Name: pre-implantation genetic testing for aneuploidy on a semiconductor based next-generation sequencing platform
Uploaded: 2022-08-17T12:50:00
Description: Watch this Scientific Journal Video about Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform at JoVE.com

We would like to thank Dr. Zhangyong Ming and Mr. Rongji Hou for their advice on LIMS expanded application. This study is supported by PLA Special Research Projects for Family Planning (17JS008, 20JSZ08), Fund of Guangxi Key Laboratory of Metabolic Diseases Research (No.20-065-76), and Guangzhou Citizen Health Science and Technology Research Project (201803010034).

All protocols and the trophectoderm (TE) biopsy (1.1.1.1 section) applied in this study were reviewed and approved by the human research ethics committee of No. 924 hospital on September 18th, 2017 (NO: PLA924-2017-59). The patients/participants provided their written informed consent to participate in this study.
1. DNA isolation from human embryo biopsy and whole genomic amplification
<ol>
	<li>Protocol for whole genome amplification9,<su...

<ol>
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Chromosomal aneuploidy of embryos is the cause of a large proportion of pregnancy loss, whether conceived naturally or in vitro fertilization (IVF). In the clinical practice of IVF, it is proposed that screening the embryo aneuploidy and transferring the euploidy embryo could improve the outcome of IVF. Fluorescence in situ hybridization is the earliest technique adopted for sex selection and PGT-A; however, this technique requires more technical expertise from laboratory personnel and is relatively lab...

