This study was supported by the General Research Fund (Ref No. 14162417) from Hong Kong, the National Natural Science Foundation of China (Ref No. 81860272), the Major Research Plan of the Provincial Science and Technology Foundation of Guangxi (Ref No. AB16380219), and the China Postdoctoral Science Foundation Grant (Ref No. 2018M630993) from China.

<ol>
	<li>Balaban, B., Urman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oocyte+morphology+on+embryo+development+and+implantation.">Effect of oocyte morphology on embryo development and implantation.</a> Reproductive BioMedicine Online. 12 (5), 608-615 (2006).</li><li>Capalbo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+standard+blastocyst+morphology,+euploidy+and+implantation:+An+observational+study+in+two+centers+involving+956+screened+blastocysts.">Correlation between standard blastocyst morphology, euploidy and implantation: An observational study in two centers involving 956 screened blastocysts.</a> Human Reproduction. 29 (6), 1173-1181 (2014).</li><li>Magli, M. C., Gianaroli, L., Ferraretti, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+abnormalities+in+embryos.">Chromosomal abnormalities in embryos.</a> Molecular and Cellular Endocrinology. 183, 29-34 (2001).</li><li>Trussler, J. L., Pickering, S. J., Ogilvie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+chromosomal+imbalance+in+human+embryos+using+comparative+genomic+hybridization.">Investigation of chromosomal imbalance in human embryos using comparative genomic hybridization.</a> Reproductive BioMedicine Online. 8 (6), 701-711 (2004).</li><li>Fragouli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+analysis+of+human+blastocysts+with+the+use+of+FISH,+CGH+and+aCGH:+Scientific+data+and+technical+evaluation.">Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: Scientific data and technical evaluation.</a> Human Reproduction. 26 (2), 480-490 (2011).</li><li>Calhaz-Jorge, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+reproductive+technology+in+Europe,+2013:+Results+generated+from+European+registers+by+ESHRE.">Assisted reproductive technology in Europe, 2013: Results generated from European registers by ESHRE.</a> Human Reproduction. 32 (10), 1957-1973 (2017).</li><li>Dyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+committee+for+monitoring+assisted+reproductive+technologies+world+report:+Assisted+reproductive+technology+2008,+2009+and+2010.">International committee for monitoring assisted reproductive technologies world report: Assisted reproductive technology 2008, 2009 and 2010.</a> Human Reproduction. 31 (7), 1588-1609 (2008).</li><li>Dahdouh, E. M., Balayla, J., Garc&#237;a-Velasco, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+chromosome+screening+improves+embryo+selection:+A+meta-analysis.">Comprehensive chromosome screening improves embryo selection: A meta-analysis.</a> Fertility and Sterility. 104 (6), 1503-1512 (2015).</li><li>Chen, M., Wei, S., Hu, J., Quan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+Comprehensive+Chromosome+Screening+Technology+Improve+IVF/ICSI+Outcomes?+A+Meta-Analysis.">Can Comprehensive Chromosome Screening Technology Improve IVF/ICSI Outcomes? A Meta-Analysis.</a> PloS One. 10 (10), e0140779 (2015).</li><li>Northrop, L. E., Treff, N. R., Levy, B., Scott, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+microarray-based+24+chromosome+aneuploidy+screening+demonstrates+that+cleavage-stage+FISH+poorly+predicts+aneuploidy+in+embryos+that+develop+to+morphologically+normal+blastocysts.">SNP microarray-based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.</a> Molecular Human Reproduction. 16 (8), 590-600 (2010).</li><li>Barbash-Hazan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preimplantation+aneuploid+embryos+undergo+self-correction+in+correlation+with+their+developmental+potential.">Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential.</a> Fertility and Sterility. 92 (3), 890-896 (2009).</li><li>Fragouli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+cytogenetic+analysis+of+the+human+blastocyst+stage.">Comprehensive cytogenetic analysis of the human blastocyst stage.</a> Fertility and Sterility. 90 (11), S36 (2008).</li><li>Fiorentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+next-generation+sequencing-based+protocol+for+24-chromosome+aneuploidy+screening+of+embryos.">Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos.</a> Fertility and Sterility. 101 (5), 1375-1382 (2014).