{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T03:01:25Z","timestamp":1787022085031,"version":"build-2736575974"},"reference-count":59,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2021,5,20]],"date-time":"2021-05-20T00:00:00Z","timestamp":1621468800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program","doi-asserted-by":"crossref","award":["2018YFC1003100"],"award-info":[{"award-number":["2018YFC1003100"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>\n                    Next Generation Sequencing (NGS) is a powerful tool getting into the field of clinical examination. Its preliminary application in pre-implantation comprehensive chromosomal screening (PCCS) of assisted reproduction (test-tube baby) has shown encouraging outcomes that improves the success rate of\n                    <jats:italic>in vitro<\/jats:italic>\n                    fertilization. However, the conventional NGS library construction is time consuming. In addition with the whole genome amplification (WGA) procedure in prior, makes the single cell NGS assay hardly be accomplished within an adequately short turnover time in supporting fresh embryo implantation. In this work, we established a concise single cell sequencing protocol, ChromInst, in which the single cell WGA and NGS library construction were integrated into a two-step PCR procedure of ~ 2.5hours reaction time. We then validated the feasibility of ChromInst for overnight PCCS assay by examining 14 voluntary donated embryo biopsy samples in a single sequencing run of Miseq with merely 13M reads production. The good compatibility of ChromInst with the restriction of Illumina sequencing technique along with the good library yield uniformity resulted superior data usage efficiency and reads distribution evenness that ensures precisely distinguish of 6 normal embryos from 8 abnormal one with variable chromosomal aneuploidy. The superior succinctness and effectiveness of this protocol permits its utilization in other time limited single cell NGS applications.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0251971","type":"journal-article","created":{"date-parts":[[2021,5,20]],"date-time":"2021-05-20T13:55:26Z","timestamp":1621518926000},"page":"e0251971","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["ChromInst: A single cell sequencing technique to accomplish pre-implantation comprehensive chromosomal screening overnight"],"prefix":"10.1371","volume":"16","author":[{"given":"Fang-Fang","family":"Gao","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shi-Ping","family":"Bo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ya-Xin","family":"Yao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhong-Li","family":"Xu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing-Yu","family":"Ding","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si-Jia","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3351-1910","authenticated-orcid":true,"given":"Jun","family":"Ren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,5,20]]},"reference":[{"issue":"6","key":"pone.0251971.ref001","doi-asserted-by":"crossref","first-page":"e17","DOI":"10.1016\/j.fertnstert.2013.02.034","article-title":"Next-generation sequencing for preimplantation genetic diagnosis","volume":"99","author":"NR Treff","year":"2013","journal-title":"Fertil Steril"},{"issue":"5","key":"pone.0251971.ref002","doi-asserted-by":"crossref","first-page":"1250","DOI":"10.1016\/j.fertnstert.2014.03.006","article-title":"Next-generation sequencing: the dawn of a new era for preimplantation genetic diagnostics","volume":"101","author":"D. Wells","year":"2014","journal-title":"Fertil Steril"},{"issue":"10","key":"pone.0251971.ref003","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1002\/pd.4665","article-title":"Validation of a semiconductor next-generation sequencing-based protocol for preimplantation genetic diagnosis of reciprocal translocations","volume":"35","author":"S Bono","year":"2015","journal-title":"Prenat Diagn"},{"issue":"6","key":"pone.0251971.ref004","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1016\/j.rbmo.2015.09.002","article-title":"Validation of next-generation sequencing for comprehensive chromosome screening of embryos.","volume":"31","author":"A Kung","year":"2015","journal-title":"Reprod Biomed Online."},{"issue":"5","key":"pone.0251971.ref005","doi-asserted-by":"crossref","first-page":"1375","DOI":"10.1016\/j.fertnstert.2014.01.051","article-title":"Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos","volume":"101","author":"F Fiorentino","year":"2014","journal-title":"Fertil Steril"},{"issue":"12","key":"pone.0251971.ref006","doi-asserted-by":"crossref","first-page":"2802","DOI":"10.1093\/humrep\/deu277","article-title":"Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles","volume":"29","author":"F Fiorentino","year":"2014","journal-title":"Hum Reprod"},{"issue":"4","key":"pone.0251971.ref007","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.jgg.2015.03.001","article-title":"The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities","volume":"42","author":"N Li","year":"2015","journal-title":"J Genet Genomics"},{"issue":"1","key":"pone.0251971.ref008","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1097\/01.AOG.0000100153.24061.45","article-title":"Increased infertility with age in men and women.","volume":"103","author":"DB Dunson","year":"2004","journal-title":"Obstet Gynecol"},{"issue":"12","key":"pone.0251971.ref009","doi-asserted-by":"crossref","first-page":"e1001356","DOI":"10.1371\/journal.pmed.1001356","article-title":"National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys.","volume":"9","author":"MN Mascarenhas","year":"2012","journal-title":"PLoS Med."