The current clinical applications of preimplantation genetic testing (PGT): acknowledging the limitations of biology and technology.

INTRODUCTION: Preimplantation Genetic Testing (PGT) is a cutting-edge test used to detect genetic abnormalities in embryos fertilized through Medically Assisted Reproduction (MAR). PGT aims to ensure that embryos selected for transfer are free of specific genetic conditions or chromosome abnormalities, thereby reducing chances for unsuccessful MAR cycles, complicated pregnancies, and genetic diseases in future children. AREAS COVERED: In PGT, genetics, embryology, and technology progress and evolve together. Biological and technological limitations are described and addressed to highlight complexity and knowledge constraints and draw attention to concerns regarding safety of procedures, clinical validity, and utility, extent of applications and overall ethical implications for future families and society. EXPERT OPINION: Understanding the genetic basis of diseases along with advanced technologies applied in embryology and genetics contribute to faster, cost-effective, and more efficient PGT. Next Generation Sequencing-based techniques, enhanced by improved bioinformatics, are expected to upgrade diagnostic accuracy. Complicating findings such as mosaicism, mt-DNA variants, variants of unknown significance, or variants related to late-onset or polygenic diseases will however need further appraisal. Emphasis on monitoring such emerging data is crucial for evidence-based counseling while standardized protocols and guidelines are essential to ensure clinical value and respect of Ethical, Legal and Societal Issues.

scMicrobe PTA: near complete genomes from single bacterial cells.

Microbial genomes produced by standard single-cell amplification methods are largely incomplete. Here, we show that primary template-directed amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard multiple displacement amplification-based approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

Targeted whole-genome recovery of single viral species in a complex environmental sample.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology.

Large-scale single-virus genomics uncovers hidden diversity of river water viruses and diversified gene profiles.

Environmental viruses (primarily bacteriophages) are widely recognized as playing an important role in ecosystem homeostasis through the infection of host cells. However, the majority of environmental viruses are still unknown as their mosaic structure and frequent mutations in their sequences hinder genome construction in current metagenomics. To enable the large-scale acquisition of environmental viral genomes, we developed a new single-viral genome sequencing platform with microfluidic-generated gel beads. Amplification of individual DNA viral genomes in mass-produced gel beads allows high-throughput genome sequencing compared to conventional single-virus genomics. The sequencing analysis of river water samples yielded 1431 diverse viral single-amplified genomes, whereas viral metagenomics recovered 100 viral metagenome-assembled genomes at the comparable sequence depth. The 99.5% of viral single-amplified genomes were determined novel at the species level, most of which could not be recovered by a metagenomic assembly. The large-scale acquisition of diverse viral genomes identified protein clusters commonly detected in different viral strains, allowing the gene transfer to be tracked. Moreover, comparative genomics within the same viral species revealed that the profiles of various methyltransferase subtypes were diverse, suggesting an enhanced escape from host bacterial internal defense mechanisms. Our use of gel bead-based single-virus genomics will contribute to exploring the nature of viruses by accelerating the accumulation of draft genomes of environmental DNA viruses.

Distinguishing recrudescence from reinfection in lymphatic filariasis.

