A One-Step Synthesis of DNA Dumbbells Tunnels Short-Read Libraries into a Long-Read Format

DNA dumbbells have many applications. For example, they can be templates for rolling-circle amplification to produce highly accurate long-read sequences of single molecules. We have developed a highly efficient and simple method for tunneling short-read Illumina sequencing libraries into long-read dumbbells. A 5-exonuclease used in Gibson Assembly converted fixed Illumina P5/P7 sequences into sticky ends. Self-looping oligonucleotides with complementary 3-overhangs were ligated to form dumbbells. The reaction proceeded in one pot and modified only end nucleotides of input libraries. Resulting dumbbells were ready for Phi29-dependent rolling circle amplification. Demonstrative readouts of full-length 16S rDNA from a standard microbial community, obtained on a Pacific Biosciences (PacBio) platform, confirmed successful tunneling of initially P5/P7-ended sequencing libraries. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on a PacBio platform.