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Next-generation sequencing library preparation from single-stranded DNA

Next-generation sequencing (NGS) of ssDNA is an attractive methodology to study ssDNA virus genomes or characterize chromatin openness states. A novel procedure allows attaching an adapter to the 3’ end of ssDNA followed by its conversion into dsDNA. For the attachment of the second adapter on the other end of now dsDNA, T4 DNA ligase-based procedure can be adopted.

This new chemo-enzymatic method named terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azidealkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation), is based on click reaction for adapter conjugation on ssDNA fragments. Firstly, nucleotide analogues Az-ddATP, Az-ddTTP, Az-ddCTP and Az-ddGTP are used for the TdT-mediated incorporation of an azide-functional group on the 3’ site of ssDNA followed by click ligation between 3’-azido and 5’-ethynyl oligodeoxyribonucleotides (ODP). The Klenow exo(-) fragment is used for the synthesis of complementary DNA resulting in the generation of dsDNA strand. T4 DNA ligase is then used to ligate the second adapter to the opposite terminal of the dsDNA. Finally, adapter-tagged products are amplified by PCR and used for Illumina sequencing.

For more details, read the original paper published in Nucleic Acids Research: Miura et al., 2018.

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