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bioRxiv · 10.1101/2024.11.04.618112

AistSeq: An In-House Tn5-Based Plasmid Sequencing Platform Using A Compact Benchtop Sequencer

Suzuki, H.·Romsuk, J.·Nakamura, A.·Moriwaki, K.·Sugano, S. S.
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Abstract

Sequence verification of plasmids is a fundamental process in synthetic biology. For plasmid sequence verification using next-generation sequencing (NGS) library preparation, Tn5 transposase is widely used. Streamlined sequencing workflow for laboratory-scale applications is important; however, recombinant Tn5 production in-house can be laborious. In this study, we demonstrated that the addition of a large soluble tag was not essential for purification and that the fusion of a His10 tag and protein A was sufficient to purify sufficient amounts of active Tn5 transposase. In addition, we evaluated exonuclease-based genomic DNA digestion for plasmid sequencing from an E. coli lysate and the data analysis pipeline of sequences derived from the Illumina iSeq100 platform for de novo assembly, reference mapping, and annotation. This study proposes a simple workflow of an in-house Tn5-based plasmid Sequencing platform using a compact benchtop sequencer (AistSeq). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/618112v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@f1546corg.highwire.dtl.DTLVardef@1dd947aorg.highwire.dtl.DTLVardef@3efb78org.highwire.dtl.DTLVardef@efbde1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Suzuki, H., Romsuk, J., Nakamura, A., Moriwaki, K., Sugano, S. S.. 2024-11-06. AistSeq: An In-House Tn5-Based Plasmid Sequencing Platform Using A Compact Benchtop Sequencer. https://doi.org/10.1101/2024.11.04.618112

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