# AistSeq: An in-house easy-to-purify Tn5-based plasmid sequencing platform using a compact benchtop sequencer

## TL;DR

This paper introduces AistSeq, a simplified in-house plasmid sequencing method using Tn5 transposase and a compact sequencer for efficient sequence verification in synthetic biology.

## Contribution

A simplified Tn5 transposase purification method and workflow for plasmid sequencing using a benchtop sequencer.

## Key findings

- A His10 tag and protein A fusion is sufficient for active Tn5 transposase production.
- Exonuclease-based DNA digestion and Illumina iSeq100 data analysis enable effective plasmid sequencing.
- AistSeq provides a streamlined, in-house plasmid sequencing platform for laboratory use.

## 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 yield active Tn5 transposase in adequate amounts. 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 easy-to-purify Tn5-based plasmid sequencing platform using a compact benchtop sequencer (AistSeq).