A high-throughput optomechanical retrieval method for sequence-verified clonal DNA from the NGS platform

Abstract

Writing DNA plays a significant role in the fields of synthetic biology, functional genomics and bioengineering. DNA clones on next-generation sequencing (NGS) platforms have the potential to be a rich and cost-effective source of sequence-verified DNAs as a precursor for DNA writing. However, it is still very challenging to retrieve target clonal DNA from high-density NGS platforms. Here we propose an enabling technology called ‘Sniper Cloning’ that enables the precise mapping of target clone features on NGS platforms and non-contact rapid retrieval of targets for the full utilization of DNA clones. By merging the three cutting-edge technologies of NGS, DNA microarray and our pulse laser retrieval system, Sniper Cloning is a week-long process that produces 5,188 error-free synthetic DNAs in a single run of NGS with a single microarray DNA pool. We believe that this technology has potential as a universal tool for DNA writing in biological sciences.

A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

“…REDI is not the only cost-effective method that leverages highthroughput sequencing to isolate sequence-perfect DNA from arraysynthesized oligo pools (Kim et al, 2012;Schwartz et al, 2012;Lee et al, 2015). For example, dial-out PCR is a method in which random tags are added to oligos and used to identify and then isolate sequence-perfect molecules via high-throughput sequencing and PCR, respectively (Schwartz et al, 2012).…”

Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide

“…Thus, an upgrade of the current optomechanical DNA retrieval system to Illumina’s HiSeq platform would eventually cover all possible combinations (4 N ; N: number of degenerate bases). We have shown the potential for target sequence retrieval based on the MiSeq platform in a previous study5.…”

“…Last, these strings consisted of (0, 1, 2, and 3) were converted to single DNA bases as described before with modification2. Following the sequencing of the degenerate oligonucleotide library, a high-throughput optomechanical system based on non-contact laser pulse5 was used to retrieve a target sequence containing beads from the 454 sequencing plate. Briefly, 454 sequencing plate (chip) is first located in the optical retrieval system.…”

Next generation gene synthesis: From microarrays to genomes

“…(A) Enzymatic error correction uses either mismatch binding proteins with filtration to remove DNA with errors, or enzymes that cleave erroneous DNA. (B) DNA that has been sequenced can be located and physically removed by micropipettes , or lasers . (C) Next generation sequencing of barcoded molecules can identify correct molecules that are then amplified with the barcode as a primer .…”

“…In a faster and more automated approach, the so‐called “sniper cloning” , Lee et al. transferred the beads with a laser pulse from the sequencing plate to a 96‐well plate.…”