Aneuploidy is the abnormality in the number of chromosomes by the presence of one or more extra chromosomes or the absence of one or more chromosomes. Embryos that carry some type of aneuploidy, such as the loss of one X chromosome (Turner syndrome), extra copies of autosomes, like trisomies of autosome 21 (Down syndrome), 13 (Patau syndrome), and 18 (Edwards syndrome), or extra sex chromosomes such as 47, XXY (Klinefelter syndrome) and 47, XXX (Triple X syndrome), can survive to term with birth defects1. Aneuploidy is the primary cause of first trimester miscarriages and in vitro fertilization (IVF) failure2. It is reported that the aneuploidy rate could range from 25.4%-84.5% through the different age layers of the natural cycle and medicated control group in IVF practice3.
Next-generation sequencing technology is becoming wildly applied in the determination of genetic information clinically; it provides practical access to genome sequence with efficiency and high throughput. Particularly, next-generation sequencing also revolutionized the diagnosis of disorders with genetic factors and tests for abnormity in the genome4. Using semiconductor sequencing technology to directly transfer chemical signals in sequencing bio-reaction into digital data, the semiconductor-based sequence system provides a direct, real-time detection to sequence data in 3-7 h5,6.
In an IVF procedure, pre-implantation genetic testing (PGT) investigates the genetic profile of the embryo before being transferred into the uterus to improve the IVF outcome and reduce the risk of genetic disorders in newborns1,7. In PGT combined with NGS techniques, genetic material extracted from less than 10 cells is amplified with whole genome amplification kits or an independently developed whole genome amplification reagent. This requires only one step in the amplification phase and does not require pre-amplification, to obtain whole-genome amplification products. Primers or panels for copy number variant and special gene loci sequencing are designed and applied in the library constructed.
A typical workflow of pre-implantation genetic testing-aneuploidy (PGT-A) in NGS involves serial procedures, and requires an intense workload of laboratory personnel8. Some misoperation caused procedure roll-back may lead to undesired loss of both time and resources of the lab. A concise and clear standard operating procedure (SOP) for PGS-NGS workflow is helpful; however, word-format protocols cannot present more detailed information on sample processing, device manipulation, and instruments' settings, which can be visualized in a video protocol. In this article, a validated workflow combined with a visualized demonstration of operating detail could offer more direct and intuitive referring protocols in PGT practice on a semiconductor sequencing platform.
The protocol here describes a method that supports batching up to 16 embryo biopsies in parallel. For larger batches, it is recommended to use a commercial kit-based protocol for semiconductor sequencing, such as Reproes-PGS.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;m Syringe Filter Unit</td>
			<td>Merkmillipore</td>
			<td>Millex-HV</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL DNA LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>30108051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mLDNA LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>30108078</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Anstart Taq Buffer (Mg2+ Plus)</td>
			<td>FAPON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Anstart Tap DNA Polymerase</td>
			<td>FAPON</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP reagent (magnetic beads for dna binding)</td>
			<td>Beckman</td>
			<td>A63881</td>
			<td>https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/pcr/a63881</td>
		</tr>
		<tr>
			<td>Cell Lysis buffer</td>
			<td>Southern Medical University</td>
			<td></td>
			<td>Cell lysis buffer containing 40 mM Tris (pH 8), 100 mM NaCl, 2 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% (v/v) Triton X-100, 5 mM sodium pyrophosphate, 2 mM &#946;-glycerophosphate, 0.1% SDS</td>
		</tr>
		<tr>
			<td>ClinVar</td>
			<td>NCBI</td>
			<td></td>
			<td>https://www.ncbi.nlm.nih.gov/clinvar/</td>
		</tr>
		<tr>
			<td>DNA elution buffer</td>
			<td>NEB</td>
			<td>T1016L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP</td>
			<td>Vazyme</td>
			<td>P031-AA</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Life Technologies</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Guangzhou Chemical Reagent Factory Thermo Fisher Scientific</td>
			<td></td>
			<td>http://www.chemicalreagent.com/</td>
		</tr>
		<tr>
			<td>Independently developed whole genome amplification reagents</td>
			<td>Southern Medical University</td>
			<td></td>
			<td>The reagents consist of the following components: 
			1. Cell Lysis 
			2. Amplification Pre-mixed solution 
			&#160;&#160;&#160; 1) Primer WGA-P2 (10 &#956;M) 
			&#160;&#160;&#160; 2) dNTP (10 mM) 
			&#160;&#160;&#160; 3) 5x Anstart Taq Buffer (Mg2+ Plus) 
			3. Amplification Enzyme 
			&#160;&#160;&#160; 1) Anstart Tap DNA Polymerase (5 U/&#956;L)</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q OT2 200 Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A26434</td>
			<td>Kit mentioned in step 4.2.8</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q Sequencing 200 Kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A26433</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Proton System</td>
			<td>Life Technologies</td>
			<td>4476610</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion Reporter Server System</td>
			<td>Life Technologies</td>
			<td>4487118</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Guangzhou Chemical Reagent Factory</td>
			<td></td>
			<td>http://www.chemicalreagent.com/</td>
		</tr>
		<tr>
			<td>Library Preparation Kit</td>
			<td>Daan Gene Co., Ltd</td>
			<td>114</td>
			<td>https://www.daangene.com/pt/certificate.html</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water</td>
			<td>Life Technologies</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo WGA-P2</td>
			<td>Sangon Biotech</td>
			<td></td>
			<td>5&#39;-ATGGTAGTCCGACTCGAGNNNN 
			NNNNATGTGG-3&#39;</td>
		</tr>
		<tr>
			<td>OneTouch 2 System</td>
			<td>Life Technologies</td>
			<td>4474779</td>
			<td>&#160;Template amplification and enrichment system</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02D-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoPLEX WGA Kit</td>
			<td>Takara Bio USA</td>
			<td>R300671</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Quality Scientific Products</td>
			<td></td>
			<td>https://www.qsptips.com/products/standard_pipette_tips.aspx</td>
		</tr>
		<tr>
			<td>Portable Mini Centrifuge LX-300</td>
			<td>Qilinbeier</td>
			<td>E0122</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q33216</td>
			<td>Fluorometer</td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer server system</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Torrent Suite Software</td>
		</tr>
		<tr>
			<td>Sequencing Reactions Universal Kit</td>
			<td>Daan Gene Co., Ltd</td>
			<td>113</td>
			<td>https://www.daangene.com/pt/certificate.html 
			This kit contains the following components: 
			1. Template Preparation Kit Set 
			 
