{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T18:23:41Z","timestamp":1793384621252,"version":"4.5.0"},"reference-count":17,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2022,2,28]],"date-time":"2022-02-28T00:00:00Z","timestamp":1646006400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["16H06328"],"award-info":[{"award-number":["16H06328"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns5:p>\n                    <ns5:bold>Background<\/ns5:bold>\n                    : 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.\n                  <\/ns5:p>\n                  <ns5:p>\n                    <ns5:bold>Methods<\/ns5:bold>\n                    : 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.\n                  <\/ns5:p>\n                  <ns5:p>\n                    <ns5:bold>Results:<\/ns5:bold>\n                    <ns5:bold\/>\n                    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.\n                  <\/ns5:p>\n                  <ns5:p>\n                    <ns5:bold>Conclusions<\/ns5:bold>\n                    : 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.\n                  <\/ns5:p>","DOI":"10.12688\/f1000research.109251.1","type":"journal-article","created":{"date-parts":[[2022,2,28]],"date-time":"2022-02-28T06:00:15Z","timestamp":1646028015000},"page":"240","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":25,"title":["Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing"],"prefix":"10.12688","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2355-5888","authenticated-orcid":false,"given":"Andrew W.","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alejandro","family":"Villar-Briones","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5293-4778","authenticated-orcid":false,"given":"Nicholas M.","family":"Luscombe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7410-6295","authenticated-orcid":false,"given":"Charles","family":"Plessy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2022,2,28]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"793","DOI":"10.1186\/s12864-019-6134-y","article-title":"Complete nontuberculous mycobacteria whole genomes using an optimized DNA extraction protocol for long-read sequencing.","volume":"20","author":"J Bouso","year":"2019","journal-title":"BMC Genomics."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"563-569","DOI":"10.1038\/nmeth.2474","article-title":"Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.","volume":"10","author":"C Chin","year":"2013 Jun","journal-title":"Nat. Methods."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"133-138","DOI":"10.1126\/science.1162986","article-title":"Real-Time DNA Sequencing from Single Polymerase Molecules.","volume":"323","author":"J Eid","year":"2009","journal-title":"Science."},{"key":"ref4","first-page":"120-124","article-title":"A comparative evaluation of four DNA extraction protocols from whole blood sample.","volume":"62","author":"M Ghaheri","year":"2016","journal-title":"Cell. Mol. Biol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"6715-6719","DOI":"10.1073\/pnas.77.11.6715","article-title":"Maternal inheritance of human mitochondrial DNA.","volume":"77","author":"R Giles","year":"1980","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"338-345","DOI":"10.1038\/nbt.4060","article-title":"Nanopore sequencing and assembly of a human genome with ultra-long reads.","volume":"36","author":"M Jain","year":"2018 Apr","journal-title":"Nat. Biotechnol."},{"key":"ref7","doi-asserted-by":"publisher","DOI":"10.1101\/2020.11.25.392936","article-title":"Robotic Search for Optimal Cell Culture in Regenerative Medicine.","author":"G Kanda","year":"2020.11.25.392936","journal-title":"bioRxiv."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"13770-13773","DOI":"10.1073\/pnas.93.24.13770","article-title":"Characterization of individual polynucleotide molecules using a membrane channel.","volume":"93","author":"J Kasianowicz","year":"1996","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref15","doi-asserted-by":"publisher","DOI":"10.5281\/zenodo.5921205","article-title":"Nanopore MinION Run Metrics and genomic DNA fragment size analysis data from automated phenol-chloroform extractions (RBI LabDroid Maholo).","author":"A Liu","year":"2022a","journal-title":"Zenodo."},{"key":"ref16","doi-asserted-by":"publisher","DOI":"10.5281\/zenodo.5733820","article-title":"Job files for automated phenol-chloroform extraction (RBI LabDroid Maholo).","author":"A Liu","year":"2022b","journal-title":"Zenodo."},{"key":"ref17","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.19189466.v1","article-title":"Video of robot-assisted organic extraction of DNA (RBI LabDroid Maholo). f1000research.com. Media.","author":"A Liu","year":"2022"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"209-217","DOI":"10.1177\/2472630320972109","article-title":"A variable scheduling maintenance culture platform for mammalian cells.","volume":"26","author":"K Ochiai","year":"2020","journal-title":"SLAS (Society for Laboratory Automation and Screening) Technol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"619-629","DOI":"10.1016\/0926-6550(63)90386-4","article-title":"Preparation of transforming deoxyribonucleic acid by phenol treatment.","volume":"72","author":"H Saito","year":"1963","journal-title":"Biochim. Biophys. Acta."