ENABLING ACCURATE NON-INVASIVE PGT-A
PG-Seq™ Rapid Non-Invasive Kit with High Correlation to Biopsied Embryos
for Illumina® & Thermo Fisher® Ion Torrent® Instruments
The PG-Seq™ Rapid Non-invasive PGT kit has been specifically developed to analyze picogram quantities of DNA (low template DNA) from spent embryo culture media or blastocoelic fluid samples for non-invasive preimplantation genetic testing. Optimization of the kit was performed using non-invasive samples provided to PerkinElmer from 15 labs around the world, each using different embryology protocols and accuracy was assessed by the degree of correlation to embryo biopsies tested with a range of commercially available PGT-A kits.
The kit is a modified version of the PG-Seq™ Rapid kit, which uses a two-step PCR to whole genome amplify and then attach indexes and sequencer-specific adapters to the template DNA, in a fast, 3-hour sample preparation workflow. The kit is available in a formats compatible with Illumina® or Thermo Fisher® Ion Torrent® sequencers. From DNA to data analysis, the kit includes all reagents required for whole genome amplification, indexing, plus PG-Find™ analysis software for automatic calling of aneuploidy and copy number variants.
4328-0010
ILLUMINA PLATFORMS
4329-0010
THERMO FISHER SCIENTIFIC ION TORRENT PLATFORMS
PG-SEQ™ RAPID NON-INVASIVE TECHNOLOGY BENEFITS
Example results from spent embryo culture media; 45,XY,-9 and 47,XX,+21, both concordant with embryo biopsy results.
“From what we’ve observed so far, the results look excellent, and we are looking forward to further developing our non-invasive PGT program.”
WGA REAGENTS
- PCR-grade H20
- WGA Polymerase
- WGA PCR Buffer
- Round 1 Primer
- dNTPs
INDEXED PRIMERS
- Indexed Primers
ADDITIONAL REAGENTS
- Purification Beads
- Resuspension Buffer
Required materials not provided
Pre-PCR Laboratory requirements
- Laminar flow cabinet
- Minicentrifuge
- Pipettes (2, 10, 20, 100, 200, 1000 µl)
- Cold block (4°C)
- Thermocycler (with hotlid & programmable ramp rate to 0.2°C/sec)
- Pipette tips (low binding, barrier filter)
- PCR thin walled reaction tube with flat cap (0.5mL or 0.2mL)
- PCR-grade 96-well plate to suit thermal cycler
- Molecular grade tubes (1.5mL)
- 96-well plate centrifuge
- Adhesive 96-well plate seals
- Vortex
Post-PCR Laboratory requirements
- Magnetic stand for 96-well plates
- 96-well plate, to suit Magnetic stand
- Adhesive 96-well plate seals
- Absolute ethyl alcohol (EtOH, undenatured) to make 80% ethanol
- Molecular grade water
- Ice
- Tris-HCl 200 mM (pH 7.0)
- PhiX Sequencing Control v3 10 nM (cat # FC-110-3001) diluted to 20 pM (Illumina)
- LabChip® GXII Touch™ nucleic acid analyzer and associated reagent kit (PerkinElmer)
- Thermo Fisher® Qubit® Fluorometer and associated reagent kit (Thermo Fisher Scientific)
- Sodium hydroxide 1N
- Illumina® Sequencer (MiSeq® Instrument or MiniSeq® Instrument)
- Illumina® Sequencer related consumables
- Illumina® Sequencer reagent kit (select from):
- Illumina® MiSeq® Reagent Kit v3 (150 cycle, cat # MS-102-3001)
- Illumina® MiSeq® Reagent Micro Kit v2 (300 cycle, cat # MS-103-1002)
- Illumina® MiniSeq® High Output Reagent Kit (75 cycles, cat # FC-420-1001)
Optional materials not provided
- Agarose gel-electrophoresis apparatus
- Electrophoresis power supply
- UV transilluminator or gel documentation instrument
- Multi-channel pipette
- Multi-channel pipette reagent reservoirs
Catalog Numbers
PG-Seq™ Rapid Non-Invasive Kit for Illumina® NGS – 4328-0010
PG-Seq™ Rapid Non-Invasive Kit for Thermo Fisher® Ion Torrent™ NGS – 4329-0010
PGT SUPPORT INFORMATION
PG-Seq™ Rapid Non-Invasive PGT Kit for Illumina® NGS Platforms
- PG-Seq™ Pooling Calculator and Final Pool Calculator
- Sequences of PG-Seq™ Rapid Kit Indexes – Excel / PDF
- Illumina® Samplesheet
PG-Seq™ Rapid Non-Invasive PGT Kit for Thermo Fisher® Ion Torrent™ NGS Platforms
- PKI PG-Seq™ Pooling Calculator and Final Pool Calculator
- Sequences of PG-Seq™ Rapid Kit Barcodes – Excel / PDF
- Samplesheets
MANUAL
- PG-Seq™ Rapid Non-Invasive Kit for Thermo Fisher® Ion Torrent™ Sequencing Manual
- PG-Seq™ Rapid Non-Invasive Kit for Illumina® Sequencing Manual
- PG-Find™ Software User Guide
APPLICATION NOTE
- Non-invasive Preimplantation Genetic Testing – Why it’s an Important New Frontier for IVF
- Non-invasive Preimplantation Genetic Testing – Embryology
FLYER
Q: How should we check the performance of our non-invasive PGT-A program?
