Postzygotic single-nucleotide mosaicisms (pSNMs) have been extensively studied in tumors and are known to play critical roles in tumorigenesis. However, the patterns and origin of pSNMs in normal organs of healthy humans remain largely unknown. Using whole-genome sequencing and ultra-deep amplicon re-sequencing, we identified and validated 164 pSNMs from 27 postmortem organ samples obtained from five healthy donors. The mutant allele fractions ranged from 1.0% to 29.7%. Inter- and intra-organ comparison revealed two distinctive types of pSNMs, with about half originating during early embryogenesis (embryonic pSNMs) and the remaining more likely to result from clonal expansion events that had occurred more recently (clonal expansion pSNMs). Compared to clonal expansion pSNMs, embryonic pSNMs had higher proportion of C>T mutations with elevated mutation rate at CpG sites. We observed differences in replication timing between these two types of pSNMs, with embryonic and clonal expansion pSNMs enriched in early- and late-replicating regions, respectively. An increased number of embryonic pSNMs were located in open chromatin states and topologically associating domains that transcribed embryonically. Our findings provide new insights into the origin and spatial distribution of postzygotic mosaicism during normal human development.

The allele fraction (AF) distribution, occurrence rate, and evolutionary contribution of postzygotic single-nucleotide mosaicisms (pSNMs) remain largely unknown. In this study, we developed a mathematical model to describe the accumulation and AF drift of pSNMs during the development of multicellular organisms. By applying the model, we quantitatively analyzed two large-scale data sets of pSNMs identified from human genomes. We found that the postzygotic mutation rate per cell division during early embryogenesis, especially during the first cell division, was higher than the average mutation rate in either male or female gametes. We estimated that the stochastic cell death rate per cell cleavage during human embryogenesis was ∼5%, and parental pSNMs occurring during the first three cell divisions contributed to ∼10% of the de novo mutations observed in children. We further demonstrated that the genomic profiles of pSNMs could be used to measure the divergence distance between tissues. Our results highlight the importance of pSNMs in estimating recurrence risk and clarified the quantitative relationship between postzygotic and de novo mutations.

Genomic mosaicism arising from postzygotic mutations has long been associated with cancer and more recently with non-cancer diseases. It has also been detected in healthy individuals including healthy parents of children affected with genetic disorders, highlighting its critical role in the origin of genetic mutations. However, most existing software for the genome-wide identification of single-nucleotide mosaicisms (SNMs) requires a paired control tissue obtained from the same individual which is often unavailable for non-cancer individuals and sometimes missing in cancer studies. Here, we present MosaicHunter (http://mosaichunter.cbi.pku.edu.cn), a bioinformatics tool that can identify SNMs in whole-genome and whole-exome sequencing data of unpaired samples without matched controls using Bayesian genotypers. We evaluate the accuracy of MosaicHunter on both simulated and real data and demonstrate that it has improved performance compared with other somatic mutation callers. We further demonstrate that incorporating sequencing data of the parents can be an effective approach to significantly improve the accuracy of detecting SNMs in an individual when a matched control sample is unavailable. Finally, MosaicHunter also has a paired mode that can take advantage of matched control samples when available, making it a useful tool for detecting SNMs in both non-cancer and cancer studies.

The roles and characteristics of postzygotic single‐nucleotide mosaicisms (pSNMs) in autism spectrum disorders (ASDs) remain unclear. In this study of the whole exomes of 2,361 families in the Simons Simplex Collection, we identified 1,248 putative pSNMs in children and 285 de novo SNPs in children with detectable parental mosaicism. Ultra‐deep amplicon resequencing suggested a validation rate of 51%. Analyses of validated pSNMs revealed that missense/loss‐of‐function (LoF) pSNMs with a high mutant allele fraction (MAF≥ 0.2) contributed to ASD diagnoses (P = 0.022, odds ratio [OR] = 5.25), whereas missense/LoF pSNMs with a low MAF (MAF<0.2) contributed to autistic traits in male non‐ASD siblings (P = 0.033). LoF pSNMs in parents were less likely to be transmitted to offspring than neutral pSNMs (P = 0.037), and missense/LoF pSNMs in parents with a low MAF were transmitted more to probands than to siblings (P = 0.016, OR = 1.45). We estimated that pSNMs in probands or de novo mutations inherited from parental pSNMs increased the risk of ASD by approximately 6%. Adding pSNMs into the transmission and de novo association test model revealed 13 new ASD risk genes. These results expand the existing repertoire of genes involved in ASD and shed new light on the contribution of genomic mosaicisms to ASD diagnoses and autistic traits.

Detection of de novo, low-frequency mutations is essential for characterizing cancer genomes and heterogeneous cell populations. However, the screening capacity of current ultrasensitive NGS methods is inadequate owing to either low-efficiency read utilization or severe amplification bias. Here, we present o2n-seq, an ultrasensitive and high-efficiency NGS library preparation method for discovering de novo, low-frequency mutations. O2n-seq reduces the error rate of NGS to 10−5–10−8. The efficiency of its data usage is about 10–30 times higher than that of barcode-based strategies. For detecting mutations with allele frequency (AF) 1% in 4.6 Mb-sized genome, the sensitivity and specificity of o2n-seq reach to 99% and 98.64%, respectively. For mutations with AF around 0.07% in phix174, o2n-seq detects all the mutations with 100% specificity. Moreover, we successfully apply o2n-seq to screen de novo, low-frequency mutations in human tumours. O2n-seq will aid to characterize the landscape of somatic mutations in research and clinical settings.

