167-A

The KiGGS survey is based on a nationally representative sample of children and adolescents 0–17 years of age with main residence in Germany. The sampling procedure was based on a two-stage protocol developed in co-operation with the Centre for Survey Research and Methodology (ZUMA), Mannheim, Germany. The study was approved by the Charité/Universitätsmedizin Berlin ethics committee and the Federal Office for the Protection of Data.
First, a systematic sample of 167 primary sample units (PSUs) was drawn from an inventory of German communities stratified according to the BIK classification system [2 ], which measures the grade of urbanization, and the geographic distribution. The number of PSUs per strata was determined using the Cox procedure for community sampling [3 (link)] with sampling probability proportional to population size. In order to ensure sufficient sample size for analyses stratifying according to residence in former East or West Germany, a disproportionate number of PSUs was included to represent former West (n = 112) and East (n = 50) Germany, and the city of Berlin (n = 5). At the second stage, an equal number of addresses (n = 24) per birth cohort were randomly selected (simple random sample) from local population registries within selected PSUs 8 weeks prior to the start of examinations. A final simple random sample was drawn at the Robert Koch Institute, including a total of 8, 9 or 10 children and adolescents per birth cohort, depending on community size. Thus, the target population per PSU consisted of 144, 162 or 180 persons eligible to be contacted and invited to participate in the study. Oversampling of children and adolescents from families with a migration nationality was used, as we expected a higher proportion of undeliverable contacts and non-respondents in this subgroup compared to children and youths from non-migrant families. The total KiGGS sample included 28,299 children and adolescents.

DNA samples obtained from the proband (II-1), his younger brother (II-2), his father (I-1) and his mother (I-2) were sequenced using target exome-based next-generation sequencing. Roche NimbleGen’s (Madison, United States) custom Sequence Capture Human Array was used to designed to capture targeted sequence, covering all exons and flanking sequence (including the 100 bp of introns) of 127 genes which is associated hereditary hearing impairment (Supplementary Table 1). The details of targeted next generation sequencing have been described in Supplementary Table 2. The 127-gene panel achieved a total of 619.167 kb of targeted sequence, covering 2,268 exons and flanking sequence. An average of 2022981 reads per sample was acquired, with approximately 85% mapping to their targets. The average mean depth for the targeted regions was 311.3 ± 56.7; 97.5 ± 0.1% of the covered exons had ≥ 30 reads. Average depth and coverage of Target genes has been described in Figure 2A. Figure 2B, is showing read depth at this causal variant in BAM file across ILDR1 locus. The procedure for preparation of libraries was consistent with standard operating protocols published previously (Yang et al., 2018 (link)). According to the standard protocol, simultaneously we sequenced 30 samples on Illumina HiSeq 2500 Analyzers (Illumina, San Diego, United States) for each pooling batch for 90 cycles (specially designed rare disease screening). We applied Illumina Pipeline software (version 1.3.4) to generate the raw data which is later used for Bioinformatic analysis. We extract the clean reads from the raw reads by using already established filtering criteria (Wei et al., 2014 (link)). Then, we selectively using at least 90 bp long clean reads for aligning to the human reference genome (Build 37) of NCBI database by using Burrows Wheeler Aligner (BWA). BWA, a multi-vision software package, generating the output file in bam format. After that, target region coverage, sequencing depth, SNP/InDel/CNV detection has been analyzed by using the bam data. Next, SOAPsnp software, Sam tools pileup software and Bioinformatic computational framework were established for identifying SNP, InDels and CNVs. Filtering criteria for a SNP or InDel has been set with at least 10 reads and >20% of total reads. SNPs are filtered out and selected for further interpretation if the frequency of the SNPs is <0.05 in dbSNP, HapMap, 1000 Genomes database, the 100 healthy reference samples (same ethnic origin with similar age and sex range) sequenced in this study. The comprehensive and detailed method of variant interpretation has been described in Figure 3A.

In spring 1997, we analyzed the bryophyte content of 34 great tit and 35 blue tit nests. We also analyzed bryophyte abundances in a total of 167 plots on the forest floor around nine of the trees with active nests (see below). Eight of the nests were included in the data set comparing blue tit and great tit nests in 1997, together with eight nests from the same nest boxes sampled in the subsequent year (1998). Sample number nine consisted of a great tit nest in a nest box analyzed for a pilot study in 1996 and a nest in the same nest box in 1998. We treated the 18 nests as independent samples. The tits were not ringed, but great tits are relatively short‐lived (Hõrak & Lebreton, 1998 (link)). Nest boxes were abundant in the study area and female great tits are known to move some distance between nest sites from 1 year to the next (Harvey et al., 1979 ).
We analyzed bryophytes in the nests when the nest lining was completed or when egg laying had started. Any egg(s) were laid aside and the nest was carefully removed from the box. The nests were then gently pried open in several places and from several angles, while care was taken not to destroy the nest, to visually estimate the proportion of each bryophyte species as a percentage of the total volume of bryophytes but later re‐calculated as a percentage of the whole nest. The nest and the eggs were then carefully returned to the nest box. There was no indication that this procedure caused any desertion.
Species abundances of bryophytes on the ground surrounding the nine nest boxes where great tits bred in both years were recorded as percentage cover in 2 × 2 m² plots. We used restricted random sampling to place 20 plots around each of the nine nest boxes within a circular area with a radius of 20 m from the nest box. The plots therefore covered 6.4% of the circle area. The circle of 1256 m² was divided into four quadrants, each with four sectors with a length of 5 m (Figure 2). In every quadrant, we placed one plot randomly in each sector and one plot randomly within the quadrant, giving five plots in each quadrant and 20 plots around each nest box. Thirteen of the plots were devoid of bryophytes, and the data set therefore consisted of 167 plots along with the 18 great tit nests. Assuming that bryophyte abundances were the same in both years, we only sampled the plots once.

The MINA53 coding sequence (A26-V464) and NO66 coding sequence (A167-N641) were sub-cloned into an expression vector pET-28b respectively and the plasmids were transformed into Escherichia coli strain BL21(DE3).^{5,24 (link)} In brief, a 6 × 10 mL overnight culture was used to inoculate 6 l of Terrific Broth media containing 100 μg mL⁻¹ kanamycin. Cultures were grown at 37 °C until the OD₆₀₀ reached ∼1.0. The temperature was adjusted to 18 °C, and expression was then induced or 18 hr with 0.5 mM isopropylthio-β-galactoside (IPTG). Cells were centrifuged, then resuspended in the lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.5 mM tris-(2-carboxyethyl)-phosphine (TCEP), and 5% glycerol in the presence of a protease inhibitor mixture 1 : 2000 (Complete, EDTA-free Protease Inhibitor Cocktail, Roche Diagnostics Ltd) and lysed by three passages through a high-pressure cell breaker (EmulsiFlex C5-Avestin) at 4 °C. The lysates were cleared by centrifugation (60 minutes, 36 000× g, 4 °C) and loaded onto a Ni NTA column. After extensively rinsing with the lysis buffer the His6-tagged MINA53 and NO66 proteins were eluted using lysis buffer containing 300 mM imidazole. The eluted fractions were further purified using an AKTA Xpress system combined with an S200 gel filtration column equilibrated in 20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5 mM tris (2-carboxyethyl)-phosphine (TCEP) and 5% glycerol. The purity was confirmed by SDS-PAGE and by mass spectrometry as reported.^{5,24 (link)}