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"43-IN6","DOI":"10.1016\/S0022-2836(59)80006-1","article-title":"A single-stranded deoxyribonucleic acid from bacteriophage \u03c6X174.","volume":"1","author":"R Sinsheimer","year":"1959","journal-title":"J. Mol. Biol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"277-288","DOI":"10.1016\/S0022-2836(61)80069-7","article-title":"The physical characterization of DNA molecules released from T2 and T4 bacteriophage.","volume":"3","author":"C Thomas","year":"1961","journal-title":"J. Mol. Biol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"143067","DOI":"10.1016\/j.scitotenv.2020.143067","article-title":"Applicability of polyethylene glycol precipitation followed by acid guanidinium thiocyanate-phenol-chloroform extraction for the detection of SARS-CoV-2 RNA from municipal wastewater.","volume":"756","author":"S Torii","year":"2021 Feb","journal-title":"Sci. Total Environ."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"33-41","DOI":"10.1016\/0076-6879(87)52007-9","article-title":"Large and small phenol extractions.","volume":"152","author":"D Wallace","year":"1987","journal-title":"Methods Enzymol."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/11-240\/v1\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/11-240\/v1\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/11-240\/v1\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,5,13]],"date-time":"2022-05-13T10:10:12Z","timestamp":1652436612000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/11-240\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,28]]},"references-count":17,"URL":"https:\/\/doi.org\/10.12688\/f1000research.109251.1","relation":{"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.120730.r125732","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.120730.r125733","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.120730.r125733","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.120730.r125732","asserted-by":"object"}],"has-preprint":[{"id-type":"doi","id":"10.1101\/2022.01.26.477939","asserted-by":"object"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,28]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/11-240\/v1#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.120730.r125733, Genki N. Kanda, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan, 08 Mar 2022, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/11-240\/v1#referee-response-125733","order":0,"name":"referee-response-125733","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Andrew W Liu<\/b>; \n<i>Posted: 11 May 2022<\/i>; We thank the reviewer for taking the time to review our work and for the helpful comments.  Regarding the reproducibility, the main source of variability we have found was in the mechanical disruption of samples. In our course of initial optimization with samples after a series of dry runs to determine the workflow, we ran robot to human comparisons with a pool of drosophila DNA for each run. We did experience variability among the three identical samples in the automated run which was the reason we decided to further optimize the protocol. The variability was in the amount of DNA recovered due to leaving larger amounts of aqueous layers behind to prevent organic contamination upon subsequent extractions and precipitations. Also, the order in which the sample was processed played a small role in the variability, as the last sample usually experienced a longer lag time, awaiting the mixing of the previous two samples.  Regarding the throughput, addition of samples beyond a total 3 have not been attempted since the lag time between first and last samples will further increase. We are considering another means to hand-mix multiple samples at the same time, but the constraints of the bench layout in front of Maholo will necessitate remapping\/reworking all current programmed protocols if this concurrent sample processing solution is to be implemented.  Regarding the purity, we ask the reviewer to read our response to the second reviewer which brought up the same comment.","URL":"https:\/\/f1000research.com\/articles\/11-240\/v1#referee-comment-8221","order":1,"name":"referee-comment-8221","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.120730.r125732, Shan Wei, Columbia University Irving Medical Center, New York City, NY, USA, 17 Mar 2022, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/11-240\/v1#referee-response-125732","order":2,"name":"referee-response-125732","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Andrew W Liu<\/b>; \n<i>Posted: 11 May 2022<\/i>; We thank the reviewer for her time to review our work and appreciate the insightful comments.  Regarding the purity, spectrophotometry readings for A260\/280 were 1.9-2.1 and A260\/230 were 2.1-2.6 upon pre-optimization runs in the initial extractions. We decided not to run those further, as the purity (trace to nil phenol contamination) did not pose an obstacle for sequencing. &nbsp; Regarding the pore occupancy on the chip and fully optimized Nanopore runs to obtain our N50 data, Nanopore pore design and library kits are continuously being upgraded. We felt that we just wanted to obtain one metric for the fragment read length and used R9 flow-cells that had gone past their expiration date. (On a more amusing note, Sanger sequencing is something I can wrap my head around but Nanopore based sequencing appears to be cryptic, seemingly advanced alien technology to this old-school technician).","URL":"https:\/\/f1000research.com\/articles\/11-240\/v1#referee-comment-8222","order":3,"name":"referee-comment-8222","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by the Japan Society for the Promotion of Science Grant-In-Aid for Scientific Research (S) [16H06328]. This work was also partly funded through OIST. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}