We physically shear the DNA by sonication using a Covaris acoustic ultrasonicator. The shearing time is varied depending on the size of the genome, with shorter times for small genomes (such as mtDNA or viral DNA) and longer times for nuclear DNA. Specific shearing parameters are given in Equipment Setup below. Other methods such as nebulization or enzymatic digestion should both be compatible with DS, but would probably require optimization. ‘Tagmentation’ methods, which involve simultaneous fragmentation of the sample DNA and ligation of sequencing adapters by a transposase (e.g., Illumina’s Nextera DNA sample prep kit), are currently incompatible with DS because they make use of an invariant transposon sequence that is incompatible with molecular barcodes. Because the DNA strands are not marked with molecular barcodes, it is impossible to identify unique DNA molecules and perform the comparison between complementary strands.