</li><li>Fiorentino, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+next-generation+sequencing+technology+for+comprehensive+aneuploidy+screening+of+blastocysts+in+clinical+preimplantation+genetic+screening+cycles.">Application of next-generation sequencing technology for comprehensive aneuploidy screening of blastocysts in clinical preimplantation genetic screening cycles.</a> Human Reproduction. 29 (12), 2802-2813 (2014).</li><li>Wells, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utilisation+of+a+rapid+low-pass+whole+genome+sequencing+technique+for+the+diagnosis+of+aneuploidy+in+human+embryos+prior+to+implantation.">Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation.</a> Journal of Medical Genetics. 51 (8), 553-562 (2014).</li><li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tale+of+three+next+generation+sequencingplatforms:+comparison+of+Ion+Torrent,+PacificBiosciences+and+Illumina+MiSeq+sequencers.">A tale of three next generation sequencingplatforms: comparison of Ion Torrent, PacificBiosciences and Illumina MiSeq sequencers.</a> BMC Genomics. 13 (1), 1-13 (2012).</li><li>Goodwin, S., McPherson, J. D., McCombie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coming+of+age:+ten+years+of+next-generation+sequencing+technologies.">Coming of age: ten years of next-generation sequencing technologies.</a> Nature Reviews Genetics. 17 (6), 333-351 (2016).</li><li>Bono, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+semiconductor+next-generation+sequencing-based+protocol+for+preimplantation+genetic+diagnosis+of+reciprocal+translocations.">Validation of a semiconductor next-generation sequencing-based protocol for preimplantation genetic diagnosis of reciprocal translocations.</a> Prenatal Diagnosis. 35 (10), 938-944 (2015).</li><li>Harton, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESHRE+PGD+Consortium/Embryology+Special+Interest+Groupbest+practice+guidelines+for+polar+body+and+embryo+biopsy+for+preimplantation+genetic+diagnosis/screening+(PGD/PGS).">ESHRE PGD Consortium/Embryology Special Interest Groupbest practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).</a> Human Reproduction. 26 (1), 41-46 (2011).</li><li>Liu, W. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+performance+of+MALBAC+and+MDA+methods+in+the+identification+of+concurrent+mutations+and+aneuploidy+screening+to+diagnose+beta-thalassaemia+disorders+at+the+single-+and+multiple-cell+levels.">The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels.</a> Journal of Clinical Laboratory Analysis. 32 (2), 1-8 (2018).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparison+of+the+performance+of+four+whole+genome+amplification+kits+on+ion+proton+platform+in+copy+number+variation+detection.">The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection.</a> Bioscience Reports. 37 (4), BSR20170252 (2017).</li><li>Munn&#233;, S., Grifo, J., Wells, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaicism:+&#8220;survival+of+the+fittest&#8221;+versus+&#8220;no+embryo+left+behind&#8221;.">Mosaicism: &#8220;survival of the fittest&#8221; versus &#8220;no embryo left behind&#8221;.</a> Fertility and Sterility. 105 (5), 1146-1149 (2016).</li><li>Del Carmen Nogales, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+of+chromosome+abnormality+affects+embryo+morphology+dynamics.">Type of chromosome abnormality affects embryo morphology dynamics.</a> Fertility and Sterility. 107 (1), 229-235 (2017).</li><li>Harton, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESHRE+PGD+consortium+best+practice+guidelines+for+amplification-based+PGD.">ESHRE PGD consortium best practice guidelines for amplification-based PGD.</a> Human Reproduction. 26 (1), 33-40 (2011).</li><li>Deleye, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shallow+whole+genome+sequencing+is+well+suited+for+the+detection+of+chromosomal+aberrations+in+human+blastocysts.">Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts.</a> Fertility and Sterility. 104 (5), 1276-1285 (2015).</li><li>Bielanska, M., Tan, S. L., Ao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+mosaicism+throughout+human+preimplantation+development+in+vitro:+incidence,+type,+and+relevance+to+embryo+outcome.">Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome.</a> Human Reproduction. 17 (2), 413-419 (2002).</li><li>Treff, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+targeted+next-generation+sequencing-based+preimplantation+genetic+diagnosis+of+monogenic+disease.">Evaluation of targeted next-generation sequencing-based preimplantation genetic diagnosis of monogenic disease.</a> Fertility and Sterility. 99 (5), 1377-1384 (2013).</li></ol>

