Biofoundry-scale DNA assembly validation using cost-effective high-throughput long read sequencing

Abstract

Biofoundries are automated high-throughput facilities specialising in the design, construction and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilised for this purpose are restriction digest or PCR followed by fragment analysis, and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package to perform in-depth analysis and generate a detailed report. Our software enables biofoundry scientists and end-users to rapidly analyse sequencing data, without specialised bioinformatics knowledge. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned or edited plasmids, using Nanopore long reads, which can serve as a useful resource for the genetics, synthetic biology and sequencing communities. Author informationAll authors contributed to the design of the sequencing quality control step and pipeline, and the preparation of the manuscript. P.V. wrote the manuscript, designed and implemented the bioinformatics pipeline and interpreted results. S.D. wrote the manuscript, implemented the laboratory protocol and interpreted results. G.S. designed the bioinformatics pipeline. R.F. wrote the manuscript and contributed to the design of the laboratory protocol and pipeline. Address: Edinburgh Genome Foundry (University of Edinburgh), Michael Swann Building, Max Born Crescent, Edinburgh, EH9 3BF, United Kingdom Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/558498v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@a819e5org.highwire.dtl.DTLVardef@3c34ceorg.highwire.dtl.DTLVardef@aef5d8org.highwire.dtl.DTLVardef@8e5d2f_HPS_FORMAT_FIGEXP M_FIG C_FIG