Roche NimbleGen Webinar Series

Exome sequencing has become a mainstay for gene discovery in Mendelian diseases and beyond. However, genetically highly heterogeneous phenotypes still present challenging targets. An example is Retinitis pigmentosa (RP), a degenerative eye disease where greater than 50 known genes explain only approximately half of the affected families. When a new gene is implicated in RP, it is usually a relatively rare cause of the disease. This situation typically requires additional studies including classic chromosomal mapping, large-scale sequencing of unrelated probands, in silico analysis of protein function and pathways, and also rapid in vivo modeling of candidate sequence variants. This webinar will draw upon our extensive experience in exome sequencing and will detail our recent identification of a novel Retinitis pigmentosa gene, DHDDS, in a single nuclear family.

Exome sequencing has become a mainstay for gene discovery in Mendelian diseases and beyond. However, genetically highly heterogeneous phenotypes still present challenging targets. An example is Retinitis pigmentosa (RP), a degenerative eye disease where greater than 50 known genes explain only approximately half of the affected families. When a new gene is implicated in RP, it is usually a relatively rare cause of the disease. This situation typically requires additional studies including classic chromosomal mapping, large-scale sequencing of unrelated probands, in silico analysis of protein function and pathways, and also rapid in vivo modeling of candidate sequence variants. This webinar will draw upon our extensive experience in exome sequencing and will detail our recent identification of a novel Retinitis pigmentosa gene, DHDDS, in a single nuclear family.

Recent collaborative efforts between Roche NimbleGen and Caliper Life Sciences have resulted in the development of an automated, optimized solution for large scale targeted resequencing sample preparation projects for Next Generation Sequencing. This webinar will demonstrate the advantages this integrated solution can provide to researchers who desire to increase throughput (up to 288 samples per week), enhance consistency, and minimize human errors in targeted resequencing projects. NimbleGen SeqCap EZ Library products enable researchers to reduce sequencing costs by allowing more samples to be multiplexed in a single sequencing run due to the combination of high capture efficiency and specificity. Learn how Roche NimbleGen and Caliper Life Sciences can enable the scale-up of your targeted resequencing sample preparation capabilities with minimal time and effort.

Recent collaborative efforts between Roche NimbleGen and Caliper Life Sciences have resulted in the development of an automated, optimized solution for large scale targeted resequencing sample preparation projects for Next Generation Sequencing. This webinar will demonstrate the advantages this integrated solution can provide to researchers who desire to increase throughput (up to 288 samples per week), enhance consistency, and minimize human errors in targeted resequencing projects. NimbleGen SeqCap EZ Library products enable researchers to reduce sequencing costs by allowing more samples to be multiplexed in a single sequencing run due to the combination of high capture efficiency and specificity. Learn how Roche NimbleGen and Caliper Life Sciences can enable the scale-up of your targeted resequencing sample preparation capabilities with minimal time and effort.

Dilated cardiomyopathy (DCM), a common primary myocardial disease, causes systolic dysfunction and heart failure, a major public health problem. Its mode of transmission is mostly autosomal dominant but the disease is extremely heterogeneous with point mutations identified in over 30 genes. However, only ~35% of DCM genetic cause can be explained by point mutations in the known DCM genes, leaving most genetic cause unknown. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes and 51 small insertion deletions; however, all were excluded based upon their presence in dbSNP, in-house population controls or their lack of segregation with those affected with DCM. Genome-wide copy number analysis using NimbleGen 2.1 M arrays identified 440 copy number variants > 1 kb, (median size ~13 kb). We then validated putative novel variants (not present in the database of genomic variants) with NimbleGen 135K custom arrays. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of BAG3 rare variants in genetic DCM, we sequenced the coding exons in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense variants that were absent in over 967 exomes sequenced at Seattle Seq, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.

Dilated cardiomyopathy (DCM), a common primary myocardial disease, causes systolic dysfunction and heart failure, a major public health problem. Its mode of transmission is mostly autosomal dominant but the disease is extremely heterogeneous with point mutations identified in over 30 genes. However, only ~35% of DCM genetic cause can be explained by point mutations in the known DCM genes, leaving most genetic cause unknown. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes and 51 small insertion deletions; however, all were excluded based upon their presence in dbSNP, in-house population controls or their lack of segregation with those affected with DCM. Genome-wide copy number analysis using NimbleGen 2.1 M arrays identified 440 copy number variants > 1 kb, (median size ~13 kb). We then validated putative novel variants (not present in the database of genomic variants) with NimbleGen 135K custom arrays. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of BAG3 rare variants in genetic DCM, we sequenced the coding exons in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense variants that were absent in over 967 exomes sequenced at Seattle Seq, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.