The authors have nothing to disclose.

Different from other sequencing chemistries, the sequencer described here uses semiconductor for the detection of nucleotides. The chip itself is an electronic device that detects hydrogen ions by polymerase-driven base incorporation17, which enables 2&#8722;4 h sequencing time of the Proton program. Besides, the chip is a microwell chip that allows the localization of one target molecule, which is different from the flow cell sequencing chemistry by other sequencer providers. This protocol is a m...

Choosing good-quality viable embryos with normal chromosome copy numbers (euploid) for transfer in assisted reproduction helps to improve pregnancy outcomes. Traditionally, the well-established morphological grading system is widely used for embryo evaluation due to its easy availability and non-invasive nature. However, it has been shown that morphological assessment can only provide limited information on embryo quality1 and implantation potential2. One fundamental reason is its inability in evaluating the chromosomal composition of the embryos.
Chromosomal aneuploidy (abnormal copy number of chromosomes) is one of the main causes leading to embryonic development arrest, implantation failure or pregnancy loss. The occurrence of aneuploidy has been well documented in human embryos, accounting for 60%&#8722;70% in cleavage-stage embryos3,4 and 50%&#8722;60% in blastocysts5. This, to some extent, has contributed to the bottleneck in improving the pregnancy rate of in-vitro fertilization (IVF) treatment, which has maintained at around 35%&#8722;40%6,7. Therefore, selecting euploid embryos for transfer is believed to be beneficial for improving pregnancy outcomes. To this end, preimplantation genetic testing for aneuploidy (PGT-A) has been further developed to investigate embryo viability using genetic approaches. There are increasing numbers of randomized controlled trials and cohort studies supporting the crucial role of PGT-A. It has been proved that the application of PGT-A decreases the miscarriage rate and increases clinical pregnancy rate and implantation rate8, ongoing pregnancy rate and live birth rate9.
Historically, different methods have been applied in PGT-A, such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), array-CGH, and single nucleotide polymorphism (SNP)-microarray. Previous studies have indicated that PGT-A for cleavage-stage embryos by FISH yields results that are poorly consistent with those obtained by comprehensive chromosomal screening (CCS) of corresponding blastocysts using 59273array-CGH or SNP-microarray5927310. These discrepancies can be attributed to chromosomal mosaicism, FISH technical artifacts, or embryonic self-correction of chromosomal segregation errors during development11. It has been widely recognized that using blastocyst trophectoderm (TE) biopsies for array-based PGT-A such as array-CGH or SNP-microarray is effective for identifying the chromosomal imbalance in embryos10,12. Recently, single-cell next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A13,14,15, which make it a promising alternative to currently available methods.
Here, we present a fast, robust, and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in human embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, using a single-cell WGA kit, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system.
Through detecting the H+ ions that are released from each deoxyribonucleoside triphosphate incorporation during DNA strand synthesis, the system transfers the chemical signals (pH change) captured by the semiconductor elements to direct digital data, which are further interpreted into DNA sequence information. Eliminating the requirement for expensive optical detection and complex sequencing reactions, this simple sequencing chemistry reduces total reagent cost and shortens the sequencing running time into 2&#8722;4 hours16. More importantly, based on the manufacturer&#8217;s performance specifications, the semiconductor sequencing platform can generate up to 15 GB sequencing data (depends on the quality of library) per run, which is significantly higher than some of the other sequencers producing only around 3&#8722;4 GB data (with 2 x 75 bp read length)17. In clinical applications of PGT-A, this platform can achieve 24 samples per chip generating up to 80 million reads17 and at least one million unique reads of each sample. The read depth can ensure that each sample has at least 0.05x whole genome coverage. The above advantages of this platform make it an ideal screening method and thus, facilitate its wide applications in PGT-A18.

