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bioRxiv · 10.1101/2022.05.01.490243

One-Step Making of DNA Dumbbells Allows Tunneling of Short-Read Libraries into a Long-Read Format

Chang, Y.-H.·Liu, H.-H.
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Abstract

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.

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Chang, Y.-H., Liu, H.-H.. 2022-05-03. One-Step Making of DNA Dumbbells Allows Tunneling of Short-Read Libraries into a Long-Read Format. https://doi.org/10.1101/2022.05.01.490243

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