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Discovery of an unusually high number of de novo mutations in sperm of older men using duplex sequencing

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.


Sample preparation 12
Sperm genomic DNA (gDNA) was extracted using the Gentra Puregene Cell kit (QIAGEN) as 13 follows: first, 25 µL of semen (~2 × 10 6 sperm cells) were centrifuged (8,000g, 20 seconds) and 14 the resulting sperm cell pellet was incubated overnight with 150 µL of cell lysis solution, 6 µL 15 of 1M DTT, and 0.5 µL of proteinase K (20mg/mL) at 37°C. RNase treatment was performed by 16 adding 0.75 µL of RNase A (4mg/mL) and incubating at 37°C for 15 minutes. For the protein 17 precipitation step, the sample was put on ice for 5 minutes. Then, 50 µL of protein 18 precipitation solution were added followed by vigorous manual shaking for 1 minute. Next, 19 the sample was centrifuged for 20 minutes at 13,000g to remove the protein precipitate. The 20 supernatant was transferred into a fresh tube and a second centrifugation step was 21 performed if the solution was still cloudy. The DNA was precipitated by adding 150 µL of 22 isopropanol (100%) and 0.25 µL of glycogen solution to the supernatant, followed by gentle 23 inversions. A centrifugation step was done (30 minutes, 13,000g at 4°C) and the supernatant 24 was discarded. DNA was washed with 150 µL of 70% ethanol, followed by a 3-minute 25 centrifugation (13,000g, 4°C). Supernatant was removed and DNA was dried at room 26 temperature for 3 minutes. Finally, DNA was resuspended in 25 µL of DNA hydration solution. 27 Genomic DNA from B-lymphocyte cell line was extracted using the same kit. Cells in cultured 28 medium (~2 × 10 6 cells) were added to a 1.5 mL microcentrifuge tube and centrifuged for 5 29 seconds at 13,000g. Supernatant was discarded and pellet was resuspended in residual 30 supernatant by vortexing vigorously. Cell lysis solution was added (300 µL) followed by a 10 1 seconds vortexing step to lyse cells. Next, 100 µL of protein precipitation solution were added 2 followed by 20 seconds of vortexing. The mixture was then centrifuged for 1 minute at 3 13,000g to pellet the proteins. Isopropanol (300 µL) was added to a clean 1.5 mL 4 microcentrifuge tube followed by the addition of the supernatant of the previous step. The 5 tube was inverted 50 times, followed by a centrifugation step (1 minute, 13,000g). At this 6 stage, DNA formed a pellet and the supernatant was carefully discarded. Then, 300 µL of 70% 7 ethanol were added and the tube was inverted several times to wash the extracted DNA. The 8 tube was centrifuged for 1 minute at 13,000g and the supernatant was removed, followed by 9 a drying step at room temperature for 5 minutes. Dried DNA was resuspended in 100 µL of 10 DNA hydration solution and was vortexed for 5 seconds at medium speed. DNA was incubated 11 overnight at 22°C with gentle shaking. Finally, samples were transferred to a storage tube. including an initial restriction digest to pre-select the target regions. In the different libraries 15 we used some minor modifications to our main protocol, either starting with distinct input 16 DNA amounts, different adapters, or amplification strategies; see Table S3. These 17 modifications should not impact the variants called. 18

Targeted enzymatic fragmentation 19
Genomic DNA (500-5,000 ng, Table S3) was subject to an overnight restriction enzyme digest 20 that targeted 5 regions of the FGFR3 gene (Table S8). Resulting fragments were expected to 21 have a length between ~300 to ~600 bp. A double size selection was performed using 22 SPRIselect beads (Beckman Coulter) in order to exclude fragments outside of this size range. 23 To the 100 µL of restriction digest solution, 93.5 µL of PCR grade water were added. Then, 24 106.5 µL of beads were added (0.55 volumes of beads), mixed by pipetting thoroughly and 25 incubated at room temperature for 5 minutes. Tubes were then placed on a magnet for 5 26 minutes and 290 µL of supernatant were transferred to a new tube. Next, 84.2 µL of beads 27 (1.0 volumes in total considering the initial bead solution) were added to the solution and 28 mixed by pipetting. The mixture was incubated at room temperature for 5 minutes. Tubes 29 were placed on a magnet and supernatant was discarded. Beads were washed twice with 80% 30 ethanol while still on the magnet. Beads were dried at room temperature and 50 µL of PCR 31 grade water were added. Beads were resuspended by pipetting and incubated at room 32 temperature for 5 minutes. Finally, tubes were placed on a magnet and the clear supernatant 1 containing the size-selected DNA was transferred to a new tube. 2

