A third-generation sequencing-based embryo abnormality detection system without family
Patent Information
- Application Number
- CN202611356109.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-09-03
- Publication Date
- 2026-10-13
AI Technical Summary
此类方案存在明显不足：流程烦琐、耗时长，需要尽量完整的家系样本；STR和SNP芯片仅能分析已知位点，而NGS读长较短，难以在单个DNA片段上获得连续多个SNP位点的相位信息，导致单体型构建可用位点受限；对于无先证者、亲属样本缺失或家系结构不完整的特殊家庭，传统连锁分析几乎无法开展，成为临床难以逾越的障碍
本发明针对胚胎活检细胞WGA产物DNA量有限、片段长度分布不均、质量异质性高及扩增偏倚明显等特点，对三代测序文库构建流程中的DNA投入量、片段筛选阈值、文库浓度及建库参数进行优化，使低起始量或质量不均一的WGA产物能够更有效地转化为适合PacBio HiFi测序的文库。该技术方案能够提高有效读段产出、目标区域覆盖深度和覆盖均一性，降低低质量片段及无效扩增片段对测序资源的消耗，从而提高胚胎WGA样本的检测成功率和结果可判读率。
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Figure CN122907131A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of detection technology, and in particular to an embryo abnormality detection system based on third-generation sequencing that does not require a family history. Background Technology
[0002] Preimplantation genetic testing (PGT) is an important method for screening healthy embryos in assisted reproductive technology. It mainly includes chromosomal aneuploidy detection (PGT-A), chromosomal structural abnormality detection (PGT-SR), and single-gene genetic disease detection (PGT-M). Due to the extremely limited number of cells obtainable from embryo biopsies, whole-genome amplification (WGA) is usually required to obtain sufficient DNA template. However, WGA products commonly suffer from amplification bias, uneven regional coverage, fragment discontinuities, and mosaic fragments, significantly interfering with downstream genetic analysis.
[0003] In the PGT-A field, direct chromosome copy number analysis of WGA products using next-generation sequencing (NGS) or gene chips has proven highly effective in clinical practice. However, for PGT-M and PGT-SR, direct detection of gene sequence variations and chromosomal structural breakpoints remains challenging, currently relying primarily on pedigree haplotype linkage analysis. This method constructs haplotypes linked to pathogenic variants by analyzing STR, SNP, or NGS data from the proband, both partners, and necessary relatives, thereby indirectly inferring the carrier status of the embryo. This approach has significant drawbacks: it is cumbersome and time-consuming, requiring a complete family sample; STR and SNP chips can only analyze known loci, while NGS reads are short, making it difficult to obtain phase information from multiple consecutive SNP loci on a single DNA fragment, thus limiting the available loci for haplotype construction; for special families without a proband, missing relative samples, or incomplete family structures, traditional linkage analysis is almost impossible, becoming a significant clinical obstacle.
[0004] To reduce reliance on family samples, current approaches include embryo reverse sequencing and single-sample haplotype assembly based on long-read sequencing. Embryo reverse sequencing requires embryos in specific carrier states as a reference, limiting its applicability. Long-read sequencing achieves single-sample haplotypes through de novo assembly of the parental whole genome, but relies on high sequencing depth and sufficiently long reads; failure is risky when these conditions are not met, and sequencing costs are high. On the other hand, even with the introduction of long-read sequencing to directly detect embryonic WGA products, several technical bottlenecks remain: the poor template integrity, uneven fragment length distribution, and branching structure of embryonic WGA products do not match the requirements for long-read sequencing library preparation, affecting effective sequencing output; chimeric reads and amplification artifacts generated during WGA are easily misjudged as real structural variations or new mutations; furthermore, there is currently a lack of standardized long-read analysis workflows for embryonic WGA products, making it difficult to simultaneously achieve direct variant detection, WGA artifact filtering, and local haplotype construction in low-volume samples.
[0005] In summary, current technologies cannot provide an efficient method that can directly and accurately interpret complex genetic variations in embryonic WGA products without relying on family history. This limits the application of PGT-M and PGT-SR in special clinical scenarios. Summary of the Invention
[0006] To achieve the above objectives, the present invention adopts the following technical solution: Third-generation sequencing-based embryo variant detection systems for probands include: The sample preprocessing and library construction module is used to screen fragments from the obtained whole genome amplification products of the embryos to be tested, recover DNA fragments with a length of 5-7 kb, and construct third-generation sequencing libraries using the recovered DNA fragments. The third-generation sequencing module is used to sequence the third-generation sequencing library and output sequencing reads; The data analysis module includes a processor and a memory. When the processor executes a computer program stored in the memory, it performs the following operations: The sequencing reads are aligned to a reference genome, and supplementary alignment sequences generated during the alignment are included in the variation analysis to filter out chimeric sequences generated during the whole genome amplification process. Based on the single nucleotide polymorphism information obtained from the variation analysis, haplotype typing was performed on the embryos, and local haplotype information of the target variation region was extracted. Based on the pre-obtained known target variant information of the parent, as well as the sequence alignment information and local haplotype information of the target variant region, the variant-carrying status of the embryo is determined.
