMs from said combined sample employing R1 and R2 sequencing primers to generate:
• i) a plurality of UID-labeled Watson strand reads that comprise:
• A) an R1 read sequence, B) a target Watson strand read, and C) a Watson sample index barcode read, and
• ii) a plurality of UID-labeled Crick strand reads that comprise:
• A) an R2 read sequence, B) a target Crick strand read, and C) a Crick sample index barcode read;
• and
• h) processing said plurality of UID-labeled Watson strand reads and said UID-labeled Crick strand reads with a computer system to identify the presence of a mutation in one of said ALDScfDFs, and identifying a mutation as present when present in said target Watson strand read and a corresponding mutation is present in said target Crick strand read.