Adapter synthesis 3
We used 3 different adapter synthesis protocols to produce Adapter 1, Adapter 2 and Adapter 4 3. Supplemental Figure S11 shows the structural differences between adapters: Adapter 1 is 5 identical to the original open-hairpin structure, whereas adapters 2 and 3 have a closed loop 6 hairpin structure that is opened up after the ligation step by the cleavage of the uracil with 7 the USER enzyme mix (New England Biolabs). It is suggested that closed loops adapters reduce 8 adapter dimers during ligation (New England Biolabs). Structurally, adapter 3 is similar to 9 adapter 2, but is 9 bp longer and contains a phosphorothioate bond before the T-overhang on 10 the 3' end such that removal of this overhang is reduced. T-tailed DS adapters were 11 synthesized with modifications to the original protocol (Kennedy et al. 2014). Oligonucleotides 12 used for each adapter are listed in Table S12. Klenow Exo-(5U/µL) (New England Biolabs). The mixture was incubated for 1 hour at 37°C. 21 Purification was done by adding 28 µl (0.5 volumes) of ammonium acetate (5 M) (Invitrogen) 22 followed by the addition of 168 µL (2 volumes) of pre-cooled 100% ethanol. The solution was 23 inverted several times until it got cloudy. Precipitation occurred while the solution was 24 incubated for 30 minutes at -20°C. The solution was then centrifuged for 30 minutes at 4°C in 25 a microcentrifuge at 14,000 rpm. Supernatant was then discarded and 1 mL of 80% ethanol 26 was added followed by a 5-minute centrifugation at 4°C, 14,000 rpm. The supernatant was 27 discarded and the tube was dried at room temperature until the pellet became transparent. 28 The pellet was resuspended in 40 µL of PCR grade water. The T-overhang was generated by 29 digestion with HpyCH4III enzyme: 47 µL of PCR grade water, 10 µL of 10× CutSmart buffer 30 (New England Biolabs), and 3 µL of HpyCH4III (5U/µL) (New England Biolabs) were added to 31 the extended adapters and incubated for 16 hours. To purify the adapters, 400 µL of PCR 32 grade water were added, followed by 250 µL of ammonium acetate (5 M). Then, 1.5 mL of 33 pre-cooled 100% ethanol were added and the solution was mixed by inverting until it got 1 cloudy. Adapters were precipitated while incubated at -20°C for 30 minutes. The solution was 2 then centrifuged for 30 minutes at 4°C, 14,000 rpm. Supernatant was discarded and 1 mL of 3 80% ethanol was added followed by a 5-minute centrifugation at 4°C, 14,000 rpm. The 4 supernatant was again discarded and the tube was dried at room temperature until the pellet 5 became transparent. Adapters were resuspended by adding 22 µL of 10mM mM TElow (Tris-6 HCl pH 8.0, 0.1 mM EDTA). 7 Adapter 2 was synthesized using the adapter 1 protocol. The only difference is that instead of 8 using oligonucleotides mws51 and mws55 in the first step, only oligonucleotide DS_Hairpin_U 9 was used, and consequently, an extra 10 µL of PCR grade water were added to the extension 10 reaction. 11 The synthesis of adapter 3 started by using oligonucleotides DS_Hairpin_U and TA-Overhang. 12 First, 10 µL of each oligonuclotide (each at 100 pmole/µL) were added to a PCR tube. The 13 phosphorylation of 5' ends was done by adding 23.5 µL of PCR grade water, 5.2 µL of T4 DNA 14 Ligase Buffer (10×) with ATP (10 mM) (New England Biolabs), and 3.3 µL of T4 Polynucleotide 15 Kinase (10U/µL), followed by an incubation at 37°C for 30 minutes. Then, the solution was 16 heated in a PCR machine at 95°C for 5 minutes. The machine was then turned off and the 17 mixture stayed inside for 1 hour to anneal the oligonucleotides. The extension of annealed 18 oligonucleotides was done by mixing 50 µL from the previous step with 26.7 µl of PCR grade 19 water, 5µL of T4 DNA Ligase Buffer (10×) with ATP (10 mM), 10 µL of dNTPs (2.5 mM), 1 µL of 20 BSA (10 mg/mL) (New England Biolabs), 0.6 µL of T4 DNA Ligase (2,000 U/µL) (New England 21 Biolabs), and 1.7 µL of T4 DNA Polymerase (New England Biolabs). The solution was incubated 22 at 12°C for 30 minutes followed by 16°C for another 30 minutes. The purification of adapters 23 was accomplished by adding 400 µL of PCR grade water followed by 250 µL of ammonium 24 acetate (5 M). Then, 1.5 mL of pre-cooled 100% ethanol were added and the solution was 25 mixed by inverting until it got cloudy. Adapters were precipitated while incubated at -20°C for 26 30 minutes. The solution was then centrifuged for 30 minutes at 4°C, 14,000 rpm. The 27 supernatant was discarded and 1 mL of 80% ethanol was added followed by a 5-minute 28 centrifugation at 4°C, 14,000 rpm. The supernatant was again discarded and the tube was 29 dried at room temperature until the pellet became transparent. Adapters were resuspended 30 by adding 22 µL of 10mM mM TElow (Tris-HCl pH 8.0, 0.1 mM EDTA). 31

