De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain of function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.
De novo mutations (DNMs) are an important player in heritable diseases and evolution, yet little is known about the different mutagenic processes in our germline given the difficulty to reliably identify ultra-low frequency variants. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ~4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified highly mutable sites with mutation frequencies 4-5 orders of magnitude higher than the genome average. Multiple mutations were found at a higher frequency, or exclusively, in older donors, suggesting that these mutations are testis exclusive mosaics expanding in the male germline with age. Also, older donors harbored more mutations associated with congenital disorders. Some mutations were found in both age groups with no significant difference, suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independently of age, the frequency and deleteriousness of the mutations in sperm were elevated compared to reports in the population. Our approach is an important strategy to identify mutations that could be associated with aberrant receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.
The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.
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