From low throughput preparation of sample libraries to keeping the sequencing pipeline full, target enrichment and library preparation for next-generation sequencing has presented significant challenges at the Core Genotyping Facility at National Cancer Institute (NCI-CGF). Through key process optimization to library preparation coupled with the incorporation of NimbleGen SeqCap EZ, a solution-based enrichment technology, NCI-CGF has overcome the throughput challenges and increased capacity to manually enrich and process multiple 96-well plates. These improvements have proven to be significant, decreasing project turn-around times from one month to one week, opening the possibility of an automated solution, and decreasing costs while increasing efficiency for next generation targeted re-sequencing projects. Also presented in this webinar are target re-sequencing results from a genomic region associated with prostate cancer risk in 78 research samples. The results of this study have generated a detailed map of common genetic variations in chr19q13.33, and these variations should facilitate fine-mapping the association signal as well as determining the contribution of this locus to prostate cancer risk and regulation of prostate specific antigen (PSA) expression.

From low throughput preparation of sample libraries to keeping the sequencing pipeline full, target enrichment and library preparation for next-generation sequencing has presented significant challenges at the Core Genotyping Facility at National Cancer Institute (NCI-CGF). Through key process optimization to library preparation coupled with the incorporation of NimbleGen SeqCap EZ, a solution-based enrichment technology, NCI-CGF has overcome the throughput challenges and increased capacity to manually enrich and process multiple 96-well plates. These improvements have proven to be significant, decreasing project turn-around times from one month to one week, opening the possibility of an automated solution, and decreasing costs while increasing efficiency for next generation targeted re-sequencing projects. Also presented in this webinar are target re-sequencing results from a genomic region associated with prostate cancer risk in 78 research samples. The results of this study have generated a detailed map of common genetic variations in chr19q13.33, and these variations should facilitate fine-mapping the association signal as well as determining the contribution of this locus to prostate cancer risk and regulation of prostate specific antigen (PSA) expression.

Epigenetic changes in DNA methylation patterns and chromatin structure play key roles in the development of disease. Examples include Angelman and Prader-Willi syndromes, where epigenetic silencing is one factor in the development of these diseases. Therefore, understanding epigenetics is critical to our future understanding of many important diseases. Methods exist to analyze DNA methylation patterns in higher eukaryotes including methylated DNA immunoprecipitation-on-chip (MeDIP-chip), an affinity based approach to enrich methylated DNA regions from genomic DNA followed by microarray analysis. Similarly, chromatin structure and DNA-binding proteins can be mapped using chromatin immunoprecipitation-on-chip (ChIP-chip) using target protein specific antibodies and microarrays. Here we introduce our new Human, Mouse and Rat 3x720K ChIP-chip and DNA Methylation Arrays which offer comprehensive coverage of gene promoters and CpG islands in a multiplex platform. These arrays offer high resolution, 100bp probe spacing through all covered regions for highly sensitive and reproducible detection of DNA methylation and target protein DNA binding. In addition, the DNA methylation arrays also include probes covering positive, negative and nonCpG control regions to aid in assessment of the overall array performance. In this presentation, we will (1) describe the array designs and content in more detail, (2) demonstrate the sensitive and reproducible detection capabilities of these new arrays, and (3) compare the performance of these multiplex arrays with the single-plex 2.1M Deluxe Promoter arrays.

Epigenetic changes in DNA methylation patterns and chromatin structure play key roles in the development of disease. Examples include Angelman and Prader-Willi syndromes, where epigenetic silencing is one factor in the development of these diseases. Therefore, understanding epigenetics is critical to our future understanding of many important diseases. Methods exist to analyze DNA methylation patterns in higher eukaryotes including methylated DNA immunoprecipitation-on-chip (MeDIP-chip), an affinity based approach to enrich methylated DNA regions from genomic DNA followed by microarray analysis. Similarly, chromatin structure and DNA-binding proteins can be mapped using chromatin immunoprecipitation-on-chip (ChIP-chip) using target protein specific antibodies and microarrays. Here we introduce our new Human, Mouse and Rat 3x720K ChIP-chip and DNA Methylation Arrays which offer comprehensive coverage of gene promoters and CpG islands in a multiplex platform. These arrays offer high resolution, 100bp probe spacing through all covered regions for highly sensitive and reproducible detection of DNA methylation and target protein DNA binding. In addition, the DNA methylation arrays also include probes covering positive, negative and nonCpG control regions to aid in assessment of the overall array performance. In this presentation, we will (1) describe the array designs and content in more detail, (2) demonstrate the sensitive and reproducible detection capabilities of these new arrays, and (3) compare the performance of these multiplex arrays with the single-plex 2.1M Deluxe Promoter arrays.

DNA Microarrays have become valuable tools for researchers in identifying biomarkers for active disease and assessing increased risk for certain genetic conditions. Roche NimbleGen, Inc. is a market leader in high-density DNA Microarrays with 2.1M probes per slide which, in a multiplex array format, provide an extremely high-quality, cost-effective, high-throughput solution for today’s microarray researcher. We proudly introduce the NimbleGen MS 200 Microarray Scanner for high resolution (down to 2 micron) scanning with a 48-slide autoloader and advanced automation capabilities that help ensure your high density arrays are analyzed with utmost precision and accuracy giving you consistent and robust data. The NimbleGen MS 200 is a state-of-the art DNA Microarray scanner optimized to provide excellent performance when used with NimbleGen arrays. With a completely isolated and ozone-protected slide magazine in addition to high-quality PMT detectors, the NimbleGen MS 200 provides the enhanced performance required to extract valuable data from even your most demanding microarray experiments.

