Analysis method and device for determining haplotypes of filial generation objects
A haplotype, object technology, applied in the fields of biomedicine and molecular cell biology, can solve the problems of false positives, inability to directly detect, false negatives, etc.
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Embodiment 1
[0181] Example 1: Embryo biopsy samples + inference of the carrier status of single-gene disease-related loci in embryos
[0182] 1) Family information: autosomal recessive methylmalonic acidemia, the causative gene MUT, the male carries the MUTc.323G>A mutation, the female carries the MUTc.729_730insTT mutation, and the male and female aborted fetuses carry the paternal mutation; Embryo samples to be tested.
[0183] 2) Extract the blastocyst trophoblast cells from the embryo, extract the gDNA of the father and mother of the embryo, which are referred to as the man and woman below, and the gDNA of the other aborted fetus from the parents of the embryo.
[0184] Directly take several blastocyst trophoblast cells and place them in 5ul lysate for single-cell whole-genome amplification by MALBAC two-step method (gene sequencing universal sample processing kit from Suzhou Xukang Medical Technology Co., Ltd., catalog number XK-028).
[0185] 3) Genotyping detection of male, female...
Embodiment 2
[0201] Embodiment 2: Embryo biopsy sample + embryo chromosome balanced translocation carrier status inference
[0202] 1) Carrier family of balanced chromosomal translocation (reciprocal translocation): the man carries it, the karyotype is 46, XY, t(4; 14) (q31.1; q21), the woman is normal, and 9 embryos to be tested.
[0203] 2) Extract gDNA from the peripheral blood of the male and female partners. The embryo samples are blastocyst trophoblast cells. After thermal lysis, the MALBAC two-step method is used to amplify the single-cell whole gene. The method is the same as in Example 1.
[0204] 3) CNV-Seq detection was performed on the embryo amplification products to detect chromosomal aneuploidy. The test results showed that embryos 1, 2, 4, 6, and 8 were normal embryos with CNV, and embryos 3, 5, and 7 were abnormal embryos with CNV (Table 2).
[0205] 4) Use CNV abnormal embryos No. 3 and No. 7 to determine the breaking point. For specific methods, refer to patent CN106834...
Embodiment 3
[0214] Example 3: Blastocyst culture fluid sample + embryo single-gene disease-related loci carrier status inference
[0215] (1) Family information: autosomal recessive β genetic disease thalassemia, the causative gene HBB, the man carries the HBBIVS-II-654C>T mutation, the woman carries the same HBB IVS-II-654C>T mutation, and the male and female children carry heterozygosity Synthetic mutation; four embryo samples to be tested. In this case, it is impossible to determine whether the heterozygous mutation of the male and female children comes from the male or the female, so the pathogenic haplotype of the male and female cannot be determined at this stage, and it needs to be deduced by the first-generation sequencing verification results of the embryos.
[0216] (2) Extract the cell-free blastocyst culture fluid from 4 extracorporeal embryos cultured to day 5 as test samples, extract the gDNA of the father and mother of the embryos, referred to below as male and female, and ...
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