all 53 scz families were recruited from the afrikaner population in south africa and heritage was established by surname and by having 4 afrikaans - speaking grandparents .
family history was obtained from the proband , each participating parent , and additional relatives as needed , by two independent raters , a nursing sister , who recorded pedigree information , and by the clinical interviewer , who inquired in detail about family history during the clinical interview . for additional cohort characteristics ,
the control cohort consisted of 22 families ( triads ) with established afrikaner heritage recruited from the afrikaner community .
paternity and maternity were confirmed prior to sequencing for all case and control families via the affymetrix genome - wide human snp array 5.0 as well as via a panel of microsatellite markers .
exome enrichment was conducted using the sureselect human all exon target enrichment system ( agilent technologies ) based on the methodology described in .
briefly , 3 ug of genomic dna was fragmented by sonication using the covaris s2 to achieve a uniform distribution of fragments with a mean size of 300 bp .
the sonicated dna was purified using agencourt s ampure xp solid phase reversible immobilization paramagnetic bead ( spri ) followed by polishing of the dna ends by removing the 3 overhangs and filling in the 5 overhangs resulting from sonication using t4 dna polymerase and klenow fragment ( new england biolabs ) .
a-base was added to the 3 end of the dna fragments using klenow fragment ( 3 to 5 exo minus ) .
this prepares the dna fragments for ligation to specialized adaptors that have a  t-base overhang at their 3ends .
the end - repaired dna with a single  a-base overhang is ligated to the illumina paired - end adaptors in a standard ligation reaction using t4 dna ligase and 2 um  4 um final adaptor concentration , depending on the dna yield following purification after the addition of the  a-base .
following ligation , the samples were purified using spri beads , quality controlled by assessment on the agilent bioanalyzer and then amplified by 6 cycles of pcr to maintain complexity and avoid bias due to amplification .
500 ng of amplified , purified dna ( dna library ) was prepared for hybridization by adding the dna library to agilent blocking reagents , denaturing at 95 c and incubating at 65 c .
hybridization buffer was added to the prepared library and the entire mix was then added to an aliquot of the agilent sureselect capture library and mixed .
the dna library and biotin - labeled capture library were hybridized by incubation at 65 c for 24 hours .
following hybridization , streptavidin coated magnetic beads were used to purify the rna : dna hybrids formed during hybridization .
the rna capture material was digested via acid hydrolysis following elution from the purification beads .
the neutralized captured dna was purified , desalted and amplified by 12 cycles of pcr using herculase ii fusion dna polymerase .
the libraries were purified following amplification and the library was assessed using the agilent bioanalyzer .
a single peak between 300  400 bp indicates a properly constructed and amplified library ready for sequencing .
final quantitation of the library was performed using the kapa biosciences real - time pcr assay and appropriate amounts of the library were loaded onto the illumina flowcell for sequencing by paired - end 50 nt sequencing on the illumina hiseq2000 instrument .
following dilution to 10 nm final concentration based on the real - time pcr and bioanalyzer results , the final library stock was then used in paired - end cluster generation at a final concentration of 6  8 pm to achieve a cluster density of 600,000/mm ( on the illumina hiseq2000 instrument ) . following cluster generation ,
raw sequencing data for each individual were mapped to the human reference genome ( build hg19 ) using the burrows - wheeler aligner ( bwa v0.5.81536 ) .
the bwa aligned sequencing reads were processed by picard ( http://picard.sourceforge.net/ ) to label the pcr duplicates .
the genome analysis toolkit ( gatk , version 5091 ) was then used to remove duplicates , perform local realignment and map quality score recalibration to produce a  cleaned  bam file for each individual .
snp calls were made by the unified genotyper module in gatk using the  cleaned  bam files in batch fashion ( 90 samples per batch ) . the resulting variant call format ( vcf , version 4.0 ) files were annotated using the genomicannotator module in gatk to identify and label the called variants that are within the targeted coding regions and overlap with known and likely benign snps reported in dbsnp v132 ( ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/vcf/v4.0/00-all.vcf.gz ) .