[0007] The embryonic whole genome amplification can be performed using methods such as multiple substitution amplification, requiring the product DNA fragment length to reach 5-10 kb.
[0008] The third-generation sequencing technology is characterized in that the sequencing platform includes at least one of a single-molecule real-time sequencing technology platform and a single-molecule nanopore sequencing technology platform.
[0009] To reduce interference from chimeric sequences in whole-genome amplification products and improve sequencing coverage uniformity and detection accuracy, library DNA fragments need to be screened and recovered before sequencing. The length of the recovered fragments should be 5-7 kb.
[0010] Bioinformatics analysis was performed on the data from third-generation sequencing, aligning the raw data to a reference genome (which could be hg19, hg38, or a T2T reference genome). Alignment software such as Minimap2 or pbmm2 was used. The analysis of variants included single nucleotide polymorphisms, small fragment insertions and deletions, copy number variations, and structural variations.
[0011] Software for detecting single nucleotide polymorphisms (SNPs) and small fragment insertion / deletion variations can include Deepvariant and Clair3. Software for detecting copy number variations can include HificNV and CNVpytorCNV. Software for detecting structural variations can include PBSV, CuteSV, and Sniffles.
[0012] To reduce interference from chimeric sequences in embryonic whole-genome amplification products, supplementary alignment sequences are included in the parameters for variant analysis. Specifically, if the software used for single nucleotide polymorphism and small fragment insertion / deletion variant analysis is Deepvariant, the parameter "keep_supplementary_alignments=true" needs to be added. The parameter settings for other software are similar.
[0013] For genes with highly homologous sequences (such as CYP21A2 and SMN1), Paraphase software is used to perform homology analysis on the sequences first, accurately distinguishing the origin of homologous sequences and obtaining accurate variation analysis results.
[0014] Based on the long-read sequence information from third-generation sequencing, the sequences are assembled de novo using software such as Hifiasm. This yields high-quality genome assembly results, which are then compared with a reference genome. This comparison with the original data provides a basis for further analysis and improves accuracy.
[0015] Based on the detection results of single nucleotide polymorphisms and small fragment insertion / deletion variations, haplotype typing of the genome of the sample to be tested can be performed. Haplotype typing software can be whatshap or hapcut2, etc.
[0016] Based on the known mutation information of the parents in the embryonic family, the alignment information of the target mutation sequence at the corresponding chromosomal location in the embryo is obtained.
[0017] Based on haplotype typing results, single nucleotide polymorphism (SNP) information upstream and downstream of the target variant is extracted to obtain haplotype typing results for the target variant. When the target variant region has poor coverage, inferences can be made based on the haplotype typing results to achieve cross-validation.
[0018] By combining the sequence alignment information of the target variant with its upstream and downstream haplotype information, the final result of the target variant detection in the embryo is output.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention addresses the limitations of embryonic biopsy cell WGA products, including limited DNA quantity, uneven fragment length distribution, high quality heterogeneity, and significant amplification bias. It optimizes the DNA input, fragment selection threshold, library concentration, and library construction parameters in the third-generation sequencing library construction process. This allows WGA products with low starting quantities or uneven quality to be more effectively converted into libraries suitable for PacBio HiFi sequencing. This technical solution improves effective read output, target region coverage depth and uniformity, and reduces the consumption of sequencing resources by low-quality fragments and invalid amplified fragments, thereby improving the detection success rate and interpretability of embryonic WGA samples.
[0020] This invention leverages the advantages of long-read sequencing, which can traverse long target regions, complex repetitive sequences, highly homologous sequences, and structural variation breakpoints, thereby improving the comprehensive detection capabilities for SNVs, Indels, CNVs, large insertions / deletions, duplications, complex rearrangements, and structural variations. Compared to traditional short-read sequencing, this invention reduces interpretation difficulties caused by multiple alignments, erroneous alignments, unclear breakpoints, or ambiguous phases in complex regions, expands the detection range of embryonic single-gene genetic disease-related variants, and improves the accuracy and interpretability of complex variant detection.
