Advanced Genomic Techniques in Sequencing of Colorectal Cancer
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ABSTRACT: We developed a method that uses allelic imbalance information in combination with phased genomic sequencing to generate mega-haplotypes that can encompass entire chromosome arms. We applied this technique to characterize aneuploidy and chromosomal alterations in colorectal cancers.
SECONDARY ACCESSION(S): PRJNA393227PRJNA393228
REPOSITORIES: dbGaP
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Project description:Filtered selection coupled with support vector machines generate functionally relevant prediction model for colorectal cancer. In this study, we built a model that uses Support Vector Machine (SVM) to classify cancer and normal samples using Affymetrix exon microarray data obtained from 90 samples of 48 patients diagnosed with CRC. From the 22,011 genes, we selected the 20, 30, 50, 100, 200, 300 and 500 genes most relevant to CRC using the Minimum-Redundancy–Maximum-Relevance (mRMR) technique. With these gene sets, an SVM model was designed using four different kernel types (linear, polynomial, radial basis function and sigmoid).
2016-02-02 | GSE77434 | GEO
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele. Total gene expression of normal breast sample from healthy controls. This submission represents transcriptome component of study.
2012-02-01 | E-GEOD-32124 | biostudies-arrayexpress
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele. Genotype of normal breast sample from healthy controls.
2012-02-01 | E-GEOD-32258 | biostudies-arrayexpress
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele.
2012-02-01 | GSE32124 | GEO
Project description:Introduction: Cis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism. Methods: We combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in-silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analysed in-vitro and in-vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2754 carriers. Results: We identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterised three cis-regulatory SNPs located at the promoter and two intronic regulatory elements, which affect the binding of the transcription factors C/EBPα, HMGA1, DBP and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele HR=0.85, 95%CI=0.72-1.00, P-trend=0.048). Conclusion: Our work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2, which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele.
2012-02-01 | GSE32258 | GEO
Project description:Aneuploidy has been well documented in blastocyst embryos, but prior studies have been limited in scale and/or lack mechanistic data. We previously reported preclinical validation of microarray 24-chromosome preimplantation genetic screening (PGS) in a 24-hour protocol. The method diagnoses chromosome copy number, structural chromosome aberrations, parental source of aneuploidy, and distinguishes certain meiotic from mitotic errors. In this study our objective was to examine aneuploidy in human blastocysts and determine correspondence of karyotypes between trophectoderm (TE) and inner cell mass (ICM). We disaggregated 51 blastocysts from seventeen couples into ICM and one or two TE fractions. The average maternal age was 31. Next, we ran 24-chromosome microarray molecular karyotyping on all of the samples, and then performed a retrospective analysis of the data. The average per-chromosome confidence was 99.95%. Approximately 80% of blastocysts were euploid. The majority of aneuploid embryos were simple aneuploid, i.e., one or two whole-chromosome imbalances. Structural chromosome aberrations, which are common in cleavage stage embryos, occurred in only three blastocysts (5.8%). All TE biopsies derived from the same embryos were concordant. Forty-nine of fifty-one (96.1%) inner cell mass (ICM) samples were concordant with TE biopsies derived from the same embryos. Discordance between TE and ICM occurred only in the two embryos with structural chromosome aberration. We conclude that trophectoderm karyotype is an excellent predictor of inner cell mass karyotype. Discordance between TE and ICM occurred only in embryos with structural chromosome aberrations. We thawed all fractions at 22°C, and then added Arcturus PicoPure Lysis Buffer (Molecular Devices, Sunnyvale, CA) to each of the biopsies. The tubes were incubated at 56°C for one hour, and then heat inactivated at 95°C for 10 minutes. DNA from the lysed biospsies was amplified using a commercial kit (GE Healthcare, Waukesha, WI) for multiple displacement amplification (MDA). MDA reactions were incubated at 30°C for 2.5 hours and then heat-inactivated at 65°C for five minutes. The amplified samples were genotyped using Illumina (San Diego, CA, USA) Infinium II genotyping microarrays (CytoSNP-12 chips) using a modified 24-hour protocol, as described previously*. Previously, we developed a genotyping microarray molecular karyotyping technology that uses parental genetic data to increase accuracy and determine mechanism and source of aneuploidy*. The algorithm uses parental genotypes and the observed distribution of unprocessed single cell microarray channel intensities to diagnose whole-chromosome imbalances and structural chromosome aberrations*. Because parental genotypes are available, the algorithm readily identifies parental source of whole-chromosome imbalances and structural chromosome aberrations. Additionally, the algorithm uses parental information, high confidence disomic single cell measurements on children, and recombination probabilities (genome.ucsc.edu) to determine the phase of the parental chromosomes. The phased data is then used to determine whether certain trisomies and uniparental disomies were mitotic or meiotic in origin*. *Johnson DS, Gemelos G, Baner J, Ryan A, Cinnioglu C, Banjevic M, Ross R, Alper M, Barrett B, Frederick J, Potter D, Behr B, Rabinowitz M. (2010). Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-hour protocol. Human Reproduction January 24 [Epub ahead of print].
2010-09-28 | E-GEOD-22864 | biostudies-arrayexpress
Project description:Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. We evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility.mosome-level allele-specific profiles of CpG methylation and chromatin accessibility.
2020-08-07 | GSE155791 | GEO
Project description:We adapted the widely used digestion of chromatin with micrococcal nuclease (MNase) followed by deep sequencing to the parasite Trypanosoma cruzi, which presents numerous singularities. In this work, we use the hybrid CL Brener strain carrying two set of chromosomes from two substantially different parental strains. The hybrid strain CL Brener is composed of the Esmeraldo-like and non Esmeraldo-like haplotypes. Additionally, part of the genome was not assembled into any of the haplotypes. Sometimes the community working in the field uses just one haplotype as reference genome for simplicity. In this work, we emphasize the importance of using its whole genome as a reference. Moreover, we extended our analysis to a clonal strain, Sylvio-X10.
2023-10-23 | GSE176341 | GEO
Project description:Using long-read nanopore sequencing, we obtained chromosome-wide phased methylomes of the active and inactive X in mouse placenta and neural stem cells (NSCs), overcoming the limitations if short-read bisulfite sequencing in allelic resolution. We also conducted quantitative analysis of methylation properties like symmetry and entropy, providing a more comprehensive view of epigenetic silencing in X chromosome inactivation. We also resolved the allele-specific genetics and epigenetics of structural macrosatellite Dxz4 and other repeats.
2024-03-12 | E-MTAB-13706 | biostudies-arrayexpress
Project description:Phenotypic variability is a hallmark of diseases involving chromosome gains and losses, such as Down Syndrome and cancer. Allelic variances have been thought to be the sole cause of this heterogeneity. Here, we systematically examine the consequences of gaining and losing single or multiple chromosomes to show that the aneuploid state causes non-genetic phenotypic variability. Yeast cell populations harboring the same defined aneuploidy exhibit heterogeneity in cell cycle progression and response to environmental perturbations, which we show to be partly due to gene copy number imbalances. Thus, subtle changes in gene expression severely impact the robustness of biological networks and cause alternate behaviors when they occur at a large scale. Because trisomic mice also exhibit variable phenotypes, we further propose that non-genetic individuality is a universal characteristic of the aneuploid state that could contribute to variability in presentation and treatment responses of diseases caused by aneuploidy.
2017-04-10 | PXD006009 | Pride