Miseqdx

DNA sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit. Sequencing was performed on the Illumina MiSeq Dx instrument using RUO (research use only) mode, using the v2 500-cycle sequencing chemistry. Illumina read quality was assessed using FastQC v0.11.7; de novo assembly was performed using CLC Genomics Workbench v12.0.3, with resulting average depth of read coverage 925x for AhMtk13a and 518x for AhMtk13b. Assembled phage genome was annotated manually using the Artemis annotation tool [72 (link)]. The putative open reading frames (ORFs) were predicted by using GenemarkS [73 (link)]. Putative functions of the ORFs were analyzed by BLASTP search and Pfam [74 (link)], and HMMER online software [75 (link)]. Prediction of tRNAs was done by using tRNAscan-SE 1.3.1 software [76 ]. Geneious software was used for multiple alignments and mapping [77 (link)]. Phage taxonomy was determined by using BLASTn results and was visualized by VIPtree [24 (link),78 (link)]. Genome sequence identity was calculated by ANI Calculator based on the OrthoANIu algorithm [79 (link)]. Phylogenetic tree was constructed by MegaX with 500 bootstrap (neighbor-joining tree) [80 (link)]. GenBank accession number for our nucleotide sequence: BankIt2526887 Aeromonas OL840900 for Phage AhMtk13a; BankIt2529090 Aeromonas OL840901 for Phage AhMtk13b.

Blastocyst trophectoderm cells were amplified using the REPLI‐g Single Cell Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. PGT‐M was conducted to test for mutated sites and linked SNPs, and Peking Jabrehoo Med Tech was used for haplotype construction. The pathogenic variants/SNPs of the target genes were amplified, followed by Sanger sequencing (Figure S1). The genotypes of the embryos were determined based on the results of pathogenic variant and haplotype analysis (Harton et al., 2011 (link)).
PGT for aneuploidy (PGT‐A) was performed with NGS‐based CNV sequencing on the Illumina MiSeqDx platform, which indicated >4 Mb CNV and 30%–70% mosaicism.

In the final step, each library was diluted to 10 nM in Low TE, and then all libraries were pooled at 10 nM and diluted to 4 nM. Finally, the pooled libraries were denatured and diluted at 12 pM and loaded on a MiSeq Reagent Nano Kit v2 (300-cycles) cartridge with 12.5 pM PhiX (1.5%) sequencing control V3 (Illumina). Libraries were then sequenced on Illumina MiseqDX (Illumina) instrumentation in RUO mode. The whole process also required the use of a Veriti 96 thermal cycler (Applied Biosystem) for the amplification of the libraries and the Dynamag-96 side (Invitrogen) for the clean-up of the libraries.

The gold standard for the identification of a female as a haemophilia carrier was genetic testing. Inversion testing, DNA sequencing and the gene dosage assay were subsequently carried out until the detection of the pathogenic mutations. Long-distance PCR or inverse-shifting PCR according to the research of Rossetti et al. was utilized for inversion tests [12 ,13 (link)]. Sanger sequencing with ABI 3130 Genetic Analyser (Applied Biosystems, Waltham, MA, USA) or next-generation sequencing using MiSeqDx^® (Illumina Inc., San Diego, CA, USA) was performed for DNA sequencing. Multiple ligation-dependent probe amplification was applied for the gene dosage assay. As for females who had wanted to undergo carrier testing, we carried out targeted gene mutation analysis for the same pathogenic mutation as their own probands. We classified the pathogenic mutations into null (inversion, nonsense, and frameshift) and non-null mutations (missense and splicing).

DNA was extracted from paraffin-embedded tissue samples of patients using a nucleic acid extraction kit (Model: FFPE DNA, Cat NO. ADx-TI01, Xiamen Aide Biomedical Technology Co., Ltd.), and the concentration of DNA was determined using the Qubit dsDNA Assay. The genomic DNA was disrupted using the covaris M220 ultrasound system. The universal kit for multigene mutation detection (Guangzhou Burning Stone Medical Laboratory Co., Ltd., item number: RS03F-12) was used for end repair, 3' end plus A, linker ligation, PCR amplification, and purification of the magnetic beads to obtain a pre-library. Then, we used the human multigene mutation detection capture probe (Lung cure ™ LK101, Lung-cure™ LK165 (Blood Version), Guangzhou Burning Stone Medical Laboratory Co., Ltd.) hybrid to capture the target area, with streptomycin affinity magnetic beads capturing the library fragments that hybridized with the probe, and we used PCR for amplification and purification to determine the size and concentration of the library fragment. The libraries were mixed, denatured, and sequenced on a genetic sequencer (Illumina MiSeq Dx) platform. After the completion of the sequencing, the sequence was analyzed and compared with the second-generation sequencing data analysis software (produced by Guangzhou Burning Stone Medical Laboratory Co., Ltd.), and the results were interpreted.