Leptin (LEP) gene as described previously [18].Plasma DNA SequencingWe prepared sequencing libraries of plasma DNA using the Paired-End Sequencing Sample Preparation Kit (Illumina) as described previously [19]. Because of the variable volume of maternal plasma accessible, we aimed to possess a fairly consistent quantity of plasma DNA input for library preparation. We thus applied 13 to 20 ng from the extracted plasma DNA for library preparation which corresponded to the amount extracted from 1.5 to two.two mL of maternal plasma. The adaptor-ligated plasma DNA was enriched by a 12-cycle PCR. We performed cluster generation on a cBot clonal amplification system (Illumina) using the TruSeq PE Cluster Generation Kit v3 (Illumina). Each and every library (each test and reference samples) was sequenced with one particular lane of a flow cell on a HiSeq 2000 sequencing method (Illumina) inside a paired-end format of 50bp62. Sequence data have been deposited in the European Genome-Phenome Archive (EGA, http://ebi.ac.uk/ega/), which can be hosted by the European Bioinformatics Institute (EBI), under the accession number EGAS00001000439.Materials and Techniques Ethical StatementThe study was authorized by the Joint Chinese University of Hong Kong ?Hospital Authority New Territories East Cluster Clinical Study Ethics Committee. We recruited pregnant females with written informed consent in the Prince of Wales Hospital, the Kwong Wah Hospital and also the Tsan Yuk Hospital in Hong Kong, plus the Asan Healthcare Center in Seoul.Sample CollectionFor situations 01, 02, and 03, maternal peripheral blood samples were collected into EDTA-containing tubes right after invasive procedures (Table 1). For cases 04, 05 and 06, maternal peripheral blood samples have been collected ahead of performing any invasive procedures. Maternal blood samples have been drawn at 12 3/7 to 28 4/7 weeks of gestation (Table 1). Among the six test samples, there were three circumstances (situations 01, 02 and 03) of fetal de novo 22q11.two microdeletion, a single case (case 04) of fetal de novo 22q11.2 microduplication (two.four Mb) and one particular case (case 05) of maternally-inherited 22q11.2 microduplication (two.four Mb). There was also 1 case (case 06) in which the mother had a balanced translocation of t(3;four)(q29;q32) and the fetus was identified to possess 3q29 microduplication (5.1 Mb) and 4q32.1-q35.two deletion (32.9 Mb). Complete karyotyping was performed along with the fetal karyotypes were further ascertained by array comparative genomic hybridization (array CGH) [16], fluorescence in situ hybridization (FISH) or possibly a combination of quantitative fluorescence PCR (QFPCR) and FISH. In addition, we collected a group of eight singleton pregnant circumstances with typical fetal karyotypes as reference controls for downstream information analysis. Table 1. Sample facts.Sequence Alignment and FilteringPaired-end reads have been aligned towards the non-repeat masked human reference genome (NCBI Make 36.Buy3-Hydroxycyclobutan-1-one 1/hg18) working with the Short Oligonucleotide Alignment System two (SOAP2) (http://http:// soap.Eugenol acetate custom synthesis genomics.PMID:35670838 org.cn/). We allowed as much as two nucleotide mismatches for each member of the paired-end reads. Only paired-end reads with both ends aligned to the exact same chromosome together with the right orientation, spanning an insert size #600 bp were included in downstream evaluation. We also removed duplicated reads which were defined as paired-end reads showing identical commence and end positions inside the human genome.Calculation on the Genomic RepresentationWe initial divided each chromosome into 100-kb bins and performed locally weighted scatterplot smoothing.