PREIMPLANTATION GENETIC TESTING MADE SIMPLE
PG-Seq™ Kit 2.0 & Technology
Go beyond the standard NGS workflows with PG-Seq™ kit 2.0, an NGS workflow solution for Preimplantation Genetic Testing for Aneuploidy (PGT-A) and Preimplantation Genetic Testing for Monogenic Disorders (PGT-M).
The PG-Seq™ kit 2.0 provides MORE than your standard NGS workflow, including:
- A robustly validated PGT-A solution that has been favorably benchmarked
against the other commercially available PGT-A kits - Higher throughput on the same sequencer with no loss on resolution
- A novel combined approach to PGT-A and PGT-M in one NGS workflow
- Amplification of cfDNA in spent culture media for non-invasive PGT-A
- A distinct embryo ID based on mtDNA
A Validated PGT-A Solution
The PG-Seq™ kit 2.0 has been validated on single cell and 5-cell aliquots using cell lines with known ploidy, including euploid, single, and double trisomies. Cell line aberrations (gains and losses) of 7-31 Mb were also included. These structural variants were detected with 98.3% sensitivity and specificity in 5-cell samples.
Higher Throughput of 48 samples on the Same Sequencer with no Loss or Compromise on Resolution
The PG-Seq™ kit 2.0 offers 1 x 75 bp read lengths, double the length offered by the Illumina® VeriSeq™ kit. This additional read length provides 15% more mapped reads per sample and 50% more coverage across the sequenced genome compared to standard 1 x 36 bp 24 sample runs, allowing increased throughput (48 samples per run) with no loss of resolution or accuracy.
| Number of Samples | Number of Mapped Reads per Sample | Smallest CNV Detectable |
|---|---|---|
| 96 | 250,000 | 10Mb |
| 48 | 500,000 | 5Mb |
| 24 | 1,000,000 | 2Mb |
| 8 | 3,000,000 | 1Mb |
| 2 | 12,000,000 | 500Kb |
| 1 | 24,000,000 | 100Kb |
“When we decided to automate our PGT workflow, we considered different alternatives. The PG-Seq™ platform offered by PerkinElmer turned out to be the best solution to our needs.”
A Novel, Combined Approach to PGT-A & PGT-M with One NGS Workflow
PG-Seq™ kit 2.0 offers one workflow that can be used to combine PGT-A & PGT-M together in a single amplification. The workflow can be used to detect relevant mutations including single nucleotide variants (SNV) with a high degree of accuracy. By using DOPlify® Whole Genome Amplification and our proprietary Targeted Sequence Enrichment (TSE) Protocol as part of PG-Seq™ kit 2.0, it is possible to obtain results with sufficient read depth (>200x) for targeted PGT-M even in a low pass NGS workflow designed for PGT-A. This streamlines lab protocols, allows PGT-A and PGT-M results to be sequenced together and keeps the cost per sample affordable.
Allele frequency percentages for three target regions analysed with three different PCR methodologies (n=10 per method). Single colour columns indicate the amplification of one allele only. No column indicates samples excluded due to read depth.
IGV screenshot of targeted HLA-A
Are You Interested in Non-invasive PGT-A?
Powered by DOPlify® technology, the PG-Seq™ kit 2.0 utilizes a unique whole genome amplification approach to successfully amplify cell free DNA in spent embryo culture media.
Our pilot study showed media/biopsy concordance for chromosomal content (ie which chromosomes were affected, not just correlation for aneuploidy) of 95%. Data recently presented at the 2017 American Society of Reproduction Meeting (ASRM) San Antonio USA, showed there was a 93% concordance rate between the biopsy and non-invasive media testing approaches.
Concordant results obtained following NI-PGT-A of spent embryo culture media from a two-step culture compared to the trophectoderm biopsy result obtained using the PG-Seq™ kit.
Evidence of maternal DNA contamination following NI-PGT-A of spent embryo culture media compared to the trophectoderm biopsy result obtained using the VeriSeq® kit (Illumina®) following continuous culture.
Generate Distinct Embryo Signatures
With PG-Seq™ kit 2.0 and its superior mtDNA coverage, it is possible to generate a distinct embryo signature based on mtDNA that can be used to determine maternal origin. Our study showed 100% embryo identification from data across multiple patients and multiple embryos across multiple cycles. The PG-Seq™ kit 2.0 has been validated and is compatible on the Illumina® MiSeq® and MiniSeq® platforms.
| Sample ID | Cycle ID | Embryo Biopsied | Embryo ID Signature |
|---|---|---|---|
| Sample 1 | Cycle 1 | 1 | ---------c-g----g------t-------a---------------- |
| 2 | ---------c-g----g------t-------a---------------- | ||
| Cycle 2 | 1 | ---------c-g----g------t-------a---------------- | |
| 2 | ---------c-g----g------t-------a---------------- | ||
| Sample 2 | Cycle 1 | 1 | ---------t- a----a------c-------g---------------- |
| 2 | ---------t- a----a------c-------g---------------- | ||
| Cycle 2 | 1 | ---------t- a----a------c-------g---------------- | |
| 2 | ---------t- a----a------c-------g---------------- | ||
| Cycle 3 | 1 | ---------t- a----a------c-------g---------------- | |
| 2 | ---------t- a----a------c-------g---------------- |
AUTOMATED PGT WORKFLOW
COLLECT EMBRYO BIOPSY
WHOLE GENOME AMPLIFICATION
with DOPLIFY® REAGENTS (included in the PG-Seq™ kit)
AUTOMATED NGS LIBRARY PREPARATION
with the SCICLONE® G3 NGS WORKSTATION & PG-SEQ™ KIT pre-plated for automated liquid handling systems
LOAD YOUR SAMPLES ON A SEQUENCER
ANALYZE THE DATA
with the PG-FIND™ SOFTWARE
Kit Contents
WGA REAGENTS
- PCR-grade Water
- Cell Lysis Enzyme
- Cell Lysis Buffer
- WGA Polymerase
- WGA PCR Buffer
- Primer
LIBRARY PREP REAGENTS
- Fragmentation Buffer
- Fragmentation Enzyme
- Ligase Buffer
- Ligase Enzyme
- Library Amplification Mix
- Library Amplification Primer
- Nuclease-free Water
- Resuspension Buffer
MISC.