End-repair/A-tailing and ligation 1
End-repair/A-tailing was done by mixing 50 µL of size-selected genomic DNA with 7 µL of 2 NEBNext Ultra II End Prep Reaction Buffer (New England Biolabs), and 3 µL of NEBNext Ultra II 3 End Prep Enzyme Mix (New England Biolabs). The mixture was incubated at 20°C for 30 4 minutes followed by 65°C for 30 minutes. 5 The ligation of adapters occurred by adding 30 µL of NEBNext Ultra II Ligation Master Mix 6 (New England Biolabs), 1 µL of NEBNext Ultra II Ligation Enhancer (New England Biolabs), and 7 2.5 µL of adapter dilution to the 60 µL of End-repair/A-tailing mix. The solution was then 8 incubated at 20°C for 15 minutes, followed by 4°C overnight. Next, 3 µL of USER (1 U/µl) (New 9 England Biolabs) were added and the solution was incubated at 37°C for 15 minutes, 10 immediately followed by AMPure XP beads (Beckman Coulter) purification. To prepare the 2.5 11 µL of adapter dilution, we first estimated the number of genomic DNA molecules was 12 estimated based on the ng present after size selection, and then the amount of adapter 13 needed to have a 20-fold excess. PCR grade water was added to fill the volume up to 2.5 µL. 14 Bead purification was performed as follows: 77.2 µL (0.8 volumes) of beads were added to the 15 96.5 µL of ligation reaction and mixed by pipetting; the mixture was incubated at room 16 temperature for 15 minutes and then placed on a magnet for 5 minutes; the supernatant was 17 discarded and beads were washed twice with 400 µL of 80% ethanol; the beads were dried at 18 room temperature and resuspended by adding 20 µL of PCR grade water; the mixture was 19 incubated at room temperature for 5 minutes and then placed on a magnet until the solution 20 was clear; the supernatant containing ligated DNA was transferred to a new tube. 21

