Fig. 3: Generation of a complex XNA template library. | Nature Communications

Fig. 3: Generation of a complex XNA template library.

From: Direct high-throughput deconvolution of non-canonical bases via nanopore sequencing and bootstrapped learning

Fig. 3

A Schematic overview of XNA synthesis process, where DNA fragments phosphorylated at the 5’-end and containing the template and barcode sequences undergo enzymatic single-nucleotide insertion of Ds at 3’-terminus. The resulting XNA fragments are then subjected to circular self-ligation followed by PCR using canonical and non-canonical base substrates to obtain the double-stranded XNA. B Figure depicting the two alternative strategies that were explored for enzymatic single-nucleotide insertion, using a 31-mer (Pa) and a 20-mer (mini-hairpin) template. C Gel electrophoresis results for single-nucleotide insertion products using the two alternative strategies. The experiment was performed once. The uncropped gel is provided as a Source Data file. D Boxplots depicting Ds insertion efficiency per pool of templates (N = 16) for the two alternative strategies. Insertion efficiencies were estimated via replacement PCR and Ion PGM sequencing. Boxplots show first and third quartiles (box), median (line), and ± 1.5× the interquartile range (whiskers). Source data are provided as a Source Data file.

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