DNA Microarrays have become valuable tools for researchers in identifying biomarkers for active disease and assessing increased risk for certain genetic conditions. Roche NimbleGen, Inc. is a market leader in high-density DNA Microarrays with 2.1M probes per slide which, in a multiplex array format, provide an extremely high-quality, cost-effective, high-throughput solution for today’s microarray researcher. We proudly introduce the NimbleGen MS 200 Microarray Scanner for high resolution (down to 2 micron) scanning with a 48-slide autoloader and advanced automation capabilities that help ensure your high density arrays are analyzed with utmost precision and accuracy giving you consistent and robust data. The NimbleGen MS 200 is a state-of-the art DNA Microarray scanner optimized to provide excellent performance when used with NimbleGen arrays. With a completely isolated and ozone-protected slide magazine in addition to high-quality PMT detectors, the NimbleGen MS 200 provides the enhanced performance required to extract valuable data from even your most demanding microarray experiments.

One of the major features associated with imprinting is the presence of parent-of-origin specific Differentially Methylated Regions (DMRs). Thus, the maternal and paternal genomes possess distinct epigenetic marks which distinguish them at imprinted loci. In order to construct an imprinting map of the human genome, we have profiled DNA methylation patterns genome-wide in rare uniparental tissues. For genome-wide studies, we have compared methylation patterns in a panel of complete hydatidiform moles, which have an exclusively paternal genetic contribution, and ovarian teratomas, which have an exclusively maternal genetic contribution. Methylated DNA was immunoprecipitated using anti 5-methyl cytidine and hybridized to Nimblegen high-density oligonucleotide tiling arrays composed of 21 million probes distributed throughout the genome, generating complete profiles of the maternal and paternal methylomes. Comparison of these profiles identifies numerous sites of methylation difference, including sites of methylation polymorphism, novel imprinted loci, and also tissue specific differences. Examination of known imprinted genes showed that many are associated with DMRs, validating this as a system for the detection of imprinting. Many novel putative imprinted loci on nearly every human chromosome were also identified. These include novel DMRs within known imprinted gene clusters, as well as chromosomal regions not previously thought to be imprinted. In order to identify novel imprinted genes specifically on chromosome 15, we have also profiled DNA methylation in cases with uniparental disomy of chromosome 15 (UPD15). Comparison of six individuals with maternal versus paternal UPD15 reveals fourteen DMRs on chromosome 15. Some novel DMRs are located outside of 15q11-q13, and are associated with genes not previously thought to be imprinted, including IGF1R at 15q26.3, which plays a fundamental role in growth regulation. To validate our array data we performed bisulfite sequencing of putative DMRs, giving base-pair resolution of these imprints and confirming the presence of parent-of-origin specific methylation marks in multiple independent samples. Our data provides the first imprinting map of the human genome, demonstrates that the number of imprinted loci in the human genome is much higher than previously thought, and suggests that imprinting may influence the phenotypes of many human diseases.

One of the major features associated with imprinting is the presence of parent-of-origin specific Differentially Methylated Regions (DMRs). Thus, the maternal and paternal genomes possess distinct epigenetic marks which distinguish them at imprinted loci. In order to construct an imprinting map of the human genome, we have profiled DNA methylation patterns genome-wide in rare uniparental tissues. For genome-wide studies, we have compared methylation patterns in a panel of complete hydatidiform moles, which have an exclusively paternal genetic contribution, and ovarian teratomas, which have an exclusively maternal genetic contribution. Methylated DNA was immunoprecipitated using anti 5-methyl cytidine and hybridized to Nimblegen high-density oligonucleotide tiling arrays composed of 21 million probes distributed throughout the genome, generating complete profiles of the maternal and paternal methylomes. Comparison of these profiles identifies numerous sites of methylation difference, including sites of methylation polymorphism, novel imprinted loci, and also tissue specific differences. Examination of known imprinted genes showed that many are associated with DMRs, validating this as a system for the detection of imprinting. Many novel putative imprinted loci on nearly every human chromosome were also identified. These include novel DMRs within known imprinted gene clusters, as well as chromosomal regions not previously thought to be imprinted. In order to identify novel imprinted genes specifically on chromosome 15, we have also profiled DNA methylation in cases with uniparental disomy of chromosome 15 (UPD15). Comparison of six individuals with maternal versus paternal UPD15 reveals fourteen DMRs on chromosome 15. Some novel DMRs are located outside of 15q11-q13, and are associated with genes not previously thought to be imprinted, including IGF1R at 15q26.3, which plays a fundamental role in growth regulation. To validate our array data we performed bisulfite sequencing of putative DMRs, giving base-pair resolution of these imprints and confirming the presence of parent-of-origin specific methylation marks in multiple independent samples. Our data provides the first imprinting map of the human genome, demonstrates that the number of imprinted loci in the human genome is much higher than previously thought, and suggests that imprinting may influence the phenotypes of many human diseases.

Audio: Developing an Imprinting Map of the Human Genome