Project description:Many studies estimate that chromosomal mosaicism within the cleavage stage human embryo is high. However, comparison of two unique methods of aneuploidy screening of blastomeres within the same embryo has not been conducted and may indicate whether mosaicism is overestimated due to technical inconsistency rather than biological phenomena. The present study investigates the prevalence of chromosomal abnormality and mosaicism found with two different single cell aneuploidy screening techniques.Thirteen arrested cleavage stage embryos were studied. Each was biopsied into individual cells (n=160). The cells from each embryo were randomized into two groups. Those destined for FISH based aneuploidy screening (n=75) were fixed, 1 cell per slide. Cells for SNP microarray based aneuploidy screening (n=85) were put into individual tubes. Microarray was significantly more reliable (96%) than FISH (83%) for providing an interpretable result (P=0.004). Markedly different results were obtained when comparing microarray and FISH results from individual embryos. Mosaicism was significantly less commonly observed by microarray (4 of 13 embryos; 31%) than by FISH (13 of 13 embryos; 100%)(P=0.0005). Although FISH evaluated fewer chromosomes per cell and fewer cells per embryo, FISH still displayed significantly more unique genetic diagnoses per embryo (3.2+0.2) than microarray (1.3+0.2)(P<0.0001). This is the first prospective, randomized, blinded, and paired comparison between microarray and FISH based aneuploidy screening. Aneuploidy and mosaicism were less common with microarray. While evaluating a smaller number of chromosomes with a proportionally smaller opportunity for finding mosaicism, FISH still had a dramatically higher level of inter-cell variation in diagnosis. SNP microarray based 24 chromosome aneuploidy screening provides more complete and consistent results than FISH. Affymetrix SNP arrays were processed according to the manufacturer's directions on DNA extracted from 13 arrested cleavage stage embryos and biopsied into individual blastomere cells. Individual blastomeres were randomized and blinded for SNP microarray analysis (n = 85). Afflymetrix SNP array analysis was successfully completed on 82 blastomeres.

Project description:Methods for haplotyping and DNA copy number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required that substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP-genotypes (AA, AB, BB), and DNA copy number profiling remains difficult as true DNA copy number aberrations have to be discriminated from WGA-artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by deciphering WGA-distorted SNP B-allele fractions, using a process we coin haplarithmisis. We demonstrate clinical precision of the method on single cells biopsied from human embryos to diagnose disease alleles genome wide, we advance and facilitate the detection of numerical and structural chromosomal anomalies in single cells, and can distinguish meiotic from mitotic segregation errors in a single assay. The samples of a reference family were applied for optimisation of single-cell genotyping using Affymetrix SNP-arrays prior to downstream analysis. Specifically, the reference family delivers genomic DNA samples isolated from peripheral blood of two siblings 'S1' and 'S2', the mother and father of these siblings, as well as of the maternal grandmother and grandfather. Of individuals ‘S1’ and ‘S2’, six EBV-transformed lymphoblastoid single cells were isolated of which three were whole-genome amplified using MDA and three using PicoPlex. These WGA-products were hybridized to Affymetrix NspI 250K SNP-arrays following the protocol as recommended by the company. Subsequently, the SNP-probe signals were interpreted by different genotyping algorithms (see data processing). Based on overall performance, it was decided to use the Dynamic Model (DM) for interpreting Affymetrix SNP-probe signals of single cells.