},{"issue":"12","key":"pone.0251971.ref010","doi-asserted-by":"crossref","first-page":"855","DOI":"10.3109\/09513590.2010.501889","article-title":"Ageing and infertility: an overview.","volume":"26","author":"J Balasch","year":"2010","journal-title":"Gynecol Endocrinol"},{"issue":"11","key":"pone.0251971.ref011","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1007\/s10815-013-0092-0","article-title":"Live birth rates and birth outcomes by diagnosis using linked cycles from the SART CORS database.","volume":"30","author":"JE Stern","year":"2013","journal-title":"J Assist Reprod Genet"},{"issue":"5","key":"pone.0251971.ref012","doi-asserted-by":"crossref","first-page":"1356","DOI":"10.1016\/j.fertnstert.2006.04.030","article-title":"Assisted reproductive technologies (ART) in Canada: 2002 results from the Canadian ART Register.","volume":"86","author":"J Gunby","year":"2006","journal-title":"Fertil Steril"},{"issue":"7","key":"pone.0251971.ref013","doi-asserted-by":"crossref","first-page":"1536","DOI":"10.1093\/humrep\/deu084","article-title":"International Committee for Monitoring Assisted Reproductive Technologies world report: Assisted Reproductive Technology 2006","volume":"29","author":"R Mansour","year":"2014","journal-title":"Hum Reprod"},{"issue":"2","key":"pone.0251971.ref014","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1016\/j.fertnstert.2014.11.004","article-title":"International committee for monitoring assisted reproductive technologies: world report on assisted reproductive technologies, 2007","volume":"103","author":"O Ishihara","year":"2015","journal-title":"Fertil Steril"},{"issue":"2","key":"pone.0251971.ref015","first-page":"233","article-title":"Assisted reproductive technology in Europe, 2011: results generated from European registers by ESHRE","volume":"31","author":"European Society of Human R","year":"2016","journal-title":"Hum Reprod"},{"issue":"3","key":"pone.0251971.ref016","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.rbmo.2014.11.015","article-title":"Impact of blastocyst biopsy and comprehensive chromosome screening technology on preimplantation genetic screening: a systematic review of randomized controlled trials.","volume":"30","author":"EM Dahdouh","year":"2015","journal-title":"Reprod Biomed Online"},{"issue":"2","key":"pone.0251971.ref017","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/S1472-6483(10)60866-8","article-title":"Chromosome abnormalities and their relationship to morphology and development of human embryos","volume":"12","author":"S. Munne","year":"2006","journal-title":"Reprod Biomed Online"},{"issue":"Suppl 3","key":"pone.0251971.ref018","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/S1472-6483(12)60113-8","article-title":"The role of preimplantation diagnosis for aneuploidies.","volume":"4","author":"L Gianaroli","year":"2002","journal-title":"Reprod Biomed Online."},{"issue":"5","key":"pone.0251971.ref019","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1097\/GCO.0b013e32832fad73","article-title":"The role of preimplantation genetic diagnosis in diagnosing embryo aneuploidy.","volume":"21","author":"S Munne","year":"2009","journal-title":"Curr Opin Obstet Gynecol"},{"issue":"4","key":"pone.0251971.ref020","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S1472-6483(10)61146-7","article-title":"FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome.","volume":"11","author":"C Rubio","year":"2005","journal-title":"Reprod Biomed Online"},{"issue":"2","key":"pone.0251971.ref021","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1016\/S0015-0282(16)57739-5","article-title":"Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities","volume":"64","author":"S Munne","year":"1995","journal-title":"Fertil Steril"},{"issue":"1","key":"pone.0251971.ref022","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.fertnstert.2008.05.029","article-title":"Preimplantation aneuploidy testing for infertile patients of advanced maternal age: a randomized prospective trial","volume":"92","author":"WB Schoolcraft","year":"2009","journal-title":"Fertil Steril"},{"issue":"5","key":"pone.0251971.ref023","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1016\/j.rbmo.2010.06.020","article-title":"Redefining advanced maternal age as an indication for preimplantation genetic screening.","volume":"21","author":"M Milan","year":"2010","journal-title":"Reprod Biomed Online"},{"issue":"6","key":"pone.0251971.ref024","doi-asserted-by":"crossref","first-page":"1695","DOI":"10.1016\/j.fertnstert.2013.07.2002","article-title":"Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization","volume":"100","author":"GL