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1.  Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders 
Neuron  2013;77(2):235-242.
SUMMARY
To characterize the role of rare complete human knockouts in autism spectrum disorders (ASD), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a two-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudo-autosomal regions on the X-chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together these results provide compelling evidence that rare autosomal and X-chromosome complete gene knockouts are important inherited risk factors for ASD.
doi:10.1016/j.neuron.2012.12.029
PMCID: PMC3613849  PMID: 23352160
2.  Exome sequencing resolves apparent incidental findings and reveals further complexity of SH3TC2 variant alleles causing Charcot-Marie-Tooth neuropathy 
Genome Medicine  2013;5(6):57.
Background
The debate regarding the relative merits of whole genome sequencing (WGS) versus exome sequencing (ES) centers around comparative cost, average depth of coverage for each interrogated base, and their relative efficiency in the identification of medically actionable variants from the myriad of variants identified by each approach. Nevertheless, few genomes have been subjected to both WGS and ES, using multiple next generation sequencing platforms. In addition, no personal genome has been so extensively analyzed using DNA derived from peripheral blood as opposed to DNA from transformed cell lines that may either accumulate mutations during propagation or clonally expand mosaic variants during cell transformation and propagation.
Methods
We investigated a genome that was studied previously by SOLiD chemistry using both ES and WGS, and now perform six independent ES assays (Illumina GAII (x2), Illumina HiSeq (x2), Life Technologies' Personal Genome Machine (PGM) and Proton), and one additional WGS (Illumina HiSeq).
Results
We compared the variants identified by the different methods and provide insights into the differences among variants identified between ES runs in the same technology platform and among different sequencing technologies. We resolved the true genotypes of medically actionable variants identified in the proband through orthogonal experimental approaches. Furthermore, ES identified an additional SH3TC2 variant (p.M1?) that likely contributes to the phenotype in the proband.
Conclusions
ES identified additional medically actionable variant calls and helped resolve ambiguous single nucleotide variants (SNV) documenting the power of increased depth of coverage of the captured targeted regions. Comparative analyses of WGS and ES reveal that pseudogenes and segmental duplications may explain some instances of apparent disease mutations in unaffected individuals.
doi:10.1186/gm461
PMCID: PMC3706849  PMID: 23806086
Exome sequencing; Whole-genome sequencing; Incidental findings; SH3TC2; Personal genomes; Precision medicine
3.  Exome Sequencing: Applications From the Lab Bench to the Clinic 
Since the initial report of targeted-enrichment (Albert et al, 2007) we have been evolving the design and utility of capture reagents and methods, while taking advantage of the parallel advances in sequencing platforms. New exome designs target a comprehensive set of coding exons from 6 different gene databases, as well as computationally predicted coding and non-coding elements: regulatory regions, and conserved UTRs. Library automation, reduction of DNA input samples, capture hybridization multiplexing and application of faster read mapping tools such as BWA, together allow a rate of >4,300 libraries/captures per month, with >40,000 exome and regional capture libraries completed to date. In addition, a fully integrated informatics and analysis pipeline (Mercury), supports all aspects of data flow and analysis from the initial data production on the sequencing instrument to annotated variant calls (SNPs and small Indels). These laboratory methods and analysis pipelines have been production hardened at the Human Genome Sequencing Center (HGSC) and have now been applied toward clinical exome sequencing. Through a joint collaboration between the Human Genome Sequencing Center and the Medical Genetics Laboratories (MGL) of the Department of Molecular and Human Genetics, clinical exome sequencing and interpretation are now provided through the CAP/CLIA certified Whole Genome Laboratory (WGL). To date, the WGL has completed exome sequencing of 650 patient samples and final interpretation completed for over 450 patients with causative deleterious mutations identified in 25% of cases. Performance has been maintained to a high standard of 95% of the exome target bases represented at 20X coverage. Overall exome performance metrics, LIMS support, variant analysis and validation of the clinical pipeline for a CAP/CLIA environment will be presented.
PMCID: PMC3635387
4.  Analysis of Rare, Exonic Variation amongst Subjects with Autism Spectrum Disorders and Population Controls 
PLoS Genetics  2013;9(4):e1003443.
We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.
Author Summary
This study evaluates association of rare variants and autism spectrum disorders (ASD) in case and control samples sequenced by two centers. Before doing association analyses, we studied how to combine information across studies. We first harmonized the whole-exome sequence (WES) data, across centers, in terms of the distribution of rare variation. Key features included filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. After filtering, the vast majority of variants calls from seven samples sequenced at both centers matched. We also evaluated whether one should combine summary statistics from data from each center (meta-analysis) or combine data and analyze it together (mega-analysis). For many gene-based tests, we showed that mega-analysis yields more power. After quality control of data from 1,039 ASD cases and 870 controls and a range of analyses, no gene showed exome-wide evidence of significant association. Our results comport with recent results demonstrating that hundreds of genes affect risk for ASD; they suggest that rare risk variants are scattered across these many genes, and thus larger samples will be required to identify those genes.
doi:10.1371/journal.pgen.1003443
PMCID: PMC3623759  PMID: 23593035
5.  Deep Resequencing and Association Analysis of Schizophrenia Candidate Genes 
Molecular psychiatry  2012;18(2):138-140.
doi:10.1038/mp.2012.28
PMCID: PMC3577417  PMID: 22472875
schizophrenia; sequencing; SNV; genetic; association; mutation; DISC1
6.  Oligogenic heterozygosity in individuals with high-functioning autism spectrum disorders 
Human Molecular Genetics  2011;20(17):3366-3375.
