PG-Seq Kit 2.02019-11-15T14:06:49+00:00

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
Zephyr G3 NGS WorkstationAutomated on Zephyr G3 NGS Workstation
Sciclone G3 NGS WorkstationsSciclone G3 NGS Workstations

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.

PG-Seg PGT-A Data

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.

Exact break points are given in the software results here
Number of SamplesNumber of Mapped Reads per SampleSmallest CNV Detectable
96250,00010Mb
48500,0005Mb
241,000,0002Mb
83,000,0001Mb
212,000,000500Kb
124,000,000100Kb

“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.”

Jakub Horák, Genetic Laboratory Director, Repromeda

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-Seqkit 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.

ado graph

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.

Dopify Workflow
igv hr

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.

PG-Seq™ Kit Media Results
spent embryo culture

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.

spent embryo culture b

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 IDCycle IDEmbryo BiopsiedEmbryo ID Signature
Sample 1Cycle 11---------c-g----g------t-------a----------------
2---------c-g----g------t-------a----------------
Cycle 21---------c-g----g------t-------a----------------
2---------c-g----g------t-------a----------------
Sample 2Cycle 11---------t- a----a------c-------g----------------
2---------t- a----a------c-------g----------------
Cycle 21---------t- a----a------c-------g----------------
2---------t- a----a------c-------g----------------
Cycle 31---------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

PG-Seq Kit

PREIMPLANTATION GENETIC TESTING

Sciclone G3 NGS and NGSx Workstation

SCICLONE® G3 NGS WORKSTATION

Doplify Kit

DOPLIFY®
KIT & TECHNOLOGY

LabChip® GX Touch™ Nucleic Acid Analyzer

LABCHIP® GX TOUCH NUCLEIC ACID ANALYZER

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

What does the PG-Seq kit 2.0 include?

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 comes with WGA and library preparation reagents in a 96-reaction kit and the PG-Find analysis software.

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

For research use only. Not for use in diagnostic procedures.