96 capillary 3730xl system

Candidate variants were confirmed using Sanger sequencing. PCR primer sequences and protocols are available upon request. Amplified fragments were sequenced using a 96‐capillary 3730xl system (Applied Biosystems).

After obtaining informed consent, 2 ml of peripheral blood was drawn from the patients and their parents. Samples from 66 unrelated patients were used for OTC molecular tests. The other 3 patients (P11-13) could not be included in the analysis as they died of acute hyperammonemic encephalopathy shortly after birth, so samples from their mothers were used for genetic test. Genomic DNA was extracted from peripheral blood leukocytes by using a blood kit (Zeesan Biotech, China). Polymerase chain reaction (PCR) was used to amplify all coding exons and exon–intron boundaries of the OTC gene using 9 pairs of synthetic oligonucleotide primers, and the primer sequences and annealing temperature were showed in Additional file 1: Table 1S. The PCR products were then purified and bidirectionally sequenced using the Applied Biosystems 96-capillary 3730XL system. Three patients underwent whole-exome sequencing (WES). The library was sequenced using Illumina HiSeq 4000 and generated 150 bp paired-end reads. Data analysis was performed as previously described [14 (link)]. Multiplex ligation-dependent probe amplification analysis (MLPA; SALSA MLPA P079 OTC kit, MRC-Holland, Amsterdam, The Netherlands) was performed for samples in which Sanger sequencing or WES failed to detect any disease-causing mutation [15 , 16 (link)].

Breakpoints were confirmed using PCR amplification and Sanger sequencing of junction fragments. PCR primer sequences and protocols are available upon request. Amplified fragments were sequenced using a 96-capillary 3730xl system (Applied Biosystems, Waltham, MA USA).