Ultra-fast multiplex small DNA sequencing on the MinION nanopore sequencing platform
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ABSTRACT:
PROVIDER: EGAS00001002650 | EGA |
REPOSITORIES: EGA
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Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
2024-06-08 | E-MTAB-14011 | biostudies-arrayexpress
Project description:Here we describe CapTrap-Seq, an experimental workflow designed to address the problem of reduced transcript end detection by long-read RNA sequencing methods, especially at the 5' ends. We apply CapTrap-Seq to profile transcriptomes of the human heart and brain and we compared the obtained results with other library preparation approaches. CapTrap-Seq is a platform-agnostic method and here tested the method by using 3 different long-read sequencing platforms: MinION (ONT), Sequel (PacBaio) and Sequel II (PacBio).
2023-12-20 | E-MTAB-13063 | biostudies-arrayexpress
Project description:Objective: To determine if embryos diagnosed as mosaic using Next-generation sequencing (NGS) have distinct gene expression profiles compared to embryos diagnosed as euploid or aneuploid. Materials and methods: Fifteen blastocysts were diagnosed as mosaic with NGS including 7 full mosaic monosomies, 5 partial mosaic monosomies, 2 full mosaic trisomies, and 1 partial mosaic trisomy. Three NGS diagnosed euploid embryos, and 25 aneuploid embryos (9 NGS, 14 aCGH, 2 Single Nucleotide Polymorphism array) were used as comparisons. Replicate samples were available for all euploid and full aneuploid embryos. Aneuploid embryos were chosen if their aneuploid chromosome corresponded with one of the mosaic embryos. Complementary DNA from the samples was created using the SMATer v4 Ultra Low Kit. RNA-seq library preparation followed by sequencing on the Illumina HiSeq 2500 with 50 nucleotide length paired end reads was performed. The STAR/2.5 aligner was used to align the reads against the hg19 ensemble reference transcriptome. Differentially expressed genes (DEGs) were calculated using DES-seq2/3.5, and p values <0.05 were considered significantly differentially expressed. Pathway analysis was performed on the top DEGs among all of the mosaic embryos with p<0.05 considered significant. Main results: The 15 mosaic embryos had transcriptomic profiles which were distinct from full aneuploid embryos involving the same chromosome. Mosaic embryos had fewer DEGs compared to aneuploid embryos of the same chromosome when using euploid embryos as controls. Mosaic embryos were grouped on principal component analysis (PCA) and distinguishable from euploid and aneuploid embryos. Pathways involving cell proliferation, differentiation, and apoptosis were the most disrupted from the presence of a mosaic cell line.
2020-05-27 | GSE151219 | GEO
Project description:Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)? Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs. What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol. Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201). Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd
2024-04-03 | GSE234338 | GEO
Project description:Array-based comparative genome hybridization. The aneuploid progeny could return to euploid state.
2015-01-07 | GSE42359 | GEO
Project description:Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, whether and how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene expression patterns have been reported: an environmental stress response (ESR) and a “common aneuploidy gene-expression” (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate and bring clarity to this controversy. We show that the CAGE signature is not an aneuploidy-specific gene expression signature but is the result of euploid control cells, but not aneuploid cells, having grown into stationary phase. Because growth into stationary phase is amongst the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells are found to exhibit the ESR. We further show that the ESR causes a selective loss of ribosomes in aneuploid cells, providing an explanation for the decreased cellular density in aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than a CAGE signature, in response to aneuploidy-induced cellular stresses that results in selective ribosome loss.
2020-06-16 | GSE146791 | GEO
Project description:Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading genetic cause of miscarriage and developmental delay in newborns. Telomere DNA deficiency is associated with genomic instability in somatic cells and may play a role in development of aneuploidy commonly found in female germ cells and human embryos. To test this hypothesis, we developed a method capable of quantifying telomere DNA in parallel with 24-chromosome aneuploidy screening from the same oocyte or embryo biopsy. Aneuploid human polar bodies possessed significantly less telomere DNA than euploid polar bodies from sibling oocytes (-3.07 fold, P=0.016). This indicates that oocytes with telomere DNA deficiency are prone to aneuploidy development during meiosis. Aneuploid embryonic cells also possessed significantly less telomere DNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blastocyst stage (-1.18 fold, P=0.340). The lack of a significant difference at the blastocyst stage was found to be due to telomere DNA normalization between the cleavage and blastocyst stage of embryogenesis and not due to developmental arrest of embryos with short telomeres. Heterogeneity in telomere length within oocytes may provide an opportunity to improve the treatment of infertility through telomere based selection of oocytes and embryos with reproductive competence.
2011-07-04 | GSE25864 | GEO
Project description:Candida albicans lab strain SC5314, which is a euploid diploid, was used to generate aneuploid fluconazole adaptors. Transcriptomes of aneuploids were compared to the euploid parent by growing cells in rich YPD broth.
2022-09-26 | E-MTAB-12185 | biostudies-arrayexpress
Project description:Array-based comparative genome hybridization. The aneuploid progeny could return to euploid state. We determined genome-wide gene copy number in the genomes of aneuploid progeny after 14, 24 and 34 days vegetative propagation in dextrose-containing rich medim.
2015-01-07 | E-GEOD-42359 | biostudies-arrayexpress
Project description:Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37ËC followed by pyronin Y for 45min at 37ËC and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid. Euploid and aneuploid were sorted with 99% purity and 1x105 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4Euploid and A4Aneuploid. Three samples of each were chosen for hybridization.
2016-07-03 | E-GEOD-74852 | biostudies-arrayexpress