Methods and compositions for cell-free cloning

A method of generating a population of product polynucleotides, wherein the method comprises:

• a. diluting a subvolume of a source sample comprising at least 1×106 candidate nucleic acid molecules to form a diluted sample having a target number of the candidate nucleic acid molecules isolated from the source sample, wherein the target number is between 10 and 400, wherein the diluted sample comprises one or more tagged candidate nucleic acid species derived from one or more of the candidate nucleic acid molecules, wherein each nucleic acid molecule of a candidate nucleic acid species has an identical nucleic acid sequence comprising the nucleic acid sequence of the one or more candidate nucleic acid molecules from which it was derived and a tag comprising a combination of at least two non-degenerate barcodes selected from a set of between 3 to 200 unique, non-degenerate barcodes;
• b. determining the sequence of at least some of the tagged candidate nucleic acid species using long read sequencing, wherein at least 1 of the tagged candidate nucleic acid species is a desired uniquely tagged nucleic acid species, wherein the desired uniquely tagged nucleic acid species comprises a sequence-perfect desired nucleic acid sequence;
• and
• c. enriching a desired uniquely tagged nucleic acid species by amplifying one or more candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species, using one or more primers that bind to one or more barcodes on the tag associated with the candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species to generate a population of product polynucleotides, wherein the sequence-perfect desired nucleic acid sequence is 1 kb to 50 kb in length, and wherein the enriching the desired uniquely tagged nucleic acid species comprises amplifying one or more tagged candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species using nested PCR with primers that bind two or more of the at least two barcodes on one side of the tagged candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species in successive PCR reactions to generate the population of product polynucleotides.