[0021] This invention establishes a dedicated bioinformatics analysis workflow for long-read data of embryonic WGA products. Through analysis modules such as sequence consistency quality control, abnormal read filtering, local haplotype assembly, and allele balance assessment, it identifies and corrects chimeric reads, branching products, and amplification bias signals generated during WGA amplification. This workflow reduces the risk of WGA amplification artifacts being misjudged as true variations or structural abnormalities, and minimizes their interference with SNV, Indel, CNV, and structural variation analysis results, thereby improving the accuracy, stability, and verifiability of embryonic genetic status interpretation.
[0022] This invention, based on the knowledge that the couple carries the target pathogenic variant or chromosomal structural abnormality, directly obtains long-read evidence near the breakpoint of the target variant region or structural variant by performing long-read sequencing on embryonic WGA products. Combined with surrounding SNP loci and local haplotype assembly results, it determines the risk of the embryo carrying the target pathogenic variant or abnormal chromosomal structure. Compared to traditional detection procedures that rely on proband, relative samples, or multiple embryo samples to construct family haplotype maps, this invention reduces the reliance on complex family haplotype construction in scenarios where there is no proband, missing relative samples, or incomplete family information. It lowers the difficulty of developing detection solutions and improves the accessibility and applicability of detection for such samples.
[0023] This invention enables aneuploidy analysis, single-gene disease target variant detection, structural variant analysis, and local haplotype analysis of embryonic WGA products within a single sequencing and analysis workflow. Compared to traditional approaches that require combining multiple detection technologies and step-by-step detection of different variant types, this invention can obtain multiple types of genetic information, including gene sequence variations, structural variations, aneuploidy, CNVs, mitochondrial variations, and de novo mutations, through a single library preparation, sequencing, and integrated analysis workflow. This reduces the need for repeated experiments, repeated library preparation, and integration of results from multiple platforms, thereby improving the consistency, standardization, and overall efficiency of the detection workflow. Attached Figure Description
[0024] Figure 1 This is a graph showing the relationship between library concentration and data output in this invention.
[0025] Figure 2 This is a diagram showing the results of embryo testing for family lineage S1.
[0026] Figure 3 This is a graph showing the SNP results for conventional PGT-M typing (blue represents the carrier chain). Figure 4 The image shows the embryo detection results at breakpoint 4p15.3 in family 2.
[0027] Figure 5 The image shows the embryo testing results at breakpoint 16q24 in family 2. Detailed Implementation
[0028] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0029] This invention provides a third-generation sequencing-based system for detecting embryonic whole-lineage genetic abnormalities without relying on probands or family pedigrees. The method involved in this system can directly and accurately determine the carrier status of target abnormalities in embryos without requiring DNA samples from probands or family members. Compared with traditional second-generation sequencing-based methods, this method has significant advantages in detection accuracy, ability to resolve complex variants, and scope of clinical applications.
[0030] Specifically, this invention constructs a third-generation sequencing detection system for embryonic whole-genome amplification products. By optimizing library construction and sequencing strategies, and combining them with a specially designed bioinformatics analysis workflow, this method can directly detect and determine embryonic variations without the need for indirect inference steps such as haplotype construction. Verification through examples shows that this method achieves highly consistent results with existing traditional detection methods based on haplotype construction, while significantly simplifying the detection process and reducing technical complexity and application barriers.
[0031] This invention discloses an embryo abnormality detection system based on third-generation sequencing without requiring a family history, comprising: The sample preprocessing and library construction module is used to screen fragments from the obtained whole genome amplification products of the embryos to be tested, recover DNA fragments with a length of 5-7 kb, and construct third-generation sequencing libraries using the recovered DNA fragments. The third-generation sequencing module is used to sequence the third-generation sequencing library and output sequencing reads; The data analysis module includes a processor and a memory. When the processor executes a computer program stored in the memory, it performs the following operations: The sequencing reads are aligned to a reference genome, and supplementary alignment sequences generated during the alignment are included in the variation analysis to filter out chimeric sequences generated during the whole genome amplification process. Based on the single nucleotide polymorphism information obtained from the variation analysis, haplotype typing was performed on the embryos, and local haplotype information of the target variation region was extracted. Based on the pre-obtained known target variant information of the parent, as well as the sequence alignment information and local haplotype information of the target variant region, the variant-carrying status of the embryo is determined.
[0032] When performing haplotype typing, the data analysis module specifically uses whatshap or hapcut2 software to extract single nucleotide polymorphism information upstream and downstream of the target variant.