A: Since the results from non-invasive PGT-A can vary between different embryology protocols, each IVF center and genetics lab should perform a side by side comparison of invasive and non-invasive PGT-A to understand their own rates of false positive, false negative, no calls, as well as sensitivity and specificity of the test.
Q: Can we analyze and report segmental chromosome changes or mosaicism with non-invasive PGT-A?
A: During development of the PG-Seq™ Rapid Non-Invasive PGT-A kit we were able to detect both segmental aneuploidies and mosaic results, however, we recommend starting with whole chromosome gains and losses first. Once your center and genetics lab are comfortable with the results and reporting parameters, you may have the confidence to increase your resolution and test sensitivity.
Q: How should spent culture media be collected and stored prior to testing?
A: Not unlike embryo biopsy material, it is critical to sample, store and ship spent culture media samples in the best possible way to maintain the integrity of the DNA in the sample. Spent culture media samples should be collected as soon as possible after the embryo has been removed from the dish, a new pipet tip should be used for each media droplet, the media droplet should be carefully placed into a labelled sterile PCR tube and the tube should be held in a biologic -20°C freezer until shipped off for testing or tested internally.
Q: Can I combine biopsy and non-invasive PGT-A samples in the same sequencing run?
A: Yes…all PGT-A kits from PerkinElmer including the PG-Seq™ 2.0, PG-Seq™ Rapid and PG-Seq™ Rapid Non-Invasive kits can be run together in a single sequencing run. This helps to reduce the sequencing cost per sample. The only precaution that needs to be taken is ensuring that bar codes from one sample are not used for other samples as some of the PerkinElmer kits share bar code sequences. If you’re combining Non-Invasive PG-Seq™ samples with other kits, a slight modification of the sample sheet is required. Please contact [email protected] for details.
Q: Why is non-invasive testing of embryos important?
A: There are a number of reasons that non-invasive PGT is important globally, a few of them can be found below…
- Presuming high levels of concordance between cells in the embryo and the SCM, it is possible to see the benefits of PGT-A without the potential harm to embryos with biopsy
- Only about 45-55% of embryos reach the blastocyst stage of development where biopsy typically takes place. Non-invasive testing that does not rely on embryos having a morphology suitable for biopsy and possibly earlier in embryo development would allow all embryos in a cohort to be tested
- Not every IVF center in the world is staffed with embryologists skilled in biopsy techniques and/or tube loading techniques capable of producing reliable invasive PGT-A results
- Some countries have strict limits on embryo manipulation so a truly non-invasive method to analyze embryos might be helpful in these territories
Q: What is the difference between spent culture media (SCM) and blastocoel fluid analysis?
A: Spent culture media analysis, as the name implies, is testing the media that the embryo was grown in while blastocoel fluid analysis is sampling the fluid inside the blastocoelic cavity of the growing blastocyst. Sampling and analysis of spent culture media is truly non-invasive to the growing embryo while sampling blastocoel fluid requires embryo manipulation to reach the blastocoelic cavity, so is semi-invasive.
Q: Where does the DNA in SCM come from?
A: Current thinking in the field is that the DNA in the blastocoel fluid and in the spent culture media come from programmed cell death through apoptosis. As more data emerges showing fairly high concordance rates between non-invasive PGT-A and biopsy-based PGT-A it appears that apoptosis is not only occurring in aneuploid cells but is occurring in euploid cells as well. There is a lot more research that needs to be done to answer this question.
Q: Are there recommendations for changing my embryology protocols for non-invasive iPGT-A?
A: Yes and no…there are no specific restrictions however during development of the PG-Seq™ Rapid Non-Invasive PGT-A kit we did note a few things that should be taken into account as you develop your plan for introducing niPGT-A into your lab.