			1.1 Template Preparation Kit: 
			Emulsion PCR buffer 
			Emulsion PCR enzyme mix 
			Template carrier solution 
			 
			1.2 Template Preparation solutions: 
			Template preparation reaction oil I 
			emulsifier breaking solution II 
			Template Preparation Reaction Oil II 
			Nuclease-free water 
			Tween solution 
			Demulsification solution I 
			Template washing solution 
			C1 bead washing solution 
			C1 bead resuspension solution 
			Template resuspension solution 
			 
			1.3 Template Preparation Materials: 
			Reagent tube I 
			connector 
			Collection tube 
			Reagent tube pipette I 
			Amplification plate 
			8 wells strip 
			Dedicated tips 
			Template preparation washing adapter 
			Template preparation filter 
			 
			2. Sequencing Kit Set 
			 
			2.1 Sequencing Kit: 
			dGTP 
			dCTP 
			dATP 
			dTTP 
			Sequencing enzyme solution 
			Sequencing primers 
			Quality control templates 
			 
			2.2&#160; Sequencing Solutions: 
			Sequencing solution II 
			Sequencing solution IIII 
			Annealing buffer 
			Loading buffer 
			Foaming agent 
			Chlorine tablets 
			C1 bead 
			 
			2.3 Sequencing Materials: 
			Reagent Tube II 
			Reagent tube cap 
			Reagent tube sipper&#160; II 
			Reagent bottle sipper 
			Reagent bottles 
			 
			3. Chip</td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution</td>
			<td>Sigma</td>
			<td>72068-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Life Technologies</td>
			<td>4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-implantation genetic testing for aneuploidy on a semiconductor based next-generation sequencing platform

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

As the sequences plan finishes after the running process in the machine, the sequence server system reports the summary with descriptive information of data generated, chip status, ISP loading rate, and library quality, as shown in Figure 2. In this results demonstration, 17.6 G data in the total base was obtained, and the overall loading rate of ISP was 88% in the total wells of the chip; the heat map showed that the sample was evenly loaded on the total area of the chip (<strong class="xfi...

Watch this Scientific Journal Video about Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform at JoVE.com