DNA amplification and targeted capture 22
The amplification of ligated DNA was done using KAPA HiFi HotStart ReadyMix (KAPA 23 Biosystems. Components and volumes used are shown in Table S9 (PCR 1) and primer 24 sequences are available in Table S10. Each library was split into multiple reactions, each with 25 an input material of approximately 240 ng. With the exceptions of 4 libraries (see Table S3), 26 the first step of amplification was 12 cycles of single primer extensions (Table S9)  volumes of AMPure XP beads were mixed with each sample. The purification procedure 31 continued as described above. DNA was eluted in 20 µL of PCR grade water. 32 The target capture was modified from Schmitt et al. (Schmitt et al. 2015). First, the purified 1 PCR 1 output was mixed with 5 µg of Cot-I DNA (Invitrogen) and 1 nmol of blocking 2 oligonucleotides mws60 and mws61 (Table S11). The mixture was then lyophilized and 3 resuspended in 3.1 µL water, 8.5 µL NimbleGen 2× Hybridization Buffer (Roche) and 3.4 µL 4 NimbleGen Hybridization Component A (Roche). Next, the mixture was heated for 10 minutes 5 at 95°C, and 3 pmol of pooled biotinylated oligos specifically designed for the targeted regions 6 (Table S11) were added at 65°C, followed by an incubation of 4 hours and 30 minutes. shortly before the hybridization reaction was completed, beads were placed on a magnet, and 17 supernatant was removed. With the beads still on the magnet, the hybridization reaction was 18 added to the beads, quickly mixed by pipetting (it is important to be fast to maintain the 19 temperature), transferred back to the PCR tube, and placed back in the PCR machine also at 20 65°C for 45 minutes. Sample were vortexed for 3 seconds every 10 minutes. After incubation, 21 captured DNA was bound to the beads and the next step is the washing which was performed 22 as following: with the tube still on the PCR machine, 75 µL of Wash Buffer I was added to the 23 mixture and then vortexed for 10 seconds; the mixture was placed on a 1.5 mL tube and then 24 placed on a magnet; the supernatant was removed and 150 µL of Stringent Wash Buffer were 25 added and mixed by pipetting; the mixture was incubated for 5 minutes at 65°C; Stringent 26 Wash Buffer wash was repeated; after the second incubation, the mixture was placed on a 27 magnet and the supernatant was removed followed by the addition of 150 µL of room 28 temperature Wash Buffer I; the mixture was vortexed for 2 minutes and placed on a magnet; 29 150 µL of Wash Buffer II were added and the mixture was vortexed for 1 minute and then 30 placed on a magnet; the supernatant was removed and 150 µL of Wash Buffer III was added 31 followed by 30 seconds of vortexing; the mixture was then placed on a magnet, the 32 supernatant was removed and 44 µL of PCR grade water were added to resuspend the beads 33 with the bound DNA. 34 The amplification of hybridized targets was done as shown in Table S9 (column PCR 2-4). PCR 1 Product was purified using 1.2 volumes of AMPure XP beads (100 µL of PCR product and 120 2 µL of beads). Apart from the different initial volumes, purification was done as described 3 before. For the final elution, 20 µL of PCR grade water was used. 4 For the second and third target captures, the same procedure was applied but only half of the 5 amounts of Cot-I DNA, blocking oligos and biotinylated oligos were used (the remaining 6 volume of the latter was replaced by PCR grade water). Also, the PCR reaction was slightly 7 different because NEBNext® Multiplex Oligos for Illumina® (New England Biolabs) were used 8 instead of NEB_mws20 primer in order to add indexes for sequencing multiple libraries in one 9 sequencing run. 10 The last step before pooling of different libraries for sequencing was a qPCR using the Library 23 Quantification kit (KAPA Biosystems), following the procedure detailed by the manufacturer. 24 The results were used to adjust the concentrations previously obtained with the fluorescence 25 method. The adjusted concentrations were then used to calculate the volumes of each library 26 that should be pooled together and sent for sequencing, following the guidelines of the 27 sequencing provider used. Then, the Variant Analyzer considers the called mutations, and re-examines the raw reads in 27 order to assign them to different tiers (Table S5). This tier classification is based mainly on the 28 number of reads per family and the proportion of the alternative allele in the consensus 29 sequence (Povysil et al. 2021). 30 High-quality tiers (tier 1.1) require at least 3 reads per family (FS>=3); whereas, second order 1 tiers (tier 1.2-2.4) allow also reads without a family (FS>= 1). Both assume that at least 75% of 2 the reads in the family carry the mutant allele. Lower quality tiers (tier 3 and 4) include also 3 other parameters such as the read position (e.g., mutants in the first or last 10bp are assigned 4 to tier 4) or lower the percentage of reads carrying the mutant allele to >50% (tier 3). 5 The Variant Analyzer output was inspected and variants with DCS coverage below 1000, 6 intronic variants, and SNPs were discarded. Only exonic variants of tier 1 were kept, together 7 with tier 2 variants that were detected more than once in different libraries. 8 Table S14 shows an example of a partial Variant Analyzer output with variants found in 9 different libraries. For this hypothetical case, we would immediately exclude variants chr4- on January 23 rd 2020 and January 21 st 2020, respectively. Variants from gnomAD were 32 remapped from GRCh37/hg19 to GRCh38/hg38 using NCBI Genome Remapping Service 1 (https://www.ncbi.nlm.nih.gov/genome/tools/remap). Exonic single-nucleotide substitutions 2 retrieved from gnomAD and COSMIC were annotated with VEP and wANNOVAR. 3 Deleteriousness analysis was performed using CADD raw scores (Rentzsch et al. 2018) 4 extracted from VEP annotation. 5 Pairwise comparison between every group of variants was done with the Mann-Whitney U 6 test using GraphPad Prism 8.2.1 (GraphPad Software). A multiple comparison correction using 7 a False Discovery Rate (FDR) approach (two-stage set-up method of Benjamini, Krieger and 8 Yekutieli) was done using the same software. 9 Germline association was investigated using ClinVar data extracted from wANNOVAR output. 10 Tumor association was investigated by consulting the presence or absence of variants in the 11 extracted COSMIC data. 12

