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.