BACKGROUND: The Global Program to Eliminate Lymphatic Filariasis (GPELF) is the largest public health program based on mass drug administration (MDA). Despite decades of MDA, ongoing transmission in some countries remains a challenge. To optimise interventions, it is critical to differentiate between recrudescence and new infections. Since adult filariae are inaccessible in humans, deriving a method that relies on the offspring microfilariae (mf) is necessary. METHODS: We developed a genome amplification and kinship analysis-based approach using Brugia malayi samples from gerbils, and applied it to analyse Wuchereria bancrofti mf from humans in Côte d'Ivoire. We examined the pre-treatment genetic diversity in 269 mf collected from 18 participants, and further analysed 1-year post-treatment samples of 74 mf from 4 participants. Hemizygosity of the male X-chromosome allowed for direct inference of haplotypes, facilitating robust maternal parentage inference. To enrich parasite DNA from samples contaminated with host DNA, a whole-exome capture panel was created for W. bancrofti. FINDINGS: By reconstructing and temporally tracking sibling relationships across pre- and post-treatment samples, we differentiated between new and established maternal families, suggesting reinfection in one participant and recrudescence in three participants. The estimated number of reproductively active adult females ranged between 3 and 11 in the studied participants. Population structure analysis revealed genetically distinct parasites in Côte d'Ivoire compared to samples from other countries. Exome capture identified protein-coding variants with ∼95% genotype concordance rate. INTERPRETATION: We have generated resources to facilitate the development of molecular genetic tools that can estimate adult worm burdens and monitor parasite populations, thus providing essential information for the successful implementation of GPELF. FUNDING: This work was financially supported by the Bill and Melinda Gates Foundation (https://www.gatesfoundation.org) under grant OPP1201530 (Co-PIs PUF & Gary J. Weil). B. malayi parasite material was generated with support of the Foundation for Barnes Jewish Hospital (PUF). In addition, the development of computational methods was supported by the National Institutes of Health under grants AI144161 (MM) and AI146353 (MM). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Rescue of Mycobacterium bovis DNA Obtained from Cultured Samples during Official Surveillance of Animal TB: Key Steps for Robust Whole Genome Sequence Data Generation.

Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of Mycobacterium bovis has recently gained track due to its high resolution to identify infection sources, characterize the pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of M. bovis DNA obtained from routinely processed animal samples; the lack of purity of M. bovis DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one M. bovis strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention.

Revealing the Genetic Diversity of Chinese Chlamydia trachomatis Strains Directly from Clinical Samples through Selective Whole-Genome Amplification.

BACKGROUND: Chlamydia trachomatis is the causative agent of most prevalent bacterial sexually transmitted infection globally. Whole-genome sequencing is essential for molecular Chlamydia surveillance; however, its application is hampered by the pathogen's low abundance in clinical specimens and the expensive, labor-intensive nature of existing enrichment methodologies for Chlamydia. METHODS: We developed a targeted whole-genome amplification tool termed SWTICH, by integrating phi29 DNA polymerase-mediated amplification with meticulously designed primer sets to enrich Chlamydia trachomatis genome, followed by whole-genome sequencing. This method underwent evaluation through testing synthetic and clinical specimens. RESULTS: SWITCH demonstrated robust ability to achieve up to 98.3% genomic coverage of Chlamydia trachomatis from as few as 26.4 genomic copies present in synthetic specimens and exhibited excellent performance across diverse Chlamydia trachomatis serovars. Utilizing SWITCH, we directly generated 21 Chlamydia genomes from 26 clinical samples, enabling us to gain insights into the genetic relationships and phylogeny of current Chlamydia strains circulating in the country. Remarkably, this study marked the first instance of generating Chinese Chlamydia genomes directly from clinical samples. CONCLUSIONS: SWITCH represents a practical, cost-efficient approach to enrich Chlamydia genome directly from clinical specimens, offering an efficient avenue for molecular surveillance of Chlamydia.

Single-cell genomics of a bloom-forming phytoplankton species reveals population genetic structure across continents.

The study of microbial diversity over time and space is fundamental to the understanding of their ecology and evolution. The underlying processes driving these patterns are not fully resolved but can be studied using population genetic approaches. Here we investigated the population genetic structure of Gonyostomum semen, a bloom-forming phytoplankton species, across two continents. The species appears to be expanding in Europe, whereas similar trends are not observed in the USA. Our aim was to investigate if populations of Gonyostomum semen in Europe and in the USA are genetically differentiated, if there is population genetic structure within the continents, and what the potential drivers of differentiation are. To this end, we used a novel method based on single-amplified genomes combined with Restriction-site Associated DNA sequencing that allows de novo genotyping of natural single-cell isolates without the need for culturing. We amplified over 900 single-cell genomes from 25 lake populations across Europe and the USA and identified two distinct population clusters, one in Europe and another in the USA. Low genetic diversity in European populations supports the hypothesized recent expansion of Gonyostomum semen on this continent. Geographic population structure within each continent was associated with differences in environmental variables that may have led to ecological divergence of population clusters. Overall, our results show that single-amplified genomes combined with Restriction-site Associated DNA sequencing can be used to analyze microalgal population structure and differentiation based on single-cell isolates from natural, uncultured samples.