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

This protocol presents the procedure from single cell amplification to final reporting data in the pre-implantation genetic testing for aneuploidy on a semiconductor based next generation sequencing platform. This procedure can help viewers have a good understanding of the experimental workflow of PGT-A sequencing and replicate this technique in a diagnostic laboratory with a semiconductor based NGS platform. Begin by pipetting 25 microliters of the whole genome amplification products and transferring them into a 1.5 milliliter centrifuge tube preloaded with 25 microliters of nuclease free water.Vortex and mix the DNA purification beads. Aliquot 50 microliters of this solution into each sample tube. Vortex and centrifuge briefly for five seconds.Set the tube for five minutes at room temperature for the DNA binding process. Insert the 1.5 milliliter centrifuge tube into a magnetic rack. Then, wait till all the magnetic beads are attracted to the side wall of the tube.Carefully transfer the supernatant into a new 1.5 milliliter centrifuge tube. Avoid pipetting out the beads. According to the original sample volume.Pipette 30 microliters of magnetic beads, vortex and centrifuge briefly for five seconds. Set the tube for five minutes at room temperature for the DNA binding process. Insert the 1.5 millimeter centrifuge tubes into the magnetic rack.Then, wait until all the magnetic beads are attracted to the side wall of the tube. Carefully remove and discard the supernatant. Avoid pipetting the beads out.Pipette 300 microliters of 70%ethanol into the 1.5 milliliter centrifuge tube. Then gently rotate the tube twice at a 180-degree angle and move the beads along the tube wall for a thorough wash. Pipette and discard the supernatant.Avoid pipetting the beads out. Repeat the wash procedure once. Place a new amplification plate in the system once the clean program is completed.Insert the collection tubes, each prefilled with 150 microliters breaking solution two into the rotor, then place the bridge on the collection tubes. Add the reaction oil to half of the tube and the emulsifier breaking solution to one third of the tube. Place a new filter for template preparation on a tube rack with the sample portal positioned upwards.Vortex the solution for five seconds and centrifuge briefly for five seconds. Then transfer the solution into the sample portal of the filter after pipetting 800 microliters three times. Centrifuge the tube to decrease bubbles before the last injection.Avoid injecting air into the filter. Inject 200 microliters of reaction oil two, following the mixed solution. Turn the sample portal of the filter downwards and replace the washing adapt with the filter.Insert the sample needle into the center hole of the rotor lid and then press the needle to the bottom. Click on the Run button on the screen and choose the program according to the corresponding kit. Then select Assisted to check all steps.Click on Next till the run is started. The run will take approximately 4.5 hours to finish. Select Next when the program finishes.The system will start a 10-minute centrifugation process. After centrifugation, click the Open Lid button and move the collection tubes to racks. If the procedure does not move to the next step in 15 minutes, press on final spin to re-centrifuge.Remove the supernatant in the collection tube, leaving 100 microliters of solution in the tube. Mix the remaining sample by pipetting and transfer the sample to the new 1.5 milliliter centrifuge tube marked OT.When pipetting the supernatant, avoid touching the bottom of the tube along the sides. Pipette 100 microliters of nuclease free water into each collection tube and transfer the solution to the OT tube washed by repeated pipetting.Add 600 microliters of nuclease free water to the OT tube to make up the total volume of one milliliter. Vortex for 30 seconds in centrifuge at 15, 500 times G for eight minutes. Gently remove the supernatant leaving 100 microliters of solution in the OT tube.Use the tip to discard the oil layer completely. Add 900 microliters of nuclease free water, vortex for 30 seconds and centrifuge at 15, 500 times G for eight minutes. Remove the supernatant until 20 microliters of the solution remains and add the template resuspending solution to make up the volume of 100 microliters.Then vortex for 30 seconds and briefly centrifuge for two seconds. Proceed to the next step within 12 hours of sample processing Load 100 microliters of the diluted library, 130 microliters of C1 beads, 300 microliters of three x template washing solution, and 300 microliters of the melt off solution to the eight well strips. Install the eight well strips on the Enrichment Module, load the pipetting tip, and set the 200 microliters centrifuge tube in the collection position.Click on Start to run the program. Pipette 55 microliters of the sample solution and inject it into the sample hole of the chip. Set the chip on the centrifuge.Keep the notch towards the outside and the sample portal inside. Then, balance it with another used chip and centrifuge for 10 minutes. Prepare the loading solution as described in the text manuscript.Blow 100 microliters of air into the foaming solution. Pipette the solution repeatedly till the bubbles are in a dense, foaming state and keep the foam volume to approximately 250 microliters. Place the chip on the bench after centrifuge.Inject 100 microliters of foam into the sample hole and remove the extruded solution into the out portal. Then, centrifuge the chip briefly for 30 seconds. Repeat this process once.Inject 100 microliters at 50%washing buffer twice and remove the extruded solution in the out portal after every injection. Then inject 100 microliters at 50%annealing buffer three times and remove the extruded solution in the out portal after every injection. Inject 65 microliters at 50%enzyme reaction buffer, avoiding bubbles and remove the extruded solution in the out portal.Stabilize the loaded chip at room temperature for five minutes and install the chip on the sequencer chip portal. Choose the Planned program check the information and start the run. The representative run obtained a total of 17.6 gigabyte data and the overall loading rate of the ion sphere particle was 88%in the total wells of the chip.The heat map confirmed even sample loading on the total chip area. 77%of the total reads were usable. All wells loaded with ISP showed 100%templates enrichment in which 78%were clonal.Out of all clonal templates 97%were qualified as the final library. The average length of the read was 177 base pairs while the median and mode were 176 and 174 base pairs, respectively. Peak counts of thymine, cytosine and adenine in the templates were approximately 76 over the qualified cutoff line of 50.In all addressable wells with live ISPs 99.5%qualified as the constructed library and 20%of polyclonal and low quality libraries were filtered out while 76.6%of ISPs were qualified for further analysis. The result of copy number variance from a single sample depicted a 182.16 megabase pairs mosaic trisomy of P 16.3 to Q 35.2 on chromosome four and a 33 point 13 megabase pairs monosomy segment of Q 11.1 to Q 13.33 on Chromosome 22. When performing fragment selection, time intervals between steps should be modified according to the size of samples in the same batch to prevent over and lack of exposure for front and rear end samples.

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