A method for preparing a plurality of transpososome complexes, comprising: a) contacting in a single reaction mixture (i) a plurality of transposases with (ii) a plurality of polynucleotides containing a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence comprises a double-stranded nucleic acid having a first attacking strand and a first non-attacking strand, wherein the first attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the first attacking strand, and (iii) a plurality of polynucleotides containing a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence comprises a double-stranded nucleic acid having a second attacking strand and a second non-attacking strand, wherein the second attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the second attacking strand; and b) forming a plurality of transpososome complexes, at least one transposome complex comprising a transposase, and a first transposon end sequence, and at least one transposome complex comprising a transposase and a second transposon end sequence. a) contacting in a single reaction mixture (i) a plurality of transposases with (ii) a plurality of polynucleotides containing a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence comprises a double-stranded nucleic acid having a first attacking strand and a first non-attacking strand, wherein the first attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the first attacking strand, and (iii) a plurality of polynucleotides containing a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence comprises a double-stranded nucleic acid having a second attacking strand and a second non-attacking strand, wherein the second attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the second attacking strand; and (i) a plurality of transposases with (ii) a plurality of polynucleotides containing a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence comprises a double-stranded nucleic acid having a first attacking strand and a first non-attacking strand, wherein the first attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the first attacking strand, and (iii) a plurality of polynucleotides containing a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence comprises a double-stranded nucleic acid having a second attacking strand and a second non-attacking strand, wherein the second attacking strand contains at least one nick or gap which is located after the sixth, eighth, tenth, fourteenth, sixteenth, eighteenth, nineteenth or twenty-seventh nucleotide from the 3' end of the second attacking strand; and b) forming a plurality of transpososome complexes, at least one transposome complex comprising a transposase, and a first transposon end sequence, and at least one transposome complex comprising a transposase and a second transposon end sequence.