A new chromosome-level genome assembly and annotation of Cryptosporidium meleagridis.

Cryptosporidium spp. are medically and scientifically relevant protozoan parasites that cause severe diarrheal illness in infants and immunosuppressed populations as well as animals. Although most human Cryptosporidium infections are caused by C. parvum and C. hominis, there are several other human-infecting species including C. meleagridis, which is commonly observed in developing countries. Here, we polished and annotated a long-read genome sequence assembly for C. meleagridis TU1867, a species which infects birds and humans. The genome sequence was generated using a combination of whole genome amplification (WGA) and long-read Oxford Nanopore Technologies sequencing. The assembly was then polished with Illumina data. The chromosome-level genome assembly is 9.2 Mbp with a contig N50 of 1.1 Mb. Annotation revealed 3,923 protein-coding genes. A BUSCO analysis indicates a completeness of 96.6% (n=446), including 430 (96.4%) single-copy and 1 (0.224%) duplicated apicomplexan conserved gene(s). The new C. meleagridis genome assembly is nearly gap-free and provides a valuable new resource for the Cryptosporidium community and future studies on evolution and host-specificity.

Simultaneous Isolation and Amplification of mRNA and Genomic DNA of a Single Cell.

Many biological or pathological processes are driven by cells difficult to identify or isolate, i.e., rare cells. Very often, these cells have elusive biology. Therefore, their detailed characterization is of utmost importance. There are many approaches that allow analysis of few or even many targets within one class of biomacromolecules/analytes (e.g., DNA, RNA, proteins, etc.) in single cells. However, due to rarity of the cells of interest, there is a great need to comprehensively analyze multiple analytes within these cells, in other words to perform multi-omics analysis. In this chapter, I describe a method to isolate, separate, and amplify total mRNA and genomic DNA of a single cells, using whole transcriptome (WTA) and whole genome amplification (WGA). These WTA and WGA products enable simultaneous analysis of transcriptome and genome of a single cell using various downstream high-throughput approaches.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

Integrative preimplantation genetic testing analysis for a Chinese family with hereditary spherocytosis caused by a novel splicing variant of SPTB.

Hereditary spherocytosis (HS), the most common inherited hemolytic anemia disorder, is characterized by osmotically fragile microspherocytic red cells with a reduced surface area on the peripheral blood smear. Pathogenic variants in five erythrocyte membrane structure-related genes ANK1 (Spherocytosis, type 1; MIM#182900), SPTB (Spherocytosis, type 2; MIM#616649), SPTA1 (Spherocytosis, type 3; MIM#270970), SLC4A1 (Spherocytosis, type 4; MIM#612653) and EPB42 (Spherocytosis, type 5; MIM#612690) have been confirmed to be related to HS. There have been many studies on the pathogenic variants and mechanisms of HS, however, studies on how to manage the transmission of HS to the next-generation have not been reported. In this study, we recruited a patient with HS. Targeted next-generation sequencing with a panel of 208 genes related to blood system diseases detected a novel heterozygous variant in the SPTB: c.300+2dup in the proband. Sanger sequencing of variant alleles and haplotype linkage analysis of single nucleotide polymorphism (SNP) based on next-generation sequencing were performed simultaneously. Five embryos were identified with one heterozygous and four not carrying the SPTB variant. Single-cell amplification and whole genome sequencing showed that three embryos had varying degrees of trisomy mosaicism. One of two normal embryos was transferred to the proband. Ultimately, a healthy boy was born, confirmed by noninvasive prenatal testing for monogenic conditions (NIPT-M) to be disease-free. This confirmed our successful application of PGT in preventing transmission of the pathogenic variant allele in the HS family.

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

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 artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, 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.

Whole-genome surveillance identifies markers of Plasmodium falciparum drug resistance and novel genomic regions under selection in Mozambique.

