Profiles of 10× Genomics sequencing results
According to previous studies[8, 9], with sequencing depth of under around 35× (120 GB raw sequencing data), N50 phase block can be over 500 kb. In this study, three carrier samples were tested. All samples except G2018001A_LSL had a sequencing depth of more than 35×, and all samples had an N50 phase block of more than 500 kb. As summarized in Table 1, the sequencing depth of each sample was 47.4×, 40.0× and 33.5× respectively. After phasing analysis, 95.9%, 94.9% and 95.6% SNPs were phased. The longest phase block could reach 5,080,496bp, 5,421,595bp and 3,620,840bp, with N50 phase block of 775,060bp, 840,402bp and 684,981bp respectively.
Table 1 10× Genomics sequencing summary
Sample
|
G2018004A_LYL
|
G2018004B_LGB
|
G2018001A_LSL
|
Input DNA Molecular Length
|
21858 bp
|
24088 bp
|
22631 bp
|
Number of Reads
|
1091.38 M
|
937.20 M
|
776.89 M
|
Mapped Reads
|
95.9%
|
94.9%
|
95.6%
|
Median Insert Size
|
365 bp
|
360 bp
|
363 bp
|
Mean Depth
|
47.4×
|
40.0×
|
33.5×
|
SNPs Phased
|
99.2%
|
98.4%
|
98.9%
|
Longest Phase Block
|
5080496 bp
|
5421595 bp
|
3620840 bp
|
N50 Phase Block
|
775060 bp
|
840402 bp
|
684981 bp
|
SNPs, Single nucleotide polymorphisms.
Haplotype phase blocks
Targeted phasing regions for respective pathogenic mutation sites in each sample were all covered by phase blocks (Table 2). For alpha thalassemia carrier samples, the corresponding phase block for G2018004A_LYL was 410.kb long and contained 309 heterozygous SNPs, while that for G2018004B_LGB was only 319.4 kb with 251 heterozygous SNPs. For NDP disorder carrier sample G2018001A_LSL, the corresponding phase block was 262.3kb long with 134 heterozygous SNPs. The carrier allele and normal allele could also be distinguished using these phase blocks (Figure 2). For alpha thalassemia carrier samples, there was a HBA gene deletion in haplotype 1, which means haplotype 1 was the carrier allele. For NDP disorder carrier sample, there was a NDP gene mutation (c.122G>A) in haplotype 2, which means haplotype 2 was the carrier allele.
Table 2 Targeted Phasing Results Summary.
Sample
|
Chr
|
Targeted Phasing Region*
|
Belonged Phase block
|
Phase Block Length
|
Contained Heterozygous SNPs
|
G2018004A_LYL
|
16
|
215400-234700
|
132167-542445
|
410.2 kb
|
309
|
G2018004B_LGB
|
16
|
215400-234700
|
223804-543224
|
319.4 kb
|
251
|
G2018001A_LSL
|
X
|
43817770
|
43752981-44015286
|
262.3 kb
|
134
|
SNPs, Single nucleotide polymorphisms; Targeted phasing region, the pathogenic mutation sites for respective samples; Belonged Phase block, the phase block that contained the mutation sites.
Informative SNPs selection
The SNPs that were heterozygous in the carriers and homozygous in their partner were considered as informative SNPs. Based on the sequence information obtained for the flanking mutations, we identified 436 informative SNPs in family 1. Among them, 286 SNPs in the target phase block were heterogeneous in the wife and homogenous in the husband; conversely, 150 SNPs were heterogeneous in the husband and homogenous in the wife. In family 2, 134 SNPs were heterogeneous in the wife. We selected 10 informative SNPs in each family to test their embryos.