[0033] The data analysis module is also used to: perform de novo assembly of the sequencing reads and compare the assembly results with the reference genome to assist in variant analysis.
[0034] The data analysis module is also used to perform homology analysis on genes with highly homologous sequences using Paraphase software.
[0035] This invention also relates to a method for detecting embryonic abnormalities without family pedigree based on third-generation sequencing, comprising the following steps: Obtain the whole genome amplification product of the embryo to be tested; Fragment screening was performed on the whole genome amplification products, and DNA fragments with a length of 5-7 kb were recovered; The recovered DNA fragments were used to construct a third-generation sequencing library and perform third-generation sequencing to obtain sequencing reads; The sequencing reads are aligned to a reference genome, and supplementary alignment sequences generated during the alignment are included in the variation analysis to filter out chimeric sequences generated during the whole genome amplification process; Based on the single nucleotide polymorphism information obtained from the variation analysis, haplotype typing was performed on the embryos, and local haplotype information of the target variation region was extracted. Based on the pre-known information about the target pathogenic variant or chromosomal structural abnormality carried by the parent, as well as the sequence alignment information and local haplotype information of the target variant region, the variant-carrying status of the embryo is determined.
[0036] Before constructing the third-generation sequencing library, the amount of DNA input and the library concentration were controlled, including: When performing whole-genome sequencing, the amount of DNA used is 1.3-1.7 μg, and the concentration after library construction is 25-35 ng / μL. In targeted capture-based sequencing, the DNA input is 1.3-1.7 μg, and the concentration after library construction is 10-20 ng / μL; For primer-based detection, the amount of DNA used is 100-200 ng, and the concentration after library construction is 5-10 ng / μL.
[0037] The inclusion of supplementary alignment sequences generated during mutation analysis to filter chimeric sequences specifically includes: performing mutation analysis using DeepVariant software and setting the parameter keep_supplementary_alignments=true to identify and subtract chimeric sequences using the supplementary alignment sequences.
[0038] The method of performing haplotype typing on embryos and extracting local haplotype information includes: performing haplotype typing on the genome of the embryos using software such as whatshap or hapcut2, and extracting single nucleotide polymorphism information upstream and downstream of the target variant to obtain local haplotype information of the target variant.
[0039] The method further includes: performing de novo assembly of the sequencing reads, and comparing the assembly results with a reference genome to help correct the results of the variation analysis.
[0040] When the target variant is located in a highly homologous sequence region, the sequence is first analyzed for homology using Paraphase software before variant analysis.
[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0042] Example 1
[0043] This embodiment provides a method for detecting embryonic variations that does not depend on the proband.
[0044] This embodiment targets families with carriers of single-gene diseases who have undergone assisted reproduction (with confirmed single-gene disease test results in the parents), and extracts MDA amplification products from offspring embryos for third-generation sequencing.
[0045] Construction of whole-genome third-generation sequencing libraries of embryonic WGA amplification products Third-generation sequencing libraries were constructed from the whole-genome amplification products of the embryos to be tested. The relationship between library concentration and data output was compared by setting different amounts of DNA input from single-cell amplification product samples. An 8kb length was used to calculate the number of moles of DNA library required for sequencing. The results showed that the highest data output per unit of DNA input was achieved when the library concentration was in the range of 25-35 ng / μL (see results below). Figure 1 ).
[0046] Sequencing performance analysis of off-machine data To evaluate the performance of sequencing data in terms of genome coverage uniformity and chimeric sequence ratio, different DNA fragment selection conditions were set for comparison. The results are shown in Table 1.
[0047] Table 1
[0048] Analysis revealed that when the average fragment length was between 3 and 7 kb, there was no significant difference in coverage uniformity and the proportion of chimeric sequences; however, when the fragment length was greater than 7 kb, coverage uniformity decreased significantly, while the proportion of chimeric sequences gradually increased. To balance detection performance and accuracy, the fragment selection criteria were ultimately determined to be 5-7 kb.
[0049] Offspring embryo testing results Based on the above optimization conditions, third-generation sequencing was performed on the target embryos S1 and S2 for whole-genome sequencing. The raw data were compared and target variants were analyzed. The results are summarized in Table 2. Specific detection results are shown below. Figure 2 .
[0050] Table 2:
[0051] This ratio represents routine PGT-M testing for families of patients with monogenic diseases.
