Lyon,  Gholson J.

O'Rawe, Jason

Cold Spring Harbor Laboratory Institutional Repository

Large	  pedigrees	  in	  human	  sequencing	  studies:	  	  toward	  a	  more	  resolved	  and	  accurate	  picture	  of	  gene8c	  disease	  Jason	  O’Rawe1,	  Yiyang	  Wu1,2,	  Alan	  Rope3,	  	  Laura	  T.	  Jimenez	  Barrón1,4,	  Jeﬀrey	  Swensen5,	  Han	  Fang1,	  David	  MiKelman6,	  Gareth	  Highnam6,	  Reid	  Robison7,	  Kai	  Wang7,8,	  Gholson	  J.	  Lyon1,2,7	  	  1Stanley	  InsUtute	  for	  CogniUve	  Genomics,	  Cold	  Spring	  Harbor	  Laboratory,	  NY,	  USA;	  2Stony	  Brook	  University,	  Stony	  Brook,	  NY,	  USA;	  3Department	  of	  Medical	  GeneUcs,	  Northwest	  Kaiser	  Permanente,	  Portland,	  OR,	  USA;	  4Centro	  de	  Ciencias	  Genomicas,	  Universidad	  Nacional	  Autonoma	  de	  Mexico,	  Cuernavaca,	  Morelos,	  MX;	  5Caris	  Life	  Sciences,	  Phoenix,	  Arizona,	  USA;	  6Gene	  by	  Gene,	  Ltd.,	  Hou-­‐ston,	  TX,	  USA	  ;	  7Utah	  FoundaUon	  for	  Biomedical	  Research,	  Salt	  Lake	  City,	  UT,	  USA,	  8Zilkha	  Neu-­‐rogeneUc	  InsUtute,	  Department	  of	  Psychiatry	  and	  PrevenUve	  Medicine,	  University	  of	  Southern	  California,	  Los	  Angeles,	  CA,	  USA	  Abstract	 Methods:  WGS was  performed on  ten  members  of  this  family using  the  Il-lumina  HiSeq2000  platform,  with  four  (the  two affected  boys  and  their  parents)  being  additionally  sequenced using the CG WGS platform. CG data analysis was performed by CG,  using  their  version  2.0  pipeline.  Multiple  variant  calling pipelines were used to detect SNVs, INDELs, STRs and CNVs.  Disease variant  prioritization was performed using ANNOVAR, Golden Helix SVS v8.1.4 and GEMINI v0.9.1, and VAAST v2.0.	Results: CG WGS covered >85% of the genome and >90% of the exome, both with 20 or more reads. Illumina WGS covered >90% of the genome with 30 reads or more and with >80% of the bases having a quality score of >30. On average, we find a 2.4 to 14.0 mean fold difference in the number of variants detected as being relevant for various disease models when using different sets of sequencing data  and analysis  pipelines.  We found a  number  of putative genetic variants and archive them here.	Background:  We  describe  a  comprehensive  whole  genome sequencing  (WGS)  study  using  the  Illumina  and  Complete Genomics (CG) sequencing platforms for one family containing two  affected  male  brothers,  aged  10  and  12,  with  severe intellectual  disability  and  very  distinctive  facial  features.  High accuracy and sensitivity is of particular importance in the context of  detecting  or  discovering  the  genetic  influencers  of  human diseases.	Fig. 1  (A) Pedigree structure of all individuals in the family that were sequenced during the course of this study. Individuals with a star next to their number indicates that their whole genomes were sequenced  with  both  the  Complete  Genomics  sequencing  and analysis pipeline as well as with Illumina sequencing. 	•  Human  sequence  variation  ranges  in  manifestation  from differences that can be detected at the single nucleotide level, to whole chromosome differences (Fig 2).	•  Conclusions:  Analyzing  multi-generational  pedigrees  using multiple orthogonal bioinformatics pipelines using two sequencing platforms can reliably reveal human sequence variants that may be important in rare disease. We have found a number of sequence variants that may play a role in the rare disease described here and highlight a variant in TAF1. Our findings are consistent with the literature  on  the  importance  of  the  TF11D  complex  in developmental delay and ID.	1bp ~200bp to 100mbFrequency per individual1010010^310^410^510^6SNVs CNVs~1 to 200bp~1 to 200bpINDELsSTRsFreeBayesGATKFreeBayesScalpelRepeatSeqScalpelPennCNV, ERDS and CGGATKCGCGCGFig. 2  A conceptual map of human sequence variation, and a list of the bioinformatics programs we used during the course of our study.	•  The two affected male brothers have severe intellectual disability, autism-like behavior, attention deficit issues, and very distinctive facial features (Fig. 1C), including broad, upturned nose, sagging cheeks,  downward  sloping  palpebral  fissures,  relative hypertelorism, high-arched palate, and prominent ears. 	•  The mother of the two affected (Fig. 1A, II-2) was shown to have 99:1 x-chromosome inactivation (Fig. 1B).	•  14 unique INDELS and SNVs were discovered using two different prioritization (Fig. 3A).   	•  The TAF1 variant arose in this family as a de-novo variant on the X-chromosome of the mother (Fig. 1A, II-2) of the two affected children (as it  is  not found in any of the other members of the family) and was then transmitted to both of them.  The mother also is the only female in the family to exhibit extreme X-chromosome skewing.  	•  The  transcription  factor  initiation  complex  TF11D has  recently been  implicated  in  playing  a  role  in  intellectual  disability  and developmental  delay,  and  TAF1  represents  the  largest  known subunit of this multi-protein complex, and this variant falls within a conserved region of the protein (Fig 3B).	Fig. 3	•  We  sought  to  identify  variants  following  de-novo,  autosomal recessive  and  x-linked  models  of  transmission  that  may  be contributing, together or alone, to the disease phenotype.  We used several  methods  to  prioritize  and  identify  possible  disease-contributory germ-line variants, in-cluding VAAST, Golden Helix SVS  v8.1.4,  ANNOVAR  (2013Aug23  version),  and  GEMINI v0.9.1.	Presentation of the phenotype Bioinformatics analysis Results •  We found  a  2.4  to  14.0  mean  fold  difference  in  the  number  of variants detected as being relevant for various disease models when using different sets of sequencing data. 	

