Massively parallel exon capture and library-free resequencing across 16 genomes.
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ABSTRACT:
SUBMITTER: Turner EH
PROVIDER: S-EPMC2703445 | biostudies-literature | 2009 May
REPOSITORIES: biostudies-literature
Publications
Massively parallel exon capture and library-free resequencing across 16 genomes.
Turner Emily H EH Lee Choli C Ng Sarah B SB Nickerson Deborah A DA Shendure Jay J
Nature methods 20090406 5
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Project description:SummaryEfficient workflows to shepherd clinically generated genomic data through the multiple stages of a next-generation sequencing pipeline are of critical importance in translational biomedical science. Here we present COSMOS, a Python library for workflow management that allows formal description of pipelines and partitioning of jobs. In addition, it includes a user interface for tracking the progress of jobs, abstraction of the queuing system and fine-grained control over the workflow. Workflows can be created on traditional computing clusters as well as cloud-based services.Availability and implementationSource code is available for academic non-commercial research purposes. Links to code and documentation are provided at http://lpm.hms.harvard.edu and http://wall-lab.stanford.edu.Contactdpwall@stanford.edu or peter_tonellato@hms.harvard.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Congenital abnormalities of the kidney and urinary tract (CAKUT) are the most frequent cause of chronic kidney disease in children, accounting for about half of all cases. Although many forms of CAKUT are likely caused by single-gene defects, mutations in only a few genes have been identified. In order to detect new contributing genes we pooled DNA from 20 individuals to amplify all 313 exons of 30 CAKUT candidate genes by PCR analysis and massively parallel exon resequencing. Mutation carriers were identified by Sanger sequencing. We repeated the analysis with 20 new patients to give a total of 29 with unilateral renal agenesis and 11 with other CAKUT phenotypes. Five heterozygous missense mutations were detected in 2 candidate genes (4 mutations in FRAS1 and 1 in FREM2) not previously implicated in non-syndromic CAKUT in humans. All of these mutations were absent from 96 healthy control individuals and had a PolyPhen score over 1.4, predicting possible damaging effects of the mutation on protein function. Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice; however, a phenotype in heterozygous carriers has not been described. Thus, heterozygous missense mutations in FRAS1 and FREM2 cause non-syndromic CAKUT in humans.
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Project description:Current major barriers for using next-generation sequencing (NGS) technologies in genetic mutation screening on an epidemiological scale appear to be the high accuracy demanded by clinical applications and high per-sample cost. How to achieve high efficiency in enriching targeted disease genes while keeping a low cost/sample is a key technical hurdle to overcome. We validated a cDNA-probe-based approach for capturing exons of a group of genes known to cause deafness. Polymerase chain reaction amplicons were made from cDNA clones of the targeted genes and used as bait probes in hybridization for capturing human genomic DNA (gDNA) fragments. The cDNA library containing the clones of targeted genes provided a readily available, low-cost, and regenerable source for producing capture probes with standard molecular biology equipment. Captured gDNA fragments by our method were sequenced by the Illumina NGS platform. Results demonstrated that targeted exons captured by our approach achieved specificity, multiplexicity, uniformity, and depth of coverage suitable for accurate sequencing applications by the NGS systems. Reliable genotype calls for various homozygous and heterozygous mutations were achieved. The results were confirmed independently by conventional Sanger sequencing. The method validated here could be readily expanded to include all-known deafness genes for applications such as genetic hearing screening in newborns. The high coverage depth and cost benefits of the cDNA-probe-based exon capture approach may also facilitate widespread applications in clinical practices beyond screening mutations in deafness genes.
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Project description:Resolution and field-of-view often represent a fundamental tradeoff in microscopy. Atomic force microscopy (AFM), in which a cantilevered probe deflects under the influence of local forces as it scans across a substrate, is a key example of this tradeoff with high resolution imaging being largely limited to small areas. Despite the tremendous impact of AFM in fields including materials science, biology, and surface science, the limitation in imaging area has remained a key barrier to studying samples with intricate hierarchical structure. Here, we show that massively parallel AFM with >1000 probes is possible through the combination of a cantilever-free probe architecture and a scalable optical method for detecting probe-sample contact. Specifically, optically reflective conical probes on a comparatively compliant film are found to comprise a distributed optical lever that translates probe motion into an optical signal that provides sub-10 nm vertical precision. The scalability of this approach makes it well suited for imaging applications that require high resolution over large areas.
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Project description:Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.
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Project description:BackgroundCell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood.ResultsHere, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV2), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance.ConclusionsOur results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.
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Project description:Short tandem repeat (STR) variants are highly polymorphic markers that facilitate powerful population genetic analyses. STRs are especially valuable in conservation and ecological genetic research, yielding detailed information on population structure and short-term demographic fluctuations. Massively parallel sequencing has not previously been leveraged for scalable, efficient STR recovery. Here, we present a pipeline for developing STR markers directly from high-throughput shotgun sequencing data without a reference genome, and an approach for highly parallel target STR recovery. We employed our approach to capture a panel of 5000 STRs from a test group of diademed sifakas (Propithecus diadema, n = 3), endangered Malagasy rainforest lemurs, and we report extremely efficient recovery of targeted loci-97.3-99.6% of STRs characterized with ≥10x non-redundant sequence coverage. We then tested our STR capture strategy on P. diadema fecal DNA, and report robust initial results and suggestions for future implementations. In addition to STR targets, this approach also generates large, genome-wide single nucleotide polymorphism (SNP) panels from flanking regions. Our method provides a cost-effective and scalable solution for rapid recovery of large STR and SNP datasets in any species without needing a reference genome, and can be used even with suboptimal DNA more easily acquired in conservation and ecological studies.
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Project description:Protein coding genes constitute only approximately 1% of the human genome but harbor 85% of the mutations with large effects on disease-related traits. Therefore, efficient strategies for selectively sequencing complete coding regions (i.e., "whole exome") have the potential to contribute to the understanding of rare and common human diseases. Here we report a method for whole-exome sequencing coupling Roche/NimbleGen whole exome arrays to the Illumina DNA sequencing platform. We demonstrate the ability to capture approximately 95% of the targeted coding sequences with high sensitivity and specificity for detection of homozygous and heterozygous variants. We illustrate the utility of this approach by making an unanticipated genetic diagnosis of congenital chloride diarrhea in a patient referred with a suspected diagnosis of Bartter syndrome, a renal salt-wasting disease. The molecular diagnosis was based on the finding of a homozygous missense D652N mutation at a position in SLC26A3 (the known congenital chloride diarrhea locus) that is virtually completely conserved in orthologues and paralogues from invertebrates to humans, and clinical follow-up confirmed the diagnosis. To our knowledge, whole-exome (or genome) sequencing has not previously been used to make a genetic diagnosis. Five additional patients suspected to have Bartter syndrome but who did not have mutations in known genes for this disease had homozygous deleterious mutations in SLC26A3. These results demonstrate the clinical utility of whole-exome sequencing and have implications for disease gene discovery and clinical diagnosis.
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