Objective: Absence of homozygosity (AOH) is a genetic characteristic known to cause human diseases mainly through autosomal recessive or imprinting mechanisms. The importance and necessity of accurate AOH detection has become more clinically significant in recent years. However, it remains a challenging task for sequencing-based methods thus far. Methods: In this study, we developed and optimized a new bioinformatic algorithm based on the assessment of minimum sequencing coverage, optimal bin size, the Z-score threshold of four types of allele count and the frequency for accurate genotyping using 28 AOH negative samples, and redefined the AOH detection cutoff value. We showed the performance of chromosome analysis by five-fold coverage whole genome sequencing (CMA-seq) for AOH identification in 27 typical prenatal/postnatal AOH positive samples, which were previously confirmed by chromosomal microarray analysis with single nucleotide polymorphism array (CMA/SNP array). Results: The blinded study indicated that for all three forms of AOH, including whole genomic AOH, single chromosomal AOH and segmental AOH, and all kinds of sample types, including chorionic villus sampling, amniotic fluid, cord blood, peripheral blood and abortive tissue, CMA-seq showed equivalent detection power to that of routine CMA/SNP arrays (750K). The subtle difference between the two methods is that CMA-seq is prone to detect small inconsecutive AOHs, while CMA/SNP array reports it as a whole. Conclusion: Based on our newly developed bioinformatic algorithm, it is feasible to detect clinically significant AOH using CMA-seq in prenatal diagnosis.

Objective: Mosaicism is a common biological phenomenon in organisms and has been reported in many types of chromosome abnormalities, including the absence of heterozygosity (AOH). Due to the detection limitations of the sequencing approach, mosaic AOH events are rarely assessed in clinical cases. Herein, we report the performance of mosaic AOH identification using a low-pass (5~8-fold) WGS method (termed ‘CMA-seq’, an abbreviation for ‘Chromosome Analysis by Sequencing’) in fetal genetic diagnosis. Methods: Thirty AOH-negative, eleven constitutional AOH, and three mosaic AOH samples were collected as training data sets to develop the algorithm and evaluate the suitable thresholds for distinguishing mosaic AOH. Twenty-four new chromosomal aberrant cases, along with sixteen constitutional AOH samples, which were previously ascertained via the SNP-array-based method, were used as a validation data set to measure the performance in terms of sensitivity and specificity of this algorithm. Results: A new statistic, ‘D-value’, was implemented to identify and distinguish constitutional and mosaic AOH events. The reporting thresholds for constitutional and mosaic AOH were also established. In the validation set consisting of 24 new cases, seven constitutional AOH cases and 1 mosaic AOH case were successfully identified, indicating that the results were consistent with those of the SNP-array-based method. The results of all sixteen constitutional AOH validation samples also met the threshold requirements. Conclusions: In this study, we developed a new bioinformatic algorithm to accurately distinguish mosaic AOH from constitutional AOH by low-pass WGS. However, due to the small sample size of the training data set, the algorithm proposed in this manuscript still needs further refinements.