Large pedigrees in human sequencing studies: toward a more resolved and accurate picture of genetic disease

Large	  pedigrees	  in	  human	  sequencing	  studies:	  	  toward	  a	  more	  resolved	  and	  accurate	  picture	  of	  gene8c	  disease	  Jason	  O’Rawe1,	  Yiyang	  Wu1,2,	  Alan	  Rope3,	  	  Laura	  T.	  Jimenez	  Barrón1,4,	  Jeﬀrey	  Swensen5,	  Han	  Fang1,	  David	  MiKelman6,	  Gareth	  Highnam6,	  Reid	  Robison7,	  Kai	  Wang7,8,	  Gholson	  J.	  Lyon1,2,7	  	  1Stanley	  InsUtute	  for	  CogniUve	  Genomics,	  Cold	  Spring	  Harbor	  Laboratory,	  NY,	  USA;	  2Stony	  Brook	  University,	  Stony	  Brook,	  NY,	  USA;	  3Department	  of	  Medical	  GeneUcs,	  Northwest	  Kaiser	  Permanente,	  Portland,	  OR,	  USA;	  4Centro	  de	  Ciencias	  Genomicas,	  Universidad	  Nacional	  Autonoma	  de	  Mexico,	  Cuernavaca,	  Morelos,	  MX;	  5Caris	  Life	  Sciences,	  Phoenix,	  Arizona,	  USA;	  6Gene	  by	  Gene,	  Ltd.,	  Hou-­‐ston,	  TX,	  USA	  ;	  7Utah	  FoundaUon	  for	  Biomedical	  Research,	  Salt	  Lake	  City,	  UT,	  USA,	  8Zilkha	  Neu-­‐rogeneUc	  InsUtute,	  Department	  of	  Psychiatry	  and	  PrevenUve	  Medicine,	  University	  of	  Southern	  California,	  Los	  Angeles,	  CA,	  USA	  Abstract Methods: WGS was performed on ten members of this family  using  the  Il-lumina  HiSeq2000  platform,  with four  (the  two  affected  boys  and  their  parents)  being additionally sequenced using the CG WGS platform. CG data analysis was performed by CG, using their version 2.0 pipeline. Multiple variant calling pipelines were used to  detect  SNVs,  INDELs,  STRs  and  CNVs.   Disease variant prioritization was performed using ANNOVAR, Golden  Helix  SVS  v8.1.4  and  GEMINI  v0.9.1,  and VAAST v2.0.Results:  CG  WGS  covered  >85%  of  the  genome  and >90%  of  the  exome,  both  with  20  or  more  reads. Illumina  WGS covered  >90% of  the  genome with  30 reads  or  more  and  with  >80% of  the  bases  having  a quality score of >30. On average, we find a 2.4 to 14.0 mean fold difference in the number of variants detected as being relevant for various disease models when using different sets of sequencing data and analysis pipelines. We  found  a  number  of  putative  genetic  variants  and archive them here.Background:  We  describe  a  comprehensive  whole genome sequencing (WGS) study using the Illumina and Complete Genomics (CG) sequencing platforms for one family containing two affected male brothers,  aged 10 and  12,  with  severe  intellectual  disability  and  very distinctive facial features. High accuracy and sensitivity is of particular importance in the context of detecting or discovering the genetic influencers of human diseases.Fig.  1   (A)  Pedigree  structure  of  all  individuals  in  the family  that  were  sequenced  during  the  course  of  this study.  Individuals  with  a  star  next  to  their  number indicates that their whole genomes were sequenced with both  the  Complete  Genomics  sequencing  and  analysis pipeline as well as with Illumina sequencing. •  Human sequence variation ranges in manifestation from differences that can be detected at the single nucleotide level, to whole chromosome differences (Fig 2).