Genetics
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Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform
Chapters
Summary August 17th, 2022
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.
Transcript
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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