<table>
	<tbody>
		<tr>
			<td>PCR tubes, 0.2 mL</td>
			<td>Axygen</td>
			<td>PCR-02D-C</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>ThermoFisher</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4, Ca2+ and Mg2+ free</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 M NaOH (1.0N) solution</td>
			<td>SIGMA-ALDRICH</td>
			<td>S2567</td>
			<td>For Melt-off solution. Molecular grade</td>
		</tr>
		<tr>
			<td>Eppendorf LoBind Tubes, 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>13-698-791</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>ELGA PURELAB Flex 3 Water Purification System or 
			Equivalent 18 M&#937; water system</td>
			<td>ThermoFisher</td>
			<td>4474524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Amplification buffer</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Amplification enzyme</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>Ion OneTouch Amplification Plate</td>
			<td></td>
			<td></td>
			<td>In kit: Ion OneTouch 2 Supplies (Part No. A26367). Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Ion PI Annealing Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MyOne Beads Capture Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer instrument</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion OneTouch Breaking Solution (black cap)</td>
			<td></td>
			<td></td>
			<td>In kit: Ion PI Hi&#8209;Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>ThermoFisher</td>
			<td>65001</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Cell extraction buffer</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-00.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Cell extraction enzyme</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>Ion PI Chip Kit v3</td>
			<td>ThermoFisher</td>
			<td>A26771</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Chip Minifuge, 230 V</td>
			<td>ThermoFisher</td>
			<td>4479673</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI dATP</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI dCTP</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI dGTP</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoQ&#8211;Temperature Dry Bath</td>
			<td>TAMAR</td>
			<td>HB-T2-A</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext dsDNA Fragmentase</td>
			<td>New England Biolabs</td>
			<td>M0348L</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext dsDNA Fragmentase</td>
			<td>New England Biolabs</td>
			<td>M0348L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI dTTP</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion OneTouch 2 Instrument</td>
			<td>ThermoFisher</td>
			<td>INS1005527</td>
			<td>ThermoFisher Catalog number: 4474778.</td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion One Touch ES</td>
			<td>ThermoFisher</td>
			<td>8441-22</td>
			<td>ThermoFisher Catalog number: 4469495. Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>51976</td>
			<td>This can be replaced by any brand&#39;s molecular grade absolute ethanol.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Extraction enzyme dilution buffer</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-01.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Extraction enzyme dilution buffer</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-02.</td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>ThermoFisher</td>
			<td>Q33216</td>
			<td>This model has been replaced by Qubit 4 Fluorometer, Catalog number: Q33226.</td>
		</tr>
		<tr>
			<td>Qubit ds DNA HS Assay kit</td>
			<td>ThermoFisher</td>
			<td>M2002-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>ThermoFisher</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI Foaming Solution</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Index for barcoding of libraries</td>
			<td>BaseCare</td>
			<td></td>
			<td>this is a in-house prepared index. Users can buy commercial product from ThermoFisher Ion Xpress Barcode Adapters Kits (Cat. No. 4474517)</td>
		</tr>
		<tr>
			<td>Ion PI Loading Buffer</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid(TM) Buffer Kit-1X Low TE Buffer</td>
			<td>ThermoFisher</td>
			<td>4389764</td>
			<td></td>
		</tr>
		<tr>
			<td>Agencour AMPure XP Kit</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet (magnetic rack)</td>
			<td>ThermoFisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI Master Mix PCR buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 17 Microcentrifuge</td>