the annotated vcf files were then filtered using the gatk variant filter module with a hard filter setting and a custom script for initial filtering .
variant calls that failed to pass the following filters were eliminated from the call set : i ) mq0 > = 4 & & ( ( mq0/(1.0 * dp ) ) > 0.1 ) ; ii ) qual <
30.0 || qd < 5.0 || hrun > 5 || sb > 0.00 ; iii ) cluster size 10 ;
iv ) contain dbsnp i d ; v ) outside the targeted regions . combined vcf files were then split into individual files and variants in each offspring were compared to variants present in parents using a custom script pipeline in order to determine the inheritance pattern and annotate de novo mutations . because the gatk unified genotyper is set to maximize the sensitivity of variant calls , it allows for a significant portion of false positives among candidate variants even following the initial filtering process . to address this issue and eliminate potential false positive calls in the offspring and false negative calls in the parents
, we took advantage of the inheritance information provided by our family design and revalidated all variants identified using the mpileup module in the sam tools ( http://samtools.sourceforge.net/ ) according to the following rules : i ) the forward reference ( fr ) count ( i.e. the number of forward reads that match the reference base at this locus ) , the reverse reference ( rr ) count ( i.e. the number of reverse reads that match the reference base at this locus ) , the forward non reference ( fnr ) count ( i.e. the number of forward reads that do not match the reference base at this locus ) and the reverse non reference ( rnf ) count ( i.e the number of reverse reads that do not match the reference base at this locus ) in the offspring must be 2 or greater ; ii ) total read depth in both parents must be 10 or greater ; iii ) both fr and rr count in both parents must be 2 or greater ; iv ) either fnr or rnr count in both parents must be 0 ; v ) the fnr and rnr count to total count ratio in the parental population ( defined as all 150 parental samples sequenced ) must be less than 1/2n , where n is the population size ; vi ) if any of rules 15 was violated , the sequence information was considered insufficient to make a de novo call at this locus . in / del calls
were made by the dindel software using one  cleaned  bam file per run .
the resulting vcf files were used to determine inheritance patterns using the same procedure described above for point mutations . to determine potential mutations at splice - donor or acceptor sites ,
gatk variant calls were made in a batch fashion ( with 90 samples per batch ) that covered each target coding region and 50 bp flanking segments in each direction .
the polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online server was used to determine the non - synonymous and synonymous nature of the mutations and predict their functional impact by further classifying them as non - tolerated ( damaging ) or benign at a given site . the grantham score for each coding variant
the phylop score for each coding variant was extracted from the  phylop46wayall  table in the ucsc table browser ( http://genome.ucsc.edu/cgi-bin/hgtables ) .
the human splicing finder ( hsf , version 2.4.1 ) software ( http://www.umd.be/hsf/ ) was used to predict potential functional impact of the mutations at splice sites .
the kolmogorov - smirnov test ( ks - test ) was used to compare the distribution of phylop and grantham scores among de novo or private inherited mutations in cases .
fisher s exact test or chi - square test with yates correction was used for the analysis of contingency tables depending on the sample sizes .
candidate de novo variants were tested using standard sanger sequencing on an abi 3730xl dna analyzer to validate presence of each mutation in the subjects and absence in the parental genomes , by designing custom primers ( sigma ) based on ~500 bp of genomic sequence flanking each variant . de novo occurrence of mutations was not confirmed in 6 out of 46 and 2 out of 9 candidate alterations in cases and controls , respectively .
all 53 scz families were recruited from the afrikaner population in south africa and heritage was established by surname and by having 4 afrikaans - speaking grandparents .
family history was obtained from the proband , each participating parent , and additional relatives as needed , by two independent raters , a nursing sister , who recorded pedigree information , and by the clinical interviewer , who inquired in detail about family history during the clinical interview . for additional cohort characteristics ,
the control cohort consisted of 22 families ( triads ) with established afrikaner heritage recruited from the afrikaner community .