- There are many embryo culture media brands available commercially and some labs make their own media. During development of the PerkinElmer PG-Seq™ Non-invasive PGT Kit we tested media from Vitrolife, Origio, Sage and Global. Our data suggests that there is no one brand or manufacturer that performs better or worse than any other.
- There are two main ‘styles’ of media available today, single step (continuous) and two-step media. During development of our kit we tested both types of media and found that the concordance rate with biopsy results was higher because the maternal cell contamination rate was lower in two-step media than in continuous. However, with proper lab technique and a keen eye toward minimizing cumulus cells before placing embryos into culture, continuous media can produce high concordance rates. We also observed that when using continuous culture media, refreshing the culture media on day 3-4 can help to reduce or eliminate the maternal DNA contamination.
- Droplet size of the media is important, and more data is needed, however, in our study we tested droplet sizes between 10 µl and 50 µl and found no significant difference in biopsy concordance at any droplet size.
- Our data suggest that it is optimal to collect spent culture media from day 5 of embryo development onwards. This collection timing allows for the maximum amount of DNA template to be available in the culture media and because there was more template and higher yield from the amplification, the results also showed better QC measures and higher concordance rates during our study.
All of the above mentioned parameters are important to note and understand as centers think about non-invasive PGT-A. The PerkinElmer PG-Seq™ Rapid Non-Invasive PGT-A kit was designed from the ground up to offer clinics the ability to choose their own embryo culture parameters. Much of the data published by the field thus far on non-invasive PGT-A requires the IVF center to make significant changes to lab protocols, some of which might not fit into the normal lab operation timings. Having a kit that has been developed for a wide variety of IVF lab conditions is unique to PerkinElmer.
Q: Does ‘hatching’ the embryo prior to sampling spent culture media improve the concordance results?
A: At this point there is not enough data to understand the answer to this question. There was a trend towards an increase in WGA yield for spent culture media from embryos that had been hatched in the media droplet, however the variability in the results from one embryo to the next and between IVF centers that we saw during development has led us to say that we need more data before we can be sure about whether hatching is necessary.
Q: How important is contamination of the spent culture media?
A: Avoiding contamination of the spent culture media is critically important to the test result. Maternal cell contamination seems to be the largest risk of contamination and can cause false negative results (an aneuploid embryo appears as a euploid female due to maternal cell contamination from cumulus cells). Changing the media around day 3-4 of embryo development minimizes maternal cell contamination but does not completely eliminate the issue without also trying to remove the initial source of contamination. Each genetic laboratory should work with all of the referring IVF centers to understand culture conditions and concordance rates between niPGT-A and biopsy-based PGT-A before offering the service to IVF couples. It is critical to include both positive and negative controls in every sequencing run for PGT-A to ensure that the media and lab are not additional sources of contamination.
Q: What recommendations does PerkinElmer have before implementing non-invasive PGT-A as a routine practice in our lab?
A: We recommend that each lab benchmarks its spent culture media results through concordance analysis side by side with biopsy-based analysis by running both systems concurrently. This will allow for precise measurement of the true concordance rate and false positive/false negative rates over multiple samples. Depending on the baseline concordance levels and your target performance parameters, you may consider changes to the culture protocol to improve concordance levels. This may include extending the amount of time the embryo spends in media before sampling, decreasing the culture droplet size, reviewing the timing of media changes, focusing on more stringent clearing of cumulus cells before culture, the use of ICSI vs. conventional insemination and introducing aseptic techniques in the lab.
Q: Can I combine blastocoel fluid testing and/or invasive PGT-A testing samples in the same sequencing run?
A: Yes…all PGT-A kits from PerkinElmer including PG-Seq™ 2.0, PG-Seq™ Rapid and PG-Seq™ Rapid Non-Invasive can be run together in a single sequencing run. The only precaution that needs to be taken is ensuring that bar codes from one sample are not used for other samples as some of the PerkinElmer kits share bar code sequences. If combining Non-Invasive PG-Seq™ samples with other kits, a slight modification of the sample sheet is required. Please contact [email protected] for details.
The shelf life of all reagents is 6 months when stored properly at -20°C. The PG-Seq™ Rapid Non-invasive PGT kit ships on ice packs.
Catalog Numbers
PG-Seq™ Rapid Non-Invasive Kit for Illumina® NGS – 4328-0010
PG-Seq™ Rapid Non-Invasive Kit for Thermo Fisher® Ion Torrent™ NGS – 4329-0010