			<td>Micro 17</td>
			<td>75002430</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>ThermoFisher</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/AM9922">AM9922</a></td>
			<td>This can be replaced by other brand.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Nuclease-free water</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>Ion OneTouch Oil bottle</td>
			<td></td>
			<td></td>
			<td>Ion PI Hi&#8209;Q OT2 Solutions 200 (Part No. A26429). Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td>Extended kit component in Sheet 3</td>
		</tr>
		<tr>
			<td>double-strand DNA standard</td>
			<td></td>
			<td></td>
			<td>This is a in-house prepared DNA standard for calibration of Qubit before quantification of library.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Preamplification buffer</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Preamplification enzyme</td>
			<td>Rubicon Genomics</td>
			<td>R30050</td>
			<td>This can be replaced by SurePlex DNA Amplification System,catalog number: PR-40-415101-03.</td>
		</tr>
		<tr>
			<td>Library Amplification Primer Mix</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td>Extended kit component in Sheet 3</td>
		</tr>
		<tr>
			<td>Ion OneTouch Reaction Filter</td>
			<td></td>
			<td></td>
			<td>Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Recovery Router</td>
			<td></td>
			<td></td>
			<td>Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>Recovery Tubes</td>
			<td></td>
			<td></td>
			<td>Extended kit component in Sheet 5</td>
		</tr>
		<tr>
			<td>ISP Resuspension Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Proton</td>
			<td>ThermoFisher</td>
			<td>DA8600</td>
			<td>This model is imported by Da An Gene Co.,LTD. of Sun Yat-Sen University from ThermoFisher and has been certified by China Food and Drug Administration for clinical application. The catalog number in ThermoFisher is 4476610.</td>
		</tr>
		<tr>
			<td>Ion PI Hi&#8209;Q Sequencing Polymerase</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI Sequencing Primer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>server for sequencer</td>
			<td>Lenovo</td>
			<td>T260</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI Sphere Particles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum PCR SuperMix High Fidelity</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene 25mm Syringe Filters</td>
			<td>ThermoFisher</td>
			<td>724-2045</td>
			<td>Pore size: 0.45&#956;m. Specifically for aqueous fluids.</td>
		</tr>
		<tr>
			<td>Ion PI Hi&#8209;Q W2 Solution</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion PI 1X W3 Solution</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion OneTouch Wash Solution C1</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The Ion PGM Hi&#8209;Q View Sequencing Kit</td>
			<td>ThermoFisher</td>
			<td>A30044</td>
			<td>Extended kit component in Sheet 2</td>
		</tr>
		<tr>
			<td>Ion Plus Fragment Library Kit</td>
			<td>ThermoFisher</td>
			<td>4471252</td>
			<td>Extended kit component in Sheet 3</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q Sequencing 200 Kit (1 sequencing run per initialization)</td>
			<td>ThermoFisher</td>
			<td>A26772</td>
			<td>Extended kit component in Sheet 4</td>
		</tr>
		<tr>
			<td>Ion PI Hi&#8209;Q OT2 200 Kit</td>
			<td>ThermoFisher</td>
			<td>A26434</td>
			<td>Extended kit component in Sheet 5</td>
		</tr>
	</tbody>
</table>

semiconductor sequencing for preimplantation genetic testing for aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Based on this modified protocol, the semiconductor sequencing platform was for the first time, applied for PGT-A. We tested on biopsies from both cleavage-stage blastomeres and blastocyst-stage embryos. It is suggested that the biopsied cells undergo WGA as soon as possible to prevent any degradation of DNA. A previous study compared the performance of different WGA methods and indicated that the method we described here had the best uniformity at the bin size of 100 KB2...

Watch this Scientific Journal Video about Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy at JoVE.com

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

semiconductor-sequencing-for-preimplantation-genetic-testing-for

Research

JoVE Journal

Genetics

9.3K Views.  Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

Video: Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the ...