IMPORTANCE: Malaria is a devastating disease caused by Plasmodium parasites. The evolution of parasite drug resistance continues to hamper progress toward malaria elimination, and despite extensive efforts to control malaria, it remains a leading cause of death in Mozambique and other countries in the region. The development of successful vaccines and identification of molecular markers to track drug efficacy are essential for managing the disease burden. We present an analysis of the parasite genome in Mozambique, a country with one of the highest malaria burdens globally and limited available genomic data, revealing current selection pressure. We contribute additional evidence to limited prior studies supporting the effectiveness of SWGA in producing reliable genomic data from complex clinical samples. Our results provide the identity of genomic loci that may be associated with current antimalarial drug use, including artemisinin and lumefantrine, and reveal selection pressure predicted to compromise the efficacy of current vaccine candidates.

DNA Polymerases for Whole Genome Amplification: Considerations and Future Directions.

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions.

SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Benchmarking of Nanopore R10.4 and R9.4.1 flow cells in single-cell whole-genome amplification and whole-genome shotgun sequencing.

Third-generation sequencing can be used in human cancer genomics and epigenomic research. Oxford Nanopore Technologies (ONT) recently released R10.4 flow cell, which claimed an improved read accuracy compared to R9.4.1 flow cell. To evaluate the benefits and defects of R10.4 flow cell for cancer cell profiling on MinION devices, we used the human non-small-cell lung-carcinoma cell line HCC78 to construct libraries for both single-cell whole-genome amplification (scWGA) and whole-genome shotgun sequencing. The R10.4 and R9.4.1 reads were benchmarked in terms of read accuracy, variant detection, modification calling, genome recovery rate and compared with the next generation sequencing (NGS) reads. The results highlighted that the R10.4 outperforms R9.4.1 reads, achieving a higher modal read accuracy of over 99.1%, superior variation detection, lower false-discovery rate (FDR) in methylation calling, and comparable genome recovery rate. To achieve high yields scWGA sequencing in the ONT platform as NGS, we recommended multiple displacement amplification with a modified T7 endonuclease Ⅰ cutting procedure as a promising method. In addition, we provided a possible solution to filter the likely false positive sites among the whole genome region with R10.4 by using scWGA sequencing result as a negative control. Our study is the first benchmark of whole genome single-cell sequencing using ONT R10.4 and R9.4.1 MinION flow cells by clarifying the capacity of genomic and epigenomic profiling within a single flow cell. A promising method for scWGA sequencing together with the methylation calling results can benefit researchers who work on cancer cell genomic and epigenomic profiling using third-generation sequencing.

Preimplantation genetic testing for hereditary hearing loss in Chinese population.

PURPOSE: To evaluate the clinical validity of preimplantation genetic testing (PGT) to prevent hereditary hearing loss (HL) in Chinese population. METHODS: A PGT procedure combining multiple annealing and looping-based amplification cycles (MALBAC) and single-nucleotide polymorphisms (SNPs) linkage analyses with a single low-depth next-generation sequencing run was implemented. Forty-three couples carried pathogenic variants in autosomal recessive non-syndromic HL genes, GJB2 and SLC26A4, and four couples carried pathogenic variants in rare HL genes: KCNQ4, PTPN11, PAX3, and USH2A were enrolled. RESULTS: Fifty-four in vitro fertilization (IVF) cycles were implemented, 340 blastocysts were cultured, and 303 (89.1%) of these received a definite diagnosis of a disease-causing variant testing, linkage analysis and chromosome screening. A clinical pregnancy of 38 implanted was achieved, and 34 babies were born with normal hearing. The live birth rate was 61.1%. CONCLUSIONS AND RELEVANCE: In both the HL population and in hearing individuals at risk of giving birth to offspring with HL in China, there is a practical need for PGT. The whole genome amplification combined with NGS can simplify the PGT process, and the efficiency of PGT process can be improved by establishing a universal SNP bank of common disease-causing gene in particular regions and nationalities. This PGT procedure was demonstrated to be effective and lead to satisfactory clinical outcomes.