Low-pass genome sequencing
Genomic DNA was extracted from prenatal specimens according to the
manufacturer’s protocol. An STR-based semi quantitative PCR assay was
used to check for maternal DNA contamination from this procedure.
Samples with >10% contamination were excluded from the
study. Finally, 200 ng of genomic DNA was randomly fragmented, and DNA
libraries were constructed by end-repaired, A-tailed, and adaptor
ligation.28
Low-pass GS was performed as previously described, with a mean coverage
of 0.06X.5, 28, 29 Mapped reads were allocated to
20-kilobase (kb) bin sizes with 5-kb sliding to identify CNVs. CNV
profiles of each chromosome were represented as log2 of the mean
sequencing reads of each sequencing bin along the chromosome. Any two
CNVs with ≥ 60% reciprocal overlap were identified as the same.
Publicly available genomic databases including 1000 genomes, DGV
(http://dgv.tcag.ca/dgv/app/home), OMIM (https://www.omim.org/),
DECIPHER (https://decipher.sanger.ac.uk/), ClinVar
(https://www.ncbi.nlm.nih.gov/clinvar/), UCSC (http://genome.ucsc.edu/),
and PubMed (https://pubmed.ncbi.nlm.nih.gov/) were used as reference CNV
sources. The pathogenicity of identified CNVs was assessed based on the
American College of Medical Genetics (ACMG)
guidelines.30 Only germline aneuploidy and pCNV were
considered in further analyses.