Harton","year":"2013","journal-title":"Fertil Steril"},{"issue":"12","key":"pone.0251971.ref025","doi-asserted-by":"crossref","first-page":"2806","DOI":"10.1093\/humrep\/den217","article-title":"Preimplantation genetic screening in women of advanced maternal age caused a decrease in clinical pregnancy rate: a randomized controlled trial","volume":"23","author":"T Hardarson","year":"2008","journal-title":"Hum Reprod"},{"issue":"5","key":"pone.0251971.ref026","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1016\/S1472-6483(10)61057-7","article-title":"Maternal age, morphology, development and chromosome abnormalities in over 6000 cleavage-stage embryos.","volume":"14","author":"S Munne","year":"2007","journal-title":"Reprod Biomed Online."},{"issue":"Suppl 1","key":"pone.0251971.ref027","doi-asserted-by":"crossref","first-page":"S29","DOI":"10.1016\/S0303-7207(01)00574-3","article-title":"Chromosomal abnormalities in embryos","volume":"183","author":"MC Magli","year":"2001","journal-title":"Mol Cell Endocrinol"},{"issue":"1","key":"pone.0251971.ref028","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/S1472-6483(10)62055-X","article-title":"Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies.","volume":"6","author":"A Kuliev","year":"2003","journal-title":"Reprod Biomed Online"},{"issue":"3","key":"pone.0251971.ref029","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1007\/s10815-014-0417-7","article-title":"In vitro fertilization with preimplantation genetic screening improves implantation and live birth in women age 40 through 43.","volume":"32","author":"HL Lee","year":"2015","journal-title":"J Assist Reprod Genet"},{"issue":"10","key":"pone.0251971.ref030","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1007\/s10815-013-0070-6","article-title":"Preimplantation genetic screening (PGS) with Comparative genomic hybridization (CGH) following day 3 single cell blastomere biopsy markedly improves IVF outcomes while lowering multiple pregnancies and miscarriages.","volume":"30","author":"MD Keltz","year":"2013","journal-title":"J Assist Reprod Genet"},{"issue":"5","key":"pone.0251971.ref031","doi-asserted-by":"crossref","first-page":"1400","DOI":"10.1016\/j.fertnstert.2012.11.041","article-title":"Preimplantation genetic screening using fluorescence in situ hybridization in patients with repetitive implantation failure and advanced maternal age: two randomized trials","volume":"99","author":"C Rubio","year":"2013","journal-title":"Fertil Steril"},{"key":"pone.0251971.ref032","unstructured":"Using a PhiX Control for HiSeq\u00ae Sequencing Runs. Available from: https:\/\/support.illumina.com.cn\/content\/dam\/illumina-marketing\/documents\/products\/technotes\/hiseq-phix-control-v3-technical-note.pdf."},{"key":"pone.0251971.ref033","unstructured":"miseq denature dilute libraries guide 15039740 v10. Available from: https:\/\/support.illumina.com.cn\/content\/dam\/illumina-support\/documents\/documentation\/system_documentation\/miseq\/miseq-denature-dilute-libraries-guide-15039740-10.pdf."},{"issue":"6114","key":"pone.0251971.ref034","doi-asserted-by":"crossref","first-page":"1622","DOI":"10.1126\/science.1229164","article-title":"Genome-wide detection of single-nucleotide and copy-number variations of a single human cell","volume":"338","author":"C Zong","year":"2012","journal-title":"Science"},{"key":"pone.0251971.ref035","unstructured":"CASAVA. Available from: http:\/\/www.illumina.com\/."},{"issue":"15","key":"pone.0251971.ref036","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: a flexible trimmer for Illumina sequence data","volume":"30","author":"AM Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"pone.0251971.ref037","unstructured":"UCSC. Available from: http:\/\/genome.ucsc.edu\/."},{"key":"pone.0251971.ref038","unstructured":"Picard Available from: http:\/\/picard.sourceforge.net\/."},{"issue":"6","key":"pone.0251971.ref039","doi-asserted-by":"crossref","first-page":"1685","DOI":"10.1016\/j.fertnstert.2014.08.015","article-title":"Validation of multiple annealing and looping-based amplification cycle sequencing for 24-chromosome aneuploidy screening of cleavage-stage embryos","volume":"102","author":"J Huang","year":"2014","journal-title":"Fertil Steril"},{"issue":"14","key":"pone.0251971.ref040","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"H Li","year":"2009","journal-title":"Bioinformatics"},{"issue":"16","key":"pone.0251971.ref041","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"H Li","year":"2009","journal-title":"Bioinformatics"},{"key":"pone.0251971.ref042","unstructured":"The Perl Programming Language. Available from: http:\/\/www.perl.org\/."},{"key":"pone.0251971.ref043","unstructured":"The R Project for Statistical Computing. Available from: http:\/\/www.r-project.org\/."