Name: semiconductor sequencing for preimplantation genetic testing for aneuploidy
Uploaded: 2019-08-25T13:00:00
Description: Watch this Scientific Journal Video about Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy at JoVE.com

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.

Ethical approval was granted by the Joint Chinese University of Hong Kong&#8213;New Territories East Cluster Clinical Research Ethics Committee (Reference Number: 2010.432). Research license was approved by the Council on Human Reproductive Technology of Hong Kong (Number R3004).
1. Whole genome amplification
<ol>
	<li>Prior to start, check the volume of magnetic beads (Table of Materials) to ensure that there is no less than 135 &#181;L (with a 20% excess) for each sample. ...

<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>

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 ...

The protocol we present here is a rapid and low-cost semi-conductor, sequencing-based methods for screening of aneuploidy in embryos. The refined protocol for performing template amplification, and the arrangements of sequencing library makes the PGTA detection reproducible, high throughput, cost efficient and time saving. The runtime of this semi-conductor sequencer is only two to four hours.Shortening the turnaround time from receiving samples to issuing reports into five days. This method provides genome sequencing for aneuploidy screening, copy number variation, and single nucleotide polymorphism callings. Due to different sequencing chemistry, the library prepared by this protocol cannot be sequenced directly on other sequencing systems.But the same sequencer, but different library preparation, it is capable to do non invasive prenatal screening. Demonstrating the procedure will be Xiangchun Guo, a technician from our laboratory. To begin sequencing, vortex each diluted library and briefly spin four times on the mini centrifuge for three seconds each time.Then, take five microliters of each library, to pool into the nucleus-free, one point five milliliter tube. Vortex the mixed library and briefly spin on a mini centrifuge for three seconds. Add 150 microliters of the breaking solution, to two new recovery tubes.Install the new recovery tubes, the recovery router and the amplification plate. Mix by inverting the oil bottle three times. Ensure that both the oil and recovery solution are at least two thirds full.Vortex the master mix PCR buffer for 30 seconds and briefly spin on a mini centrifuge for three seconds. Vortex the sphere particles and the mixed library for one minute and briefly spin on a mini centrifuge for three seconds. Next, prepare a 2400 microliter ligation mix, by adding 172 microliters of nuclease-free water, eight microliters of the mixed library, 120 microliters of the enzyme mix, and 100 microliters of the sphere particles, to the tube containing 2000 microliters of the master mix PCR buffer.Set a pipette to 800 microliters and load the ligation mix to the reaction filter through the sample port. Invert the oil bottle three times and use a P1000 pipette to add 200 microliters of the reaction oil to the reaction filter. Select the program Proton and then select the assisted button, to ensure that the device has been set up correctly, by following the instructions on the monitor.Then click Next to start the program. Load 100 microliters of the emulsion PCR product sample, 130 microliters of the washed beads, 300 microliters of the ES wash solution, and 300 microliters of the melt-off solution into the eight tube strip. Place the eight tube strip onto the enrichment system.Install a pipette tip and a new zero point two milliliter tube and start the program. Centrifuge the zero point two milliliter tube at 15, 500 times G for five minutes. Discard the supernatant, and keep ten microliters of the enrichment product.Add 200 microliters of nuclease-free water to the tube and mix by vortexing. Centrifuge the zero point two milliliter tube again. Discard the supernatant and keep 10 microliters of the enrichment product.Add 90 microliters of nuclease-free water to the tube and mix by vortexing. To prepare the template, vortex the positive control and spin briefly. Add five microliters of the positive control to 100 microliters of the enrichment product.Vortex and centrifuge at 15, 500 times G for five minutes. Discard the supernatant and keep 10 microliters of the template. Next, add 20 microliters of the sequencing primer, and 15 microliters of the annealing buffer to the template tube.Vortex the tube and briefly spin on a mini centrifuge for three seconds. Incubate the tube in a thermal cycler with a heated lid. Then add 10 microliters of the loading buffer to the tube.Mix by pipetting up and down. Mix the 55 microliters of sample by pipetting up and down and load the sample to the loading well of the chip. Keep the used pipette tip and the zero point two milliliter PCR tube.Place the chip onto the chip mini centrifuge when some sample enter the chip. Check the position and centrifuge the chip for 10 minutes. Prepare 50%annealing buffer by adding 500 microliters of annealing buffer and 500 microliters of nuclease-free water into a one point five milliliter tube.Prepare the flushing solution, by adding 500 microliters of annealing buffer and 500 microliters of 100%2-propanol into a one point five milliliter tube. Then, prepare the foaming mixture, by adding 49 microliters of the 50%annealing buffer and one microliter of the foaming solution to two new one point five milliliter tubes. Pipette air into one of the foaming mixtures, and load 120 microliters of the bubbles into the loading well.Transfer the excessive expelled liquid from the exit well to the loading well, and centrifuge the chip for 30 seconds on the chip mini centrifuge. Repeat the process for the second foaming mixture. Add 55 microliters of the 50%annealing buffer to the kept at zero point two milliliter tube.Use the kept pipette tip to pipette up and down. Load all 55 microliters of the annealing buffer to the loading well. Centrifuge the chip for 30 seconds on the designated chip mini centrifuge.Load 100 microliters of the flushing solution into the chip loading well and discard the expelled liquid from the exit well. Repeat this loading step once. Load 100 microliters of the 50%annealing buffer, into the chip loading well.Repeat this loading step for a total of three times. Add 60 microliters of the 50%annealing buffer and six microliters of the sequencing enzyme into a new one point five milliliter tube. Mix by pipetting up and down.Slowly pipette 65 microliters of this mixed solution into the chip loading well, avoiding bubbles. Keep the chip away from light and incubate at room temperature for five minutes. Finally, after the incubation, immediately load the chip onto the sequencer and click Start The Sequencing Run on the screen to start sequencing.A retrospective statistical analysis on 186 cleavage stage and 1135 blastocyst stage embryos resulted in over 95%WGA success rates in both types of samples. Sequencing quality control failure rates were 3.4%in the cleavage stage group and only 1.9%in the blastocyst stage group. Beside aneuploidy, the sequencing data can be used for other analysis.For example, copy number variations with a size less than five megabase, or mycondrial DNA analysis, depending on the sequencing depth and quality. With the improvement in copy number calling, researchers can further investigate the future of chromosomal mosaicism in human early stage embryos.

Research

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Genetics

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Pooled CRISPR-Based Genetic Screens in Mammalian Cells

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