PG-Seq Rapid v2 kit

mtDetect™ is a simple and intuitive tool that provides excellent internal control for sample traceability. It detects positional changes in embryos within the same run and compares the mitochondrial SNP profile in repeated biopsies with those of their siblings from previous runs, establishing the same origin of the samples. For us, this is a mandatory step before proceeding with sample analysis.

Dr. Marta Rodríguez de Alba
Jefe Asociado - Servicio de Genética
Hospital Universitario Fundación Jiménez Díaz (grupo QuirónSalud).

Sample Traceability in PGT-A

Sample traceability is crucial to ensure reliable embryo classification. This process involves tracking from the initial collection of the biopsy through to the final analysis.

For sample collection we recommend PCR tubes free of DNAse, Rnase, human genomic DNA and compatible with automation. Tubes with datamatrix code such as Cap2™ tubes, will facilitate sample tracking.

To track samples individually we take advantage of the mitochondrial genome.

The mitochondrial genome is maternally derived and contains single nucleotide variants that can be used to distinguish between individuals. Revvity has shown that correct grouping of sibling embryos can be achieved in >98% of cases using mitochondrial DNA SNV profiling. The new mtDetect™ web app extracts mitochondrial DNA information from data obtained during the standard PG-Seq™ Rapid v2 workflow enabling the monitoring of external DNA contamination and the ability to detect sample swapping and mislabeled samples.

Figure 1 shows 90% coverage of the mtDNA genome using the PG-Seq Rapid v2 kit without any protocol modification. Embryo biopsies processed with the PG-Seq™ Rapid v2 kit analyzed with mtDetect™ web app can successfully be identified and grouped based on their mitochondrial DNA SNV profiles. Identity determination allows sample tracking and can assist in the identification and monitoring of possible external sample contamination along with mislabeled samples.

Figure 1: IGV coverage track of the full mitochondrial genome (chrM: 1-16364bp) from a PG-Seq Rapid v2 amplified 5-cell sample with 1,000,000 sequencing reads shows 90% coverage of the mtDNA genome.

Easy-to-Use, Highly Customizable Analysis Software

PG-Find supports self-reference and reference-based analysis options to suit different lab preferences. Includes flexible visualization tools—customizable chromosome display, raw vs. smoothed data toggle, multiple result image outputs, and unlimited color options. Users can adjust event calling thresholds to designate mosaicism as well as 1 or 2 copy gains and losses. A user-friendly overview screen enables quick interpretation of individual results and observation of broader data trends (Figure 2).

Figure 2. Examples of different visualizations that are used during routine analysis of data generated with PG-Seq™ Rapid v2
 

Figure 3. CNV resolution can be adjusted easily for each sample. Detailed information on the length and position of each even is provided.

Compatibility with Element® Biosciences sequencing platform

PG-Seq™ Rapid v2 libraries can be run native on the AVITI™ sequencer. Results obtained are concordant with those generated at the Miseq and no differences are observed in terms of quality and read per sample requirement at 10Mb resolution (Figure 4)

Figure 4: Example of data generated on AVITI from a GenQA sample (47XY, +chr16) and visualized at 10 Mb resolution.

Compatibility with MGI® sequencing platform

PG-Seq™ Rapid v2 libraries can be run on MGI® sequencing platforms. Results obtained are concordant with those generated on Illumina sequencers and no differences are observed in terms of quality and read per sample requirement at 10Mb resolution (Figure 5)

Figure 5: Example of data generated on MGI and visualized at 10 Mb resolution. Sample was processed at LifeCell International Pvt Ltd, Chennai

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