PGT-M
A total of 10 blastocysts were biopsied for WGA; 5 for each family. Biopsy trophectoderm cells from 10 blastocysts were successfully amplified. Next, PCR analysis that spanned the mutations was performed. In total, 10 SNPs within phase block of the mutations were successfully analyzed in each family. Next, the linkage analysis with the informative SNPs in 10 embryos of two families was performed. The linkage analysis showed that Embryo1-1, Embryo1-2, Embryo1-5 (Table 3) Embryo2-3, and Embryo2-4 (Table 4) inherited the normal haplotypes. However, Embryo1-3, Embryo2-1, Embryo2-2 and Embryo2-5 inherited an affected haplotype from the mother (solid square box ), and Embryo1-4 inherited affected haplotypes from the parents (solid square box and dotted square box).
Table 3 Ten SNP markers were selected to construct the haplotypes of embryos in family 1
Chr
|
POS
|
M
|
F
|
Embryo1-1
|
Embryo1-2
|
Embryo1-3
|
Embryo1-4
|
Embryo1-5
|
chr16
|
210977
|
C T
|
C C
|
T C
|
T C
|
C C
|
C C
|
T C
|
chr16
|
211090
|
A G
|
A A
|
G A
|
G A
|
A A
|
A A
|
G A
|
chr16
|
211167
|
A G
|
A A
|
G A
|
G A
|
A A
|
A A
|
G A
|
chr16
|
316945
|
A G
|
A A
|
G A
|
G A
|
A A
|
A A
|
G A
|
chr16
|
377623
|
G A
|
G G
|
A G
|
A G
|
G G
|
G G
|
A G
|
chr16
|
377644
|
G C
|
G G
|
C G
|
C G
|
G G
|
G G
|
C G
|
chr16
|
503947
|
AT AT
|
A AT
|
AT A
|
AT A
|
AT A
|
AT AT
|
AT A
|
chr16
|
514407
|
A A
|
C A
|
A C
|
A C
|
A C
|
A A
|
A C
|
chr16
|
539093
|
G G
|
C G
|
G C
|
G C
|
G C
|
G G
|
G C
|
chr16
|
539140
|
C C
|
A C
|
C A
|
C A
|
C A
|
C C
|
C A
|
POS, position; M, Mother ; F, Father; solid square box, affected haplotype from the mother; dotted square box, affected haplotype from the father
Table 4 Ten SNP markers were selected to construct the haplotypes of embryos in family 2
Chr
|
POS
|
M
|
F
|
Embryo2-1
|
Embryo2-2
|
Embryo2-3
|
Embryo2-4
|
Embryo2-5
|
chrX
|
43764085
|
A T
|
T -
|
A -
|
A -
|
T T
|
T -
|
A -
|
chrX
|
43767329
|
G A
|
A -
|
G -
|
G -
|
A A
|
A -
|
G -
|
chrX
|
43791371
|
C T
|
T -
|
C -
|
C -
|
T T
|
T -
|
C -
|
chrX
|
43807671
|
A C
|
C -
|
A -
|
A -
|
C C
|
C -
|
A -
|
chrX
|
43817770
|
T C
|
C -
|
T -
|
T -
|
C C
|
C -
|
T -
|
chrX
|
43824858
|
G A
|
A -
|
G -
|
G -
|
A A
|
A -
|
G -
|
chrX
|
43824916
|
C G
|
G -
|
C -
|
C -
|
G G
|
G -
|
C -
|
chrX
|
43824993
|
A G
|
G -
|
A -
|
A -
|
G G
|
G -
|
A -
|
chrX
|
43829396
|
G A
|
A -
|
G -
|
G -
|
A A
|
A -
|
G -
|
chrX
|
43833438
|
G A
|
A -
|
G -
|
G -
|
A A
|
A -
|
G -
|
POS, position; M, Mother ; F, Father; solid square box, affected haplotype from the mother;
Clinical Outcome
Embryo1-2 and Embryo2-3 were transferred to their mothers’ uterus, resulting in pregnancy in both families. Unfortunately, an unexplained abortion occurred in family 1 during the first trimester. However, amniocentesis was performed in family 2 at 16 weeks of gestation to verify the accuracy of the PGT-M. After 38 weeks of pregnancy, the family 2 got a healthy baby girl.