Methods for obtaining correctly assembled nucleic acids

A cell-free method of identifying and amplifying a correctly assembled nucleic acid comprising a sequence of interest, comprising:

• (i) ligating adapters to the 5′ and 3′ ends of a plurality of assembled nucleic acids, wherein the adapters comprise an adapter primer binding site and an adapter capture sequence;
• (ii) binding the plurality of assembled nucleic acids to beads, wherein the beads comprise a capture oligonucleotide attached to the surface of the beads that specifically hybridizes to the adapter capture sequence;
• (iii) performing clonal amplification of the plurality of assembled nucleic acids bound to the beads using the capture oligonucleotide as a first primer and a second primer that hybridizes to the adapter primer binding site, wherein amplicons produced by clonal amplification of the assembled nucleic acids are bound to the beads, wherein the beads are compartmentalized during clonal amplification such that the amplicons on each bead are separated from other amplicons on other beads so that the amplicons cannot migrate from one bead to another bead;
• (iv) adding a bead-specific index sequence to free 3′ ends of each clonally amplified assembled nucleic acid and amplicons thereof using split-pool enzymatic DNA synthesis (EDS) to produce a plurality of indexed assembled nucleic acids bound to the beads, wherein the bead-specific index sequence for each bead comprises a different unique identifier sequence;
• (v) adding a reverse priming site to the free 3′ ends of each indexed assembled nucleic acid bound to the beads using EDS;
• (vi) amplifying the plurality of indexed assembled nucleic acids bound to the beads using a primer that hybridizes to the adapter primer binding site and a primer that hybridizes to the reverse priming site added by EDS;
• (vii) sequencing at least a portion of the amplified indexed assembled nucleic acids to identify a correctly assembled nucleic acid comprising the sequence of interest;
• (viii) identifying the bead-specific index sequence of the correctly assembled nucleic acid comprising the sequence of interest;
• and
• (ix) performing nucleic acid amplification using a primer that hybridizes to the bead-specific index sequence of the correctly assembled nucleic acid comprising the sequence of interest, wherein the correctly assembled nucleic acid comprising the sequence of interest is selectively amplified.