Project description:Methods for haplotyping and DNA copy number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required that substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP-genotypes (AA, AB, BB), and DNA copy number profiling remains difficult as true DNA copy number aberrations have to be discriminated from WGA-artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by deciphering WGA-distorted SNP B-allele fractions, using a process we coin haplarithmisis. We demonstrate clinical precision of the method on single cells biopsied from human embryos to diagnose disease alleles genome wide, we advance and facilitate the detection of numerical and structural chromosomal anomalies in single cells, and can distinguish meiotic from mitotic segregation errors in a single assay. Here we provide two sample sets, including (1) a reference family delivering samples applied for the development and optimization of single-cell genotyping, QC-metrics, haplarithmisis and the siCHILD algorithm. Specifically, the reference family delivers genomic DNA samples isolated from peripheral blood of two siblings 'S1' and 'S2', the mother and father of these siblings, as well as of the maternal grandmother and grandfather. Of individuals ‘S1’ and ‘S2’, six EBV-transformed lymphoblastoid single cells were also isolated, of which three were whole-genome amplified using MDA and three using PicoPlex. These multi-cell genomic DNA samples and single-cell WGA-products were hybridized to Illumina HumanCytoSNP12-v2.1 SNP-arrays following: (i) the protocol as recommended by the company and/or (ii) a modified rapid protocol as described below. Subsequently, the SNP-probe signals were interpreted by two different genotyping algorithms (GenCall and GenoSNP). Based on overall performance, we decided to use GenCall for interpreting Illumina SNP-probe signals of single-cell and multi-cell DNA samples. (2) A set of 12 families undergoing genetic diagnosis (for Mendelian disorders or translocation chromosomes) of preimplantation embryos following in vitro fertilization. In general, each family delivers genomic DNA samples isolated from peripheral blood of the two parents as well as a sibling and/or other close relatives; the specific kinships for each family are given in the 'description' column. In addition, of each family single blastomeres biopsied from preimplantation embryos obtained via in vitro fertilization of the parental gametes are also provided. All single-cell genomes were amplified by MDA. All the samples were hybridized to Illumina HumanCytoSNP12-v2.1 SNP-arrays following the rapid protocol. This data was used for clinical validation of the siCHILD algorithm.

Project description:We conducted a genome-wide survey of genes in Ae. aegypti females that are transcriptionally responsive upon challenge with dengue virus (serotype-2). The array was designed with 60-mer oligos specific to 16,092 gene transcripts of gene build AaegL1.1 (www. vectorbase.org). The hybridizations were performed at NimbleGen. We provided total RNA purified from the infected and control samples to NimbleGen. To identify dengue-specific transcription response (DTR) genes of MS and MR females upon infection with DENV2-JAM1409, a total of 15 independent samples (12 test samples and 3 control samples) were used for array hybridizations. These consisted of four DENV2 infected samples (MS-3hr, MR-3hr, MS-18hr and MR-18hr, post-infection, respectively) and a control sample that consisted of RNA isolated at the same time points following uninfected blood meals and pooled across strains and time of sampling. Three independent biological replicates were prepared for each of the above five samples were used for hybridizations. The Ae. aegypti genes responsive to dengue infection were identified in MOYO-S (MS) and MOYO-R (MR) females, upon challenging them with DENV serotype-2, by a genome-wide transcriptome analysis carried out using the NimbleGen oligonucleotide microarray format. We profiled gene expressions of Aedes aegypti mosquitoes, strain MOYO-S (MS, susceptible to dengue virus) and MOYO-R (MR, refractory to dengue virus) at 3 hr and 18 hr after infection with dengue virus (seroptype-2). The test samples were prepared in parallel to the control samples. The control samples were derived from females fed with normal blood that was not mixed with dengue (mock infection). The test samples were prepared from females that were fed with blood mixed with dengue virus (infectious meal). A total of 12 test samples were prepared that represented three biological replicates of dengue infected MS females at 3hr and 18 post-infection times; and MR females at 3hr and 18hr post-infection times. The test samples were compared to a common uninfected control that was prepared by pooling equal amount RNA of the individual control of both strains and both post-infection time points (MS at 3hr, MS at 18 hr, MR at 3hr and MR at 18hr). The control-pool strategy ensured us to identify genes that were responsive in response to dengue infection but not due to genetic differences between the two strains or developmental changes between the two post-infection time points. A total of 3 control pools were used in our array expression studies. Control-1 was prepared from RNA isolated from individual controls set up along with each of the test samples (MS at 3hr, MS at 18 hr, MR at 3hr and MR at 18hr) for the first biological replication. Similarly, control-2 was prepared from individual controls set up along with each of the test samples (MS at 3hr, MS at 18 hr, MR at 3hr and MR at 18hr) for the second biological replication. And control-3 was prepared from individual controls set up along with each of the test samples (MS at 3hr, MS at 18 hr, MR at 3hr and MR at 18hr) for the third biological replication. Thus, the three control pools represented three biological replicates corresponding to the three biological replicates of the test samples.

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Single Cell Whole Genome Amplification Technique Impacts the Accuracy of SNP Microarray Based Genotyping and Copy Number Analyses

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