- 1 x 96 reaction format
- Purification Beads
Required materials not provided
- Laminar flow cabinet
- Microcentrifuge
- Pipettes (2, 10, 20, 100, 200, 1000 µl)
- Thermocycler (with hotlid & programmable ramp rate to 25°C/sec)
- Pipette tips (low binding, barrier filter)
- PCR thin walled reaction tube with flat cap (0.5mL or 0.2mL)
- Molecular grade tubes (1.5mL)
- 96 well PCR plate, to suit thermocycler
- Adhesive 96 well plate seal
- Vortex
- 96 well plate centrifuge
- Magnetic stand -96
- 80% Ethanol
- LabChip® GXII Touch™ Nucleic Acid Analyzer and associated reagent kit or Qubit® Fluorometer and associated reagent kit (recommended: Qubit™ 1X dsDNA HS Assay Kit)
- Sodium hydroxide 1N
- Illumina® Sequencer (MiSeq® Instrument or MiniSeq® Instrument)
- Illumina® Sequencer related consumables
- Illumina® Sequencer reagent kit
- MiSeq® Reagent Kit v3 (150 cycle, cat # MS-102-3001)
- MiSeq® Reagent Micro Kit v2 (300 cycle, cat # MS-103-1002)
- MiniSeq® High Output Reagent Kit (75 cycles, Cat # FC-420-1001)
Optional materials not provided
- Multi-channel pipette
- Agarose gel-electrophoresis apparatus
- Electrophoresis power supply
- UV transilluminator or gel documentation instrument
Catalog numbers:
PG-Seq™ 2.0 Manual Version – 4320-0020
PG-Seq™ 2.0 Automation pre-plated for Sciclone – 4323-0030
NGS SUPPORT INFORMATION
- Sequences of PG-Seq™ 2.0 Kit Indexes – Excel / PDF
- PKI PG-Seq™ Pooling Calculator and Final Pool Calculator
- Samplesheet_Template
MANUAL
BROCHURES
TECHNICAL NOTES
Publications that Cite Using the PG-Seq Kit 2.0 for Preimplantation Genetic Testing
Mai, A. D., Harton, G. L., Quang, V. N., Van, H. N., Thi, N. H., Thuy, N. P., . . . Quoc, Q. T. (2020). Development and clinical application of a preimplantation genetic testing for monogenic disease (PGT-M) for beta thalassemia in Vietnam. Journal of Assisted Reproduction and Genetics. doi:10.1007/s10815-020-02006-y.
The PG-Seq™ kit 2.0 includes WGA reagents, library preparation reagents including 96 unique barcodes and purification beads and PG-Find™ software.
How many reactions do I get in a single PG-Seq™ kit 2.0
96.
What is the maximum throughput for the PG-Seq™ kit 2.0?
96.
Does the PG-Seq™ kit 2.0 include primers for the TSE protocol?
No, customers are required to supply their own primer sets.
Can the PG-Seq™ kit 2.0 amplify day 3 biopsies and polar bodies?
Yes.
How long does it take to process from WGA to sequencer (lab time) for 24, 48 and 96 samples?
24 samples: ~7 hours | 48 samples: ~7.5 hours | 96 samples: ~9.5 hours
How long does the sequencing time take for the v3 and v2 chemistry Illumina® MiSeq® reagent kits?
V3 1×75 cycle: ~ 9 hours | V2 1×75 cycle: ~5 hours
Does the sequencer reagent kit come with the PG-Seq™ kit 2.0?
No, this needs to be purchased from Illumina® or an Illumina® distributor.
What reagent kits do I need for the sequencer?
Illumina® MiSeq® v3 150 cycle kit (MS-102-3001) | Illumina® MiSeq® v2 Micro 300 cycle kit (MS-103-1002)
/or/
Illumina® MiniSeq® High output 75 cycle kit (FC-420-1001)
Can I use the PG-Find™ software to view point mutations and SNPs?
No, PerkinElmer recommends using IGV software to view point mutations and SNPs.
What equipment do I need for the PG-Seq™ kit 2.0?
Standard molecular biology equipment and a thermocycler with a programmable ramp rate (please also refer to the kit manual for more information).
At what temperature do I store the kit components?
WGA reagents should be stored at -20°C in the clean room. Library preparation reagents (including the adaptor plate) should be stored at -20°C in the general lab with purification beads stored at 4°C in the general lab. Resuspension buffer & H2O stored at room temperature.
What buffer should I use to transfer the biopsy in and what size tubes shall I use?
Recommended cell transfer buffers include 10 mM Tris-HCl (pH 8.0) (no EDTA) and PBS (Mg2+, Ca2+ free and BSA free) in 0.2 ml or 0.5 ml PCR tubes.
The PG-Seq™ kit 2.0 also comes in an automation version with the library preparation reagents pre-plated ready to use on the Sciclone® G3 NGSx workstation.
The shelf life of all reagents is 6 months when stored properly at -20°C. The PG-Seq™ kit 2.0 ships on dry ice.
Catalog numbers:
PG-Seq™ 2.0 Manual Version – 4320-0020
PG-Seq™ 2.0 Automation pre-plated for Sciclone® G3 NGSx workstation – 4323-0030