Sensitivity evaluation 13
In order to assess the ability of our adapted DS approach to detect low-and ultralow-14 frequency variants, we sequenced four libraries with a mixture of genomic DNA containing the 15 FGFR3 variants c.742C>T, c.746C>G, c.749C>G, c.1620C>A, and wild-type DNA at different 16 ratios. The ratio of each variant to WT was 1:10, 1:100, 1:1000, and 1:10,000 (one ratio per 17 library) and it was calculated considering the heterozygosity of the genomic samples 18 containing the variants analyzed. Variant calling and frequency calculations were done as 19 described above. 20

Droplet Digital PCR 21
Site-specific assays were designed using the Droplet digital PCR Assay design tool from BioRad 22 (available at: https://www.bio-rad.com/digital-assays). For site-specific assay information see 23 Table S13. 24 Individual 20 µL ddPCR reactions were prepared by adding 10 µL of 10× SuperMix for probes 25 (no dUTP), 2 µL of genomic DNA (~125ng/µL; total of ~72500 genomes), 6.7 µL of Nucleic Acid 26 free water, 1 µL of primers and probes mix (900nM/probe and 100nM/primer), and 0.3 µL of 27 restriction enzyme (CviQI or MseI; 10U/µL). Note that for each sample 4 replicates were 28 carried out (total of ~300,000 genomes). The digest occurred at room temperature for 15 29 minutes prior to transferring the samples into the cartridges. 70 µL of droplet generation oil 30 for probes and a sealing gasket were added and droplets were formed in the droplet 1 generator. Next, the newly formed droplets were transferred to a 96-well plate and sealed for 2 5 seconds at 180°C. PCR was ran as follows: 10 minutes at 95°C, 40 cycles of: 30 seconds at 3 94°C and 1 minute at 53/55°C (depending on the target site, see Table S13), and a final step of 4 10 minutes at 98°C. Each step had a ramp rate of 2°C and the lid was heated to 105°C. End-5 point analysis was done in the droplet reader. Results were analyzed using QuantaSoft 6 Analysis Pro Software version 1.7.4 (Bio-Rad Laboratories Inc.). 7

K o n g _ a ll
K o n g _ c p g _ t r a n s it io n K o n g _ c p g _ t r a n s v e r s io n K o n g _ n o n _ c p g _ t r a n s it io n K o n g _ n o n _ c p g _ t r a n s v e r s io n a ll c p g _ t r a n s it io n c p g _ t r a n s v e r s io n n o n _ c p g _ t r a n s it io n n o n _ c p g _ t r a n s v e r s io   domains (IgI-III n=32, TM_Inter-domain n=15, TK n=28). Note that the mutational spectra are based on the relative mutation 4 count, therefore every variant is considered only once although they can be present in multiple libraires. The cosine similarities 5 between the spectra can be found in Supplemental Table S7. For the mutational spectra of the databases COSMIC v94 and 6 gnomAD v3.1.1, all variants except for indels of the targeted regions were downloaded. Next, we compared them to the spectra of 7 the duplex sequencing data using the measure of relative counts.