Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing

摘要

<ns5:p> <ns5:bold>Background</ns5:bold> : Automation has increasingly become more commonplace in the research laboratory workspace. The introduction of articulated robotic arms allows the researcher more flexibility in the tasks a single piece of automated machinery can perform. We set out to incorporate automation in processing of genomic DNA organic extractions to increase throughput and limit researchers to the exposure of organic solvents. </ns5:p> <ns5:p> <ns5:bold>Methods</ns5:bold> : In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute's Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. </ns5:p> <ns5:p> <ns5:bold>Results:</ns5:bold> <ns5:bold/> We achieved routine extraction of high molecular weight genomic DNA (&gt;100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. </ns5:p> <ns5:p> <ns5:bold>Conclusions</ns5:bold> : We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run is included in this report. </ns5:p>