Methods and compositions for preparing polynucleotides

A method of forming amplification products of a target polynucleotide, comprising:

• (a) contacting a target polynucleotide present in a polynucleotide sample with transposomes to yield a plurality of tagged fragments, wherein individual transposomes comprise a transposase complexed with a transposon sequence having a transposon element, wherein a given tagged fragment of the plurality comprises the transposon sequence joined to the 5′ end of a segment of the target polynucleotide;
• (b) subjecting said plurality of tagged fragments to an extension reaction using extension primers to yield extension products, wherein individual extension primers have a segment at a 3′ end exhibiting sequence complementarity to a tagged fragment and a segment at a 5′ end lacking sequence complementarity to the tagged fragment, and wherein a given extension product comprises (i) a sequence of the given tagged fragment and a complement of an extension primer sequence, or (ii) a complement of the given tagged fragment sequence and the extension primer sequence, wherein the transposon sequence or the extension primer comprises a methylated cytosine, wherein the methylated cytosine stabilizes a primer-dimer formed by the extension primers;
• and
• (c) amplifying the extension products using a primer pair to yield amplification products, wherein the primer pair includes a first primer comprising the transposon sequence or a portion thereof and a second primer comprising the sequence of the segment at the 5′ end of the extension primer or a portion thereof, wherein individual amplification products comprise a single copy of the transposon sequence or a complement thereof.