[0052] 1. Family monotypic construction: (1) Sample genotyping: Large-scale SNP genotyping was performed on samples from both spouses of patients with single-gene disease and control subjects; (2) Determine effective SNP sites: Select SNP sites that are heterozygous in carriers of single-gene diseases, homozygous in their spouses, and homozygous in probands; Select effective SNPs that cover the range of 2Mb upstream and downstream of the pathogenic mutation; (3) Constructing the reference sample haplotype in the family haplotype: Collect the effective SNPs determined in step (2), obtain the haplotype covering the region of pathogenic variation through family linkage analysis, and obtain the reference sample haplotype in the family haplotype.
[0053] 2. Differentiate between embryos carrying mutated chromosomes and those with normal chromosomes: The haplotype information of the pathogenic mutation region in the daughter embryo (blastocyst) cells was compared with the haplotype information of the reference sample to distinguish whether the embryo carried the pathogenic mutation. The detection results and atlas are shown in Table 3. Figure 3 .
[0054] Table 3
[0055] Results Comparison The results of this embodiment were compared with those of Comparative Example 1 (conventional PGT-M family linkage analysis method). The haplotype results obtained by this method were consistent with the PGT-M results: offspring S1 was FMR1: c.-128_-126CGG
[29] , and offspring S2 was FMR1: c.-128_-126CGG
[72] . This variant is located in a high GC region of the genome and is difficult to detect directly by next-generation sequencing. This method does not require the construction of a family haplotype, that is, it does not require the detection of SNP information of both parents, nor does it require the SNP results of the reference sample. By directly detecting the embryo sample, the accurate determination of the target variant in the embryo is achieved.
[0056] Example 2: Balanced Translocation Family This example focuses on a family of carriers of balanced chromosomal translocations who underwent assisted reproduction (parental karyotype testing results are known). Family variation information is shown in Table 2. No reference sample was available for haplotype typing in this family. MDA amplification products from offspring embryos were extracted and subjected to third-generation sequencing; the results are shown in Table 4. Figure 4 and Figure 5 .
[0057] according to Figure 4 and Figure 5 The sequencing data showed that the breakpoint of the balanced translocation t(4;16)(p15.3;q24) was directly detected in embryo S1, while this breakpoint was not detected in embryo S2. Therefore, embryo S1 was determined to be a carrier of the balanced translocation, and embryo S2 was determined to be normal.
[0058] Table 4
[0059] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A third-generation sequencing-based embryo abnormality detection system without family pedigree, characterized in that, include: The sample preprocessing and library construction module is used to screen fragments from the obtained whole genome amplification products of the embryos to be tested, recover DNA fragments with a length of 5-7 kb, and construct third-generation sequencing libraries using the recovered DNA fragments. The third-generation sequencing module is used to sequence the third-generation sequencing library and output sequencing reads; The data analysis module includes a processor and a memory. When the processor executes a computer program stored in the memory, it performs the following operations: The sequencing reads are aligned to a reference genome, and supplementary alignment sequences generated during the alignment are included in the variation analysis to filter out chimeric sequences generated during the whole genome amplification process. Based on the single nucleotide polymorphism information obtained from the variation analysis, haplotype typing was performed on the embryos, and local haplotype information of the target variation region was extracted. Based on the pre-obtained known target variant information of the parent, as well as the sequence alignment information and local haplotype information of the target variant region, the variant-carrying status of the embryo is determined.
2. The system according to claim 1, characterized in that, When performing haplotype typing, the data analysis module specifically uses whatshap or hapcut2 software to extract single nucleotide polymorphism information upstream and downstream of the target variant.
3. The system according to claim 1, characterized in that, The data analysis module is also used to: perform de novo assembly of the sequencing reads and compare the assembly results with the reference genome to assist in variant analysis.
4. The system according to claim 1, characterized in that, The data analysis module is also used to perform homology analysis on genes with highly homologous sequences using Paraphase software.
5. The system according to claim 1, characterized in that, Before constructing the third-generation sequencing library, the amount of DNA input and the library concentration were controlled, including: When performing whole-genome sequencing, the amount of DNA used is 1.3-1.7 μg, and the concentration after library construction is 25-35 ng / μL; In targeted capture-based sequencing, the DNA input is 1.3-1.7 μg, and the concentration after library construction is 10-20 ng / μL; For primer-based detection, the amount of DNA used is 100-200 ng, and the concentration after library construction is 5-10 ng / μL.
6. The system according to claim 1, characterized in that, The inclusion of supplementary alignment sequences generated during variant analysis to filter out chimeric sequences generated during the whole-genome amplification process specifically includes: using DeepVariant software for variant analysis and setting the parameter keep_supplementary_alignments=true to identify and subtract chimeric sequences using the supplementary alignment sequences.