•  Conclusions:  Analyzing  multi-generational  pedigrees using multiple orthogonal bioinformatics pipelines using two  sequencing  platforms  can  reliably  reveal  human sequence variants that may be important in rare disease. We have found a number of sequence variants that may play  a  role  in  the  rare  disease  described  here  and highlight a variant in TAF1. Our findings are consistent with  the  literature  on  the  importance  of  the  TF11D complex in developmental delay and ID.1bp ~200bp to 100mbFrequency per individual1010010^310^410^510^6SNVs CNVs~1 to 200bp~1 to 200bpINDELsSTRsFreeBayesGATKFreeBayesScalpelRepeatSeqScalpelPennCNV, ERDS and CGGATKCGCGCGFig. 2  A conceptual map of human sequence variation, and a list of the bioinformatics programs we used during the course of our study.•  The two affected male brothers have severe intellectual disability,  autism-like behavior,  attention deficit  issues, and very distinctive facial features (Fig. 1C), including broad, upturned nose, sagging cheeks, downward sloping palpebral  fissures,  relative  hypertelorism,  high-arched palate, and prominent ears. •  The  mother  of  the  two  affected  (Fig.  1A,  II-2)  was shown  to  have  99:1  x-chromosome  inactivation  (Fig. 1B).•  14 unique INDELS and SNVs were discovered using two different prioritization (Fig. 3A).   •  The  TAF1  variant  arose  in  this  family  as  a  de-novo variant  on  the  X-chromosome of  the  mother  (Fig.  1A, II-2) of the two affected children (as it is not found in any of  the  other  members  of  the  family)  and  was  then transmitted to both of them.  The mother also is the only female in the family to exhibit extreme X-chromosome skewing.  •  The  transcription  factor  initiation  complex  TF11D  has recently been implicated in playing a role in intellectual disability and developmental delay, and TAF1 represents the largest known subunit of this multi-protein complex, and  this  variant  falls  within  a  conserved  region  of  the protein (Fig 3B).Fig. 3•  We  sought  to  identify  variants  following  de-novo, autosomal recessive and x-linked models of transmission that may be contributing, together or alone, to the disease phenotype.   We  used  several  methods  to  prioritize  and identify possible disease-contributory germ-line variants, in-cluding VAAST, Golden Helix SVS v8.1.4, ANNOVAR (2013Aug23 version), and GEMINI v0.9.1.Presentation of the phenotype Bioinformatics analysis Results •  We found a 2.4 to 14.0 mean fold difference in the number of variants detected as being relevant for vari-ous disease models when using different sets of sequencing data. 

Large pedigrees in human sequencing studies: toward a more resolved and accurate picture of genetic disease

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Cold Spring Harbor Laboratory Institutional Repository