Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2',3'-dideoxynucleotides

Abstract

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2',3'-dideoxynucleotide (dd^ONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of dd^ONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate dd^ONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.

Fig. 1. Comparison of library preparation strategies.

Previously reported workflow: a schematic representation of chemical ribose-to-ribose ligation: i) templated click reaction; ii) non-templated click reaction, b workflow for NGS library preparation using ribose-to-ribose ligation. Our reported workflow: c schematic ligation, PEX/termination by dd^ONNTP and d NGS library preparation workflow using dd^ONNTP.

Fig. 2. The general principles of chemical conjugation of nucleic acids.

a Previously reported ribose-to-ribose conjugated analogues, b our proposed nucleobase-phosphate conjugation analogues, c synthesis of azido group-bearing ddNTPs, d oligo-tethered ddNTP derivatives and e dd^ONNTP enzymatic incorporation and read-through.

Fig. 3. dd^ONNTP incorporation by DNA polymerases.

Electropherograms showing PEX results obtained using various DNA polymerases with either dd^N3NTPs, dNTPs, or dd^ONNTPs in the presence of the following templates: a Dup^A, b Dup^T, c Dup^G, d Dup^C. NC - negative control.

Fig. 4. Semi-targeted sequencing of the M13mp18 viral genome.

a A schematic overview of library preparation with dd^ONNTPs. b M13mp18 genome coverage. The reads concentrated at two loci with one terminus of sequenced inserts fixed at the specific priming sites. Another terminus corresponds to the stochastic positions of dd^ONUTP incorporation. The orange and blue lines represent technical replicates. c Base composition of sequenced reverse reads. The dominance of A base at the first position indicates a seamless copying of the template around the linker position.