Methods and materials for assessing nucleic acids

A method of preparing a duplex sequencing library, the method comprising:

• a) attaching 3′ adapters to 3′ ends of a plurality of both Watson and Crick strands of a plurality of double-stranded DNA fragments of an analyte DNA sample obtained from cell-free DNA (cfDNA), whereby the plurality of Watson and Crick strands are UID labeled and uniquely identified,
• wherein a first strand of the 3′ adapters comprises, in the 5′-3′ direction,
• (i) a first segment,
• (ii) a single stranded UID (ssUID) sequence from a set of ssUIDs,
• (iii) an annealing site for the universal 5′ adapter, and
• (iv) a universal 3′ adapter sequence, and
• wherein the second strand of the 3′ adapters comprises, in the 5′ to 3′ direction,
• (i) a segment complementary to the first segment, and
• (ii) a 3′ blocking group,
• b) annealing universal 5′ adapters to the annealing sites, wherein the universal 5′ adapters comprise, in the 5′ to 3′ direction,
• (i) a universal 5′ adapter sequence that is not complementary to the universal 3′ adapter sequence, and
• (ii) a sequence complementary to the annealing site for the universal 5′ adapter;
• c) extending the universal 5′ adapters across ssUID sequences and first segments, thereby generating complements of the ssUID sequences and first segments to obtain extended universal 5′ adapters;
• and
• d) covalently linking the 3′ end of the complements of the first segments to the 5′ ends of the Watson and Crick strands of the double-stranded cfDNA fragments having 3′ adapters attached to the 3′ ends, thereby attaching extended universal 5′ adapters to the 5′ ends of the Watson and Crick strands to obtain adapter-double-stranded cell-free DNA fragments (ADScfDFs) for the Watson and Crick strands;
• and
• e) amplifying the ADScfDFs with a first universal primer complementary to the universal 3′ adapter sequence and a second universal primer complementary to the complement of the universal 5′ adapter sequence to obtain a duplex sequencing library.