Genome-Wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

Xia, Yuntao; Gonzales-Pena, Veronica; Klein, David; Luquette, Joe J; Puzon, Liezl; Siddiqui, Noor; Reddy, Vikrant; Park, Peter J.; Behr, Barry; Gawad, Charles

doi:10.1101/2021.07.06.451077

Cited by 5 publications

(7 citation statements)

References 32 publications

(32 reference statements)

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“…Mitochondrial DNA is known to be exclusively inherited from the mother. We have already confirmed it using our amplified DNA in previous studies (Xia et al, 2021).…”

Section: Resultssupporting

confidence: 82%

First clinical validation of whole genome screening on standard trophectoderm biopsies of preimplantation embryos

Xia¹,

Chandramohan²,

Chertman³

et al. 2022

Preprint

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Whole genome screening is currently not available in preimplantation genetic testing (PGT) due to poor performance of whole genome amplification methods. Here, we present the first validation study using our latest whole genome amplification (WGA) protocol, which yields amplified DNA with comparable quality to genomic DNA when perfoming whole genome sequencing assays. We started by validating PGT for aneuploidy (PGT-A) using our WGA protocol on cell lines and donated human embryos, where amplification success rates were >99.9% and 96.2% respectively. Accuracy on both was >99.9% on aneuploidy status. We next validated whole genome sequencing using amplified and genomic DNA from the Genome in the Bottle reference sample NA12878 to demonstrate accuracy, specificity, sensitivity, and precision of our WGA methods. Amplified DNA and genomic DNA are comparable in this case, with 99.99% accuracy, 99.99% specificity, 98.0% sensitivity and 98.1% precision with our WGA protocol. We additionally examined variant calls between biopsies and the remaining embryos from which they were derived. Again, 99.99% accuracy, 99.99% specificity, 98.1% sensitivity and 98.0% precision were observed. Mitochondrial heteroplasmy between biopsies and embryos was validated, and 99.9% accuracy, 100% specificity, 99.1% sensitivity and 100% precision were observed. Finally, we demonstrated the ability to improve the accuracy of certain types of mendelian variants in our data to close to 100% by leveraging parental and a born child’s genome to perform linkage analysis.

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“…Mitochondrial DNA is known to be exclusively inherited from the mother. We have already confirmed it using our amplified DNA in previous studies (Xia et al, 2021).…”

Section: Resultssupporting

confidence: 82%

First clinical validation of whole genome screening on standard trophectoderm biopsies of preimplantation embryos

Xia¹,

Chandramohan²,

Chertman³

et al. 2022

Preprint

Self Cite

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“…The introduction of PTA greatly improved the accuracy of single-cell WGA, leading to rapid adoption in the field 17,18,34–36 . However, bioinformatic tools making optimal use of the potential of PTA have been lacking.…”

Section: Discussionmentioning

confidence: 99%

“…PTA was performed using the ResolveDNA Whole Genome Amplification Kit (BioSkryb Genomics) according to the manufacturer’s protocol. Instead of 10 minutes cell lysis on ice as indicated in the protocol, lysis was performed by 5 minutes incubation on ice followed by 5 minutes incubation at room temperature to maximize DNA denaturation as previously described 34 . DNA samples from bulk AML and bulk MSCs (for germline control) were isolated using the DNeasy DNA Micro Kit (QIAGEN) or DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Middelkamp

Manders

Peci

et al. 2023

Preprint

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Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole genome sequencing (WGS), but still generate hundreds of artefacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolkit (PTATO), to accurately detect single base substitutions, small insertions and deletions (indels) and structural variants in PTA-based WGS data. PTATO includes a machine learning approach to distinguish PTA-artefacts from true mutations with high sensitivity (up to 90% for base substitution and 95% for indels), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that many hematopoietic stem and progenitor cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS technologies, have normal somatic single base substitution burdens, but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

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“…In all cases where the depth of coverage exceeded 5X, we could correctly detect the SNV. A prior study that conducted WGS on embryos to identify de novo pathogenic variants for known diseases and polygenic risk score analysis 46 did not account for allelic dropout or drop-in issues related to amplification as they did not include parental sequencing information in their analysis. An advantage of our method is that it can be performed both in a direct and indirect fashion using haplarithmisis principles, to be certain of our pathogenic variant of interest.…”

Section: Discussionmentioning

confidence: 99%

Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

Janssen,

Koeck,

Essers

et al. 2023

Preprint

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High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied fromin vitrofertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devised a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA (37 embryos, 18 families, 25 indications), shallow sequencing-based PGT (10 embryos, 3 families), and PCR-based PGT for mitochondrial DNA (10 embryos, 2 families), our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by 4-fold, and reducing wet-lab turn-around-time by 2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution.

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Genome-Wide Disease Screening in Early Human Embryos with Primary Template-Directed Amplification

Cited by 5 publications

References 32 publications

First clinical validation of whole genome screening on standard trophectoderm biopsies of preimplantation embryos

First clinical validation of whole genome screening on standard trophectoderm biopsies of preimplantation embryos

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

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