Autism spectrum disorders (ASDs) are a heterogeneous group of neuro-developmental disorders. While significant progress has been made in the identification of genes and copy number variants associated with syndromic autism, little is known to date about the etiology of idiopathic non-syndromic autism. Sanger sequencing of 21 known autism susceptibility genes in 339 individuals with high-functioning, idiopathic ASD revealed de novo mutations in at least one of these genes in 6 of 339 probands (1.8%). Additionally, multiple events of oligogenic heterozygosity were seen, affecting 23 of 339 probands (6.8%). Screening of a control population for novel coding variants in CACNA1C, CDKL5, HOXA1, SHANK3, TSC1, TSC2 and UBE3A by the same sequencing technology revealed that controls were carriers of oligogenic heterozygous events at significantly (P < 0.01) lower rate, suggesting oligogenic heterozygosity as a new potential mechanism in the pathogenesis of ASDs.
doi:10.1093/hmg/ddr243
PMCID: PMC3153303  PMID: 21624971
7.  Somatic mutations affect key pathways in lung adenocarcinoma 
Ding, Li | Getz, Gad | Wheeler, David A. | Mardis, Elaine R. | McLellan, Michael D. | Cibulskis, Kristian | Sougnez, Carrie | Greulich, Heidi | Muzny, Donna M. | Morgan, Margaret B. | Fulton, Lucinda | Fulton, Robert S. | Zhang, Qunyuan | Wendl, Michael C. | Lawrence, Michael S. | Larson, David E. | Chen, Ken | Dooling, David J. | Sabo, Aniko | Hawes, Alicia C. | Shen, Hua | Jhangiani, Shalini N. | Lewis, Lora R. | Hall, Otis | Zhu, Yiming | Mathew, Tittu | Ren, Yanru | Yao, Jiqiang | Scherer, Steven E. | Clerc, Kerstin | Metcalf, Ginger A. | Ng, Brian | Milosavljevic, Aleksandar | Gonzalez-Garay, Manuel L. | Osborne, John R. | Meyer, Rick | Shi, Xiaoqi | Tang, Yuzhu | Koboldt, Daniel C. | Lin, Ling | Abbott, Rachel | Miner, Tracie L. | Pohl, Craig | Fewell, Ginger | Haipek, Carrie | Schmidt, Heather | Dunford-Shore, Brian H. | Kraja, Aldi | Crosby, Seth D. | Sawyer, Christopher S. | Vickery, Tammi | Sander, Sacha | Robinson, Jody | Winckler, Wendy | Baldwin, Jennifer | Chirieac, Lucian R. | Dutt, Amit | Fennell, Tim | Hanna, Megan | Johnson, Bruce E. | Onofrio, Robert C. | Thomas, Roman K. | Tonon, Giovanni | Weir, Barbara A. | Zhao, Xiaojun | Ziaugra, Liuda | Zody, Michael C. | Giordano, Thomas | Orringer, Mark B. | Roth, Jack A. | Spitz, Margaret R. | Wistuba, Ignacio I. | Ozenberger, Bradley | Good, Peter J. | Chang, Andrew C. | Beer, David G. | Watson, Mark A. | Ladanyi, Marc | Broderick, Stephen | Yoshizawa, Akihiko | Travis, William D. | Pao, William | Province, Michael A. | Weinstock, George M. | Varmus, Harold E. | Gabriel, Stacey B. | Lander, Eric S. | Gibbs, Richard A. | Meyerson, Matthew | Wilson, Richard K.
Nature  2008;455(7216):1069-1075.
Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.
doi:10.1038/nature07423
PMCID: PMC2694412  PMID: 18948947
8.  Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus 
BMC Microbiology  2007;7:99.
Background
Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear.
Results
We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified.
Conclusion
USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.
doi:10.1186/1471-2180-7-99
PMCID: PMC2222628  PMID: 17986343
9.  Paradoxical DNA Repair and Peroxide Resistance Gene Conservation in Bacillus pumilus SAFR-032 
PLoS ONE  2007;2(9):e928.
Background
Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, γ-radiation, H2O2, desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species.
Principal Findings
The genome of B. pumilus SAFR-032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks several genes that function in UV or H2O2 resistance in other Bacillus species.
Significance
This study identifies several candidate genes for further research into UV and H2O2 resistance. These findings will help explain the resistance of B. pumilus and are applicable to understanding sterilization survival strategies of microbes.
doi:10.1371/journal.pone.0000928
PMCID: PMC1976550  PMID: 17895969
10.  Complete Genome Sequence of Rickettsia typhi and Comparison with Sequences of Other Rickettsiae 
Journal of Bacteriology  2004;186(17):5842-5855.
Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.
doi:10.1128/JB.186.17.5842-5855.2004
PMCID: PMC516817  PMID: 15317790
11.  Deep resequencing reveals excess rare recent variants consistent with explosive population growth 
Nature Communications  2010;1:131-.
Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.
To fully catalogue rare genetic variation in humans, many samples need to be examined. In this study, Coventry et al. resequenced two genes, KCNJ11 and HHEX, in 13,715 humans, and concluded that most of the sequence variation arose recently and that variation is greater than expected.
doi:10.1038/ncomms1130
PMCID: PMC3060603  PMID: 21119644

Results 1-11 (11)