Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Autor:	Li W; SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA., Miller D; SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA., Liu X; SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA., Tosi L; Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA., Chkaiban L; Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA., Mei H; SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA., Hung PH; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA., Parekkadan B; Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA., Sherlock G; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA., Levy SF; SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA.; Present Address: BacStitch DNA, Los Altos, CA, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Oct 18. Date of Electronic Publication: 2023 Oct 18.
DOI:	10.1101/2023.10.13.562064
Abstrakt:	Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel. Competing Interests: Conflict of interest statement WL, DM, XL, and SFL are inventors on patent applications related to this work. GS and SFL are co-founders of a company, BacStitch DNA, that is commercializing this technology. DM, HM, and SFL are leaving Stanford to become employees of BacStitch DNA.
Databáze:	MEDLINE
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