On the cover: The cover image, by Yanmei Dou et al., is based on the Research Article Postzygotic single‐nucleotide mosaicisms contribute to the etiology of autism spectrum disorder and autistic traits and the origin of mutations, Pages 1002–1013. DOI 10.1002/humu. 23284

Genomic mosaicism in parental gametes and peripheral tissues is an important consideration for genetic counseling. We studied a Chinese cohort affected by a severe epileptic disorder, Dravet syndrome (DS). There were 56 fathers who donated semen and 15 parents who donated multiple peripheral tissue samples. We used an ultra-sensitive quantification method, micro-droplet digital PCR (mDDPCR), to detect parental mosaicism of the proband’s pathogenic mutation in SCN1A, the causal gene of DS in 112 families. Ten of the 56 paternal sperm samples were found to exhibit mosaicism of the proband’s mutations, with mutant allelic fractions (MAFs) ranging from 0.03% to 39.04%. MAFs in the mosaic fathers’ sperm were significantly higher than those in their blood (p = 0.00098), even after conditional probability correction (p’ = 0.033). In three mosaic fathers, ultra-low fractions of mosaicism (MAF < 1%) were detected in the sperm samples. In 44 of 45 cases, mosaicism was also observed in other parental peripheral tissues. Hierarchical clustering showed that MAFs measured in the paternal sperm, hair follicles and urine samples were clustered closest together. Milder epileptic phenotypes were more likely to be observed in mosaic parents (p = 3.006e-06). Our study provides new insights for genetic counseling.

Objective: To investigate the clinical phenotypes and the mutant allele proportion of parents with SCN1A gene mutation mosaicism of Dravet syndrome (DS) children, thus to provide guidance for family reproduction and prenatal diagnosis. Method: The clinical data and peripheral blood DNA samples of DS patients with a SCN1A gene mutation proved by Sanger sequencing were collected prospectively from February 2005 to November 2016 in Department of Pediatrics, Peking University First Hospital. The same mutation was searched in parents and other available relatives. Parental somatic mosaicism was confirmed and quantified by Ion Torrent Personal Genome Machine (PGM) and Raindrop droplet digital PCR (ddPCR). The families were followed up and prenatal diagnosis was provided. Result: Mosaicisms of SCN1A gene mutation in parents were identified in 5.2% (30 out of 575) DS families. Seventeen were fathers and thirteen were mothers. The mutant allele proportion ranged from 1.7% to 32.9% by PGM and from 0.82% to 34.51% by ddPCR, respectively. In 30 parents with somatic mosaicism, thirteen were asymptomatic, ten had a history of febrile seizures (FS), five with epilepsy, one with febrile seizure plus and one had a history of afebrile seizure. Four families had two children with DS. Three siblings of the probands were confirmed genetically with the same pathogenic mutation. One deceased sister of the proband was assumed to have the same pathogenic mutation because she matched DS diagnosis after medical history review despite no blood sample. Two families received prenatal diagnosis. One second pregnancy was terminated because the fetus inherited the mutation as the mother's wish. Conclusion: Sanger sequencing detects parents of some children with DS are SCN1A mutation mosaics. PGM and ddPCR can be used for accurate quantification of mutant mosaics, which can provide accurate guidance for family genetic counseling.

The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling.

Bioinformatics is a fast-growing interdisciplinary field in which the demand for quality education exceeds the supply, especially in developing regions and countries. A massive open online course (MOOC) is a new model for education that delivers videotaped lectures and other course materials over the Internet for all interested persons around the globe to learn for free. Here we present our MOOC “Bioinformatics: Introduction and Methods,” which is the second bioinformatics MOOC in the world and one of the first batch of seven MOOCs from China. In the first two runs of this bilingual MOOC, more than 30,000 students with diverse backgrounds registered from 110 countries and regions. In this manuscript, we present the content design of the MOOC, the demographic profiles and learning patterns of the students, the requirement for English support, and feedback from on-campus students. We offer a few suggestions to other scientists who may be interested in creating a MOOC. We also remember the S* course, a successful open online bioinformatics course that ran from 2001 to 2007, long before the current wave of MOOCs. We believe that MOOC education has great potential to enhance global bioinformatics education.

Postzygotic single-nucleotide mutations (pSNMs) have been studied in cancer and a few other overgrowth human disorders at whole-genome scale and found to play critical roles. However, in clinically unremarkable individuals, pSNMs have never been identified at whole-genome scale largely due to technical difficulties and lack of matched control tissue samples, and thus the genome-wide characteristics of pSNMs remain unknown. We developed a new Bayesian-based mosaic genotyper and a series of effective error filters, using which we were able to identify 17 SNM sites from ∼80× whole-genome sequencing of peripheral blood DNAs from three clinically unremarkable adults. The pSNMs were thoroughly validated using pyrosequencing, Sanger sequencing of individual cloned fragments, and multiplex ligation-dependent probe amplification. The mutant allele fraction ranged from 5%-31%. We found that C→T and C→A were the predominant types of postzygotic mutations, similar to the somatic mutation profile in tumor tissues. Simulation data showed that the overall mutation rate was an order of magnitude lower than that in cancer. We detected varied allele fractions of the pSNMs among multiple samples obtained from the same individuals, including blood, saliva, hair follicle, buccal mucosa, urine, and semen samples, indicating that pSNMs could affect multiple sources of somatic cells as well as germ cells. Two of the adults have children who were diagnosed with Dravet syndrome. We identified two non-synonymous pSNMs in SCN1A, a causal gene for Dravet syndrome, from these two unrelated adults and found that the mutant alleles were transmitted to their children, highlighting the clinical importance of detecting pSNMs in genetic counseling.