Characterizing sensitivity and coverage of clinical WGS as a diagnostic test for genetic disorders.

BACKGROUND: Due to its reduced cost and incomparable advantages, WGS is likely to lead to changes in clinical diagnosis of rare and undiagnosed diseases. However, the sensitivity and breadth of coverage of clinical WGS as a diagnostic test for genetic disorders has not been fully evaluated. METHODS: Here, the performance of WGS in NA12878, the YH cell line, and the Chinese trios were measured by assessing their sensitivity, PPV, depth and breadth of coverage using MGISEQ-2000. We also compared the performance of WES and WGS using NA12878. The sensitivity and PPV were tested using the family-based trio design for the Chinese trios. We further developed a systematic WGS pipeline for the analysis of 8 clinical cases. RESULTS: In general, the sensitivity and PPV for SNV/indel detection increased with mean depth and reached a plateau at an ~ 40X mean depth using down-sampling samples of NA12878. With a mean depth of 40X, the sensitivity of homozygous and heterozygous SNPs of NA12878 was > 99.25% and > 99.50%, respectively, and the PPV was 99.97% and 98.96%. Homozygous and heterozygous indels showed lower sensitivity and PPV. The sensitivity and PPV were still not 100% even with a mean depth of ~ 150X. We also observed a substantial variation in the sensitivity of CNV detection across different tools, especially in CNVs with a size less than 1 kb. In general, the breadth of coverage for disease-associated genes and CNVs increased with mean depth. The sensitivity and coverage of WGS (~ 40X) was better than WES (~ 120X). Among the Chinese trios with an ~ 40X mean depth, the sensitivity among offspring was > 99.48% and > 96.36% for SNP and indel detection, and the PPVs were 99.86% and 97.93%. All 12 previously validated variants in the 8 clinical cases were successfully detected using our WGS pipeline. CONCLUSIONS: The current standard of a mean depth of 40X may be sufficient for SNV/indel detection and identification of most CNVs. It would be advisable for clinical scientists to determine the range of sensitivity and PPV for different classes of variants for a particular WGS pipeline, which would be useful when interpreting and delivering clinical reports.

Preimplantation genetic testing for a family with usher syndrome through targeted sequencing and haplotype analysis.

BACKGROUND: Preimplantation genetic testing for monogenic defects (PGT-M) has been available in clinical practice. This study aimed to validate the applicability of targeted capture sequencing in developing personalized PGT-M assay. METHODS: One couple at risk of transmitting Usher Syndrome to their offspring was recruited to this study. Customized capture probe targeted at USH2A gene and 350 kb flanking region were designed for PGT-M. Eleven blastocysts were biopsied and amplified by using multiple displacement amplification (MDA) and capture sequencing. A hidden Markov model (HMM) assisted haplotype analysis was performed to deduce embryo's genotype by using single nucleotide polymorphisms (SNPs) identified in each sample. The embryo without paternal rare variant was implanted and validated by conventional prenatal or postnatal diagnostic means. RESULTS: Four embryos were diagnosed as free of father's rare variant, two were transferred and one achieved a successful pregnancy. The fetal genotype was confirmed by Sanger sequencing of fetal genomic DNA obtained by amniocentesis. The PGT-M and prenatal diagnosis results were further confirmed by the molecular diagnosis of the baby's genomic DNA sample. The auditory test showed that the hearing was normal. CONCLUSIONS: Targeted capture sequencing is an effective and convenient strategy to develop customized PGT-M assay.

Applying targeted next generation sequencing to dried blood spot specimens from suspicious cases identified by tandem mass spectrometry-based newborn screening.

BACKGROUND: Tandem mass spectrometry (TMS)-based newborn screening has been proven successful as one of the public healthcare programs, although the practicability has not yet been specifically addressed. METHODS: Sixty residual dried blood spot (DBS) specimens from confirmation/diagnosis-insufficient cases discovered by TMS screening were analyzed by targeted next generation sequencing (TNGS) assay. RESULTS: In total, 26, 11, 9, and 14 cases were diagnosed as positive, high risk, low risk, and negative, respectively. CONCLUSIONS: Applying the DBS-based TNGS assay for the accurate and rapid diagnosis of inborn errors of metabolism (IEMs) is feasible, competent, and advantageous, enabling a simplified TMS screening-based, TNGS assay-integrated newborn screening scheme highlighting an efficient, executable, and one-step screening-to-diagnosis workflow.