},{"key":"pone.0251971.ref044","article-title":"CNSA: a data repository for archiving omics data.","author":"X Guo","year":"2020","journal-title":"Database: the journal of biological databases and curation"},{"issue":"9","key":"pone.0251971.ref045","doi-asserted-by":"crossref","first-page":"2003","DOI":"10.1093\/humrep\/15.9.2003","article-title":"Healthy births and ongoing pregnancies obtained by preimplantation genetic diagnosis in patients with advanced maternal age and recurrent implantation failure","volume":"15","author":"S Kahraman","year":"2000","journal-title":"Hum Reprod"},{"issue":"2","key":"pone.0251971.ref046","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.fertnstert.2005.02.027","article-title":"Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages","volume":"84","author":"S Munne","year":"2005","journal-title":"Fertil Steril"},{"issue":"10","key":"pone.0251971.ref047","doi-asserted-by":"crossref","first-page":"e0140779","DOI":"10.1371\/journal.pone.0140779","article-title":"Can Comprehensive Chromosome Screening Technology Improve IVF\/ICSI Outcomes? A Meta-Analysis.","volume":"10","author":"M Chen","year":"2015","journal-title":"PLoS One."},{"key":"pone.0251971.ref048","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1186\/s13039-015-0143-6","article-title":"Application of next-generation sequencing for 24-chromosome aneuploidy screening of human preimplantation embryos.","volume":"8","author":"H Zheng","year":"2015","journal-title":"Mol Cytogenet."},{"issue":"3\u20134","key":"pone.0251971.ref049","first-page":"57","article-title":"Comparative study of aCGH and Next Generation Sequencing (NGS) for chromosomal microdeletion and microduplication screening.","volume":"8","author":"CD Russo","year":"2014","journal-title":"J Prenat Med"},{"key":"pone.0251971.ref050","doi-asserted-by":"crossref","first-page":"37","DOI":"10.4137\/CMRH.S10852","article-title":"Preimplantation genetic screening: a practical guide.","volume":"7","author":"PR Brezina","year":"2013","journal-title":"Clin Med Insights Reprod Health."},{"issue":"1","key":"pone.0251971.ref051","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.ijgo.2016.04.007","article-title":"Comparison of perinatal outcomes following fresh and frozen-thawed blastocyst transfer.","volume":"135","author":"N Pereira","year":"2016","journal-title":"Int J Gynaecol Obstet"},{"issue":"5","key":"pone.0251971.ref052","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.1016\/j.fertnstert.2015.01.045","article-title":"Freeze-all policy: fresh vs. frozen-thawed embryo transfer","volume":"103","author":"M Roque","year":"2015","journal-title":"Fertil Steril"},{"issue":"6","key":"pone.0251971.ref053","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1056\/NEJMoa1513873","article-title":"Fresh versus Frozen Embryos for Infertility in the Polycystic Ovary Syndrome","volume":"375","author":"ZJ Chen","year":"2016","journal-title":"N Engl J Med"},{"issue":"6","key":"pone.0251971.ref054","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1016\/j.fertnstert.2016.02.027","article-title":"In vitro fertilization outcomes after fresh and frozen blastocyst transfer in South Asian compared with white women","volume":"105","author":"MS Shah","year":"2016","journal-title":"Fertil Steril"},{"key":"pone.0251971.ref055","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1186\/s13039-016-0238-8","article-title":"A pilot proof-of-principle study to compare fresh and vitrified cycle preimplantation genetic screening by chromosome microarray and next generation sequencing.","volume":"9","author":"GC Ma","year":"2016","journal-title":"Mol Cytogenet."},{"issue":"3","key":"pone.0251971.ref056","doi-asserted-by":"crossref","first-page":"718","DOI":"10.1016\/0888-7543(92)90147-K","article-title":"Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer","volume":"13","author":"H Telenius","year":"1992","journal-title":"Genomics"},{"issue":"6","key":"pone.0251971.ref057","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1101\/gr.180501","article-title":"Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification","volume":"11","author":"FB Dean","year":"2001","journal-title":"Genome Res"},{"issue":"25","key":"pone.0251971.ref058","doi-asserted-by":"crossref","first-page":"14676","DOI":"10.1073\/pnas.93.25.14676","article-title":"Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA","volume":"93","author":"VG Cheung","year":"1996","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"5","key":"pone.0251971.ref059","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1002\/humu.22541","article-title":"A robust approach for blind detection of balanced chromosomal rearrangements with whole-genome low-coverage sequencing","volume":"35","author":"Z Dong","year":"2014","journal-title":"Human mutation"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0251971","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,27]],"date-time":"2022-12-27T23:48:49Z","timestamp":1672184929000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0251971"}},"subtitle":[],"editor":[{"given":"Wei","family":"Cui","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2021,5,20]]},"references-count":59,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5,20]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0251971","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,20]]}}}