paternity and maternity were confirmed prior to sequencing for all case and control families via the affymetrix genome - wide human snp array 5.0 as well as via a panel of microsatellite markers .
exome enrichment was conducted using the sureselect human all exon target enrichment system ( agilent technologies ) based on the methodology described in .
briefly , 3 ug of genomic dna was fragmented by sonication using the covaris s2 to achieve a uniform distribution of fragments with a mean size of 300 bp .
the sonicated dna was purified using agencourt s ampure xp solid phase reversible immobilization paramagnetic bead ( spri ) followed by polishing of the dna ends by removing the 3 overhangs and filling in the 5 overhangs resulting from sonication using t4 dna polymerase and klenow fragment ( new england biolabs ) .
a-base was added to the 3 end of the dna fragments using klenow fragment ( 3 to 5 exo minus ) .
the end - repaired dna with a single  a-base overhang is ligated to the illumina paired - end adaptors in a standard ligation reaction using t4 dna ligase and 2 um  4 um final adaptor concentration , depending on the dna yield following purification after the addition of the  a-base .
following ligation , the samples were purified using spri beads , quality controlled by assessment on the agilent bioanalyzer and then amplified by 6 cycles of pcr to maintain complexity and avoid bias due to amplification .
500 ng of amplified , purified dna ( dna library ) was prepared for hybridization by adding the dna library to agilent blocking reagents , denaturing at 95 c and incubating at 65 c .
hybridization buffer was added to the prepared library and the entire mix was then added to an aliquot of the agilent sureselect capture library and mixed .
the dna library and biotin - labeled capture library were hybridized by incubation at 65 c for 24 hours .
following hybridization , streptavidin coated magnetic beads were used to purify the rna : dna hybrids formed during hybridization .
the rna capture material was digested via acid hydrolysis following elution from the purification beads .
the neutralized captured dna was purified , desalted and amplified by 12 cycles of pcr using herculase ii fusion dna polymerase .
the libraries were purified following amplification and the library was assessed using the agilent bioanalyzer .
a single peak between 300  400 bp indicates a properly constructed and amplified library ready for sequencing .
final quantitation of the library was performed using the kapa biosciences real - time pcr assay and appropriate amounts of the library were loaded onto the illumina flowcell for sequencing by paired - end 50 nt sequencing on the illumina hiseq2000 instrument .
following dilution to 10 nm final concentration based on the real - time pcr and bioanalyzer results , the final library stock was then used in paired - end cluster generation at a final concentration of 6  8 pm to achieve a cluster density of 600,000/mm ( on the illumina hiseq2000 instrument ) . following cluster generation ,
raw sequencing data for each individual were mapped to the human reference genome ( build hg19 ) using the burrows - wheeler aligner ( bwa v0.5.81536 ) .
the bwa aligned sequencing reads were processed by picard ( http://picard.sourceforge.net/ ) to label the pcr duplicates .
the genome analysis toolkit ( gatk , version 5091 ) was then used to remove duplicates , perform local realignment and map quality score recalibration to produce a  cleaned  bam file for each individual .
snp calls were made by the unified genotyper module in gatk using the  cleaned  bam files in batch fashion ( 90 samples per batch ) . the resulting variant call format (
vcf , version 4.0 ) files were annotated using the genomicannotator module in gatk to identify and label the called variants that are within the targeted coding regions and overlap with known and likely benign snps reported in dbsnp v132 ( ftp://ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/vcf/v4.0/00-all.vcf.gz ) .
the annotated vcf files were then filtered using the gatk variant filter module with a hard filter setting and a custom script for initial filtering .
variant calls that failed to pass the following filters were eliminated from the call set : i ) mq0 > = 4 & & ( ( mq0/(1.0 * dp ) ) > 0.1 ) ; ii ) qual < 30.0 || qd
< 5.0 || hrun > 5 || sb > 0.00 ; iii ) cluster size 10 ; iv ) contain dbsnp i d ; v ) outside the targeted regions . combined vcf files were then split into individual files and variants in each offspring were compared to variants present in parents using a custom script pipeline in order to determine the inheritance pattern and annotate de novo mutations . because the gatk unified genotyper is set to maximize the sensitivity of variant calls
, it allows for a significant portion of false positives among candidate variants even following the initial filtering process . to address this issue and eliminate potential false positive calls in the offspring and false negative calls in the parents , we took advantage of the inheritance information provided by our family design and revalidated all variants identified using the mpileup module in the sam tools ( http://samtools.sourceforge.net/ ) according to the following rules : i ) the forward reference ( fr ) count ( i.e. the number of forward reads that match the reference base at this locus ) , the reverse reference ( rr ) count ( i.e. the number of reverse reads that match the reference base at this locus )
, the forward non reference ( fnr ) count ( i.e. the number of forward reads that do not match the reference base at this locus ) and the reverse non reference ( rnf ) count ( i.e the number of reverse reads that do not match the reference base at this locus ) in the offspring must be 2 or greater ; ii ) total read depth in both parents must be 10 or greater ; iii ) both fr and rr count in both parents must be 2 or greater ; iv ) either fnr or rnr count in both parents must be 0 ; v ) the fnr and rnr count to total count ratio in the parental population ( defined as all 150 parental samples sequenced ) must be less than 1/2n , where n is the population size ; vi ) if any of rules 15 was violated , the sequence information was considered insufficient to make a de novo call at this locus . in / del calls
were made by the dindel software using one  cleaned  bam file per run .
the resulting vcf files were used to determine inheritance patterns using the same procedure described above for point mutations . to determine potential mutations at splice - donor or acceptor sites ,
gatk variant calls were made in a batch fashion ( with 90 samples per batch ) that covered each target coding region and 50 bp flanking segments in each direction .
the polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online server was used to determine the non - synonymous and synonymous nature of the mutations and predict their functional impact by further classifying them as non - tolerated ( damaging ) or benign at a given site . the grantham score for each coding variant
the phylop score for each coding variant was extracted from the  phylop46wayall  table in the ucsc table browser ( http://genome.ucsc.edu/cgi-bin/hgtables ) .
the human splicing finder ( hsf , version 2.4.1 ) software ( http://www.umd.be/hsf/ ) was used to predict potential functional impact of the mutations at splice sites .
the kolmogorov - smirnov test ( ks - test ) was used to compare the distribution of phylop and grantham scores among de novo or private inherited mutations in cases .
fisher s exact test or chi - square test with yates correction was used for the analysis of contingency tables depending on the sample sizes .
candidate de novo variants were tested using standard sanger sequencing on an abi 3730xl dna analyzer to validate presence of each mutation in the subjects and absence in the parental genomes , by designing custom primers ( sigma ) based on ~500 bp of genomic sequence flanking each variant .
de novo occurrence of mutations was not confirmed in 6 out of 46 and 2 out of 9 candidate alterations in cases and controls , respectively .
 despite high heritability , a large fraction of cases with schizophrenia do not have a family history of the disease ( sporadic cases ) . here , we examine the possibility that rare de novo protein - altering mutations contribute to the genetic component of schizophrenia by sequencing the exome of 53 sporadic cases , 22 unaffected controls and their parents . 
 we identified 40 de novo mutations in 27 patients affecting 40 genes including a potentially disruptive mutation in dgcr2 , a gene removed by the recurrent schizophrenia - predisposing 22q11.2 microdeletion . 
 comparison to rare inherited variants revealed that the identified de novo mutations show a large excess of nonsynonymous changes in cases , as well as a greater potential to affect protein structure and function . 
 our analysis reveals a major role of de novo mutations in schizophrenia and also a large mutational target , which together provide a plausible explanation for the high global incidence and persistence of the disease . 
METHODS
Cohorts
Exome library construction
Library capture and sequencing
Exome data analysis for 
Statistics
None
Supplementary Material