9. ASSESSMENT OF ANEUPLOIDY AND MOSAICISM CONCORDANCE BETWEEN DIFFERENT TROPHECTODERM BIOPSY SITES AND THE INNER CELL MASS EVALUATED WITH NEXT-GENERATION SEQUENCING

2019 ◽  
Vol 39 ◽  
pp. e19-e20
Author(s):  
M.A. Tufekci ◽  
M. Cetinkaya ◽  
C. Cinar Yapan ◽  
Y. Kumtepe Colakoglu ◽  
H. Yelke ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Trophectoderm Biopsy ◽  
Next Generation ◽  
Generation Sequencing

243 GENE EXPRESSION PROFILING BY NEXT-GENERATION SEQUENCING OF cDNA SAMPLES FROM INNER CELL MASS AND TROPHECTODERM OF A SINGLE-DAY 12 PORCINE EMBRYO

2010 ◽  
Vol 22 (1) ◽  
pp. 279
Author(s):  
S. C. Isom ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Next Generation Sequencing ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Next Generation ◽  
Sequencing Technologies ◽  
Cdna Fragments ◽  
Generation Sequencing

Traditional microarray approaches to gene expression profiling often require RNA or cDNA amplification when working with extremely small or valuable tissue samples.This process is generally viewed as being undesirable because there is potential for bias to be introduced during amplification. Very recently, the so-called next-generation sequencing technologies were adapted for use in global gene expression profiling. Herein we report our efforts to apply these sequencing technologies to assess relative transcript abundances in pre-implantation-stage porcine embryos, without additional nucleic acid amplification before sequencing. As a proof-of-principle experiment, we have isolated total RNA from the embryonic disc (inner cell mass; ICM) and a small piece of trophectoderm (TE) from a Day 12 in vivo-produced embryo, which were estimated to be composed of 500 to 1000 cells each. The RNA was reverse transcribed using oligo-dT priming followed by second-strand cDNA synthesis. The double-stranded cDNA was then randomly sheared by sonication, and 10 ng of double-stranded cDNA fragments was used for sample preparation before sequencing. Prepared cDNA fragments (at 7 picomolar concentrations) were submitted for sequencing using the Illumina/Solexa platform as recommended. The millions of short (36 bp) reads generated by Illumina sequencing for each sample were then aligned to the swine UniGene database from NCBI, allowing for zero or one mismatches. Relative transcript abundances between cell types were profiled by considering the read counts for a given UniGene member as a percentage of the total number of reads generated for each cell type. It was demonstrated that approximately 11 000 and 9000 UniGene members were represented by a normalized average of 5 or more short reads per lane (0.001% of the total) in the ICM and TE samples, respectively. As expected, pluripotency factors, chromatin remodeling components, and cell-cell communication molecules were overrepresented in the ICM sample as compared with the TE sample. Conversely, epithelial determinants, ion transporters, and components of the steroid biosynthesis pathways were more abundant in the TE sample than in the ICM sample. Relative abundances of representative transcripts in these samples were verified by quantitative RT-PCR. In conclusion, we demonstrate the utility of next-generation sequencing technologies for gene expression profiling using even minute tissue samples and show that such analyses are possible even in species without a sequenced genome.

scholarly journals Transcriptome of the Inner Cell Mass and Trophectoderm in Bovine Blastocyst as Determined by Next Generation Sequencing.

2012 ◽  
Vol 87 (Suppl_1) ◽  
pp. 191-191
Author(s):  
Manabu Ozawa ◽  
Miki Sakatani ◽  
Savita Shanker ◽  
JiQiang Yao ◽  
William G. Farmerie ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Next Generation ◽  
Generation Sequencing ◽  
Bovine Blastocyst

74 Analysis of chromosomal abnormality of bovine IVF embryos based on next-generation sequencing

2020 ◽  
Vol 32 (2) ◽  
pp. 163
Author(s):  
M. Okada ◽  
Y. Nagai ◽  
S. Matoba ◽  
Y. Sakuraba ◽  
S. Sugimura
Keyword(s):  
Next Generation Sequencing ◽  
Reference Genome ◽  
Chromosomal Abnormalities ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Copy Number Variations ◽  
P Value ◽  
Next Generation ◽  
Generation Sequencing

It has been suggested that bovine IVF embryos have a higher frequency of occurrence of chromosomal abnormalities than invivo-fertilised embryos, which may explain low pregnancy success, but the details have not been clarified (Yao et al. 2018 Sci. Rep. 8, 7460). In this study, chromosomal aneuploidy in blastocysts of bovine IVF and invivo-fertilised was analysed by copy number variations (CNVs) based on next-generation sequencing. The IVF bovine embryos were cultured in well of-the-well culture dishes (LinKID micro25: Dai Nippon Printing) containing 125µL of CR1aa supplemented with 5% calf serum at 38.5°C in 5% O2 and 5% CO2 for 8 days after insemination. Invitro development of embryos was monitored using time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970-78). Invivo embryos were produced by collection of a superstimulated Japanese Black cow. Embryos that reached the blastocyst stage were divided into inner cell mass (ICM) and trophectoderm (TE) fractions by a micromanipulator with a blade. The TE and ICM samples were biopsied individually from 10 IVF and 4 invivo-derived embryos, and extracted DNA was amplified using the SurePlex DNA amplification System (Illumina). The whole-genome amplified DNA libraries were sequenced using MiSeq (Illumina). The sequencing reads were mapped onto the Bos taurus reference genome ARS-UCD1.2, obtained from the National Center for Biotechnology Information. In all 29 autosomal chromosomes and the X chromosome, CNV analysis was performed by CNV-seq (Xie and Tammi 2009 BMC Bioinformatics 10, 80). Male or female Japanese Black cattle DNA sequence was used for the reference genome. The parameter of CNV-seq was run with P-value=0.001, log2=0.6, and window size=1M. Four IVF embryos showed chromosomal duplications or deletions in either ICM- or TE-cell samples (4/10, 40%). The CNV loci between ICM and TE cells were relatively similar in each embryo. One of them was a code 1-expanded blastocyst with normal cleavage. Interestingly, CNV was not identified in another code 1-expanded blastocyst that underwent direct cleavage from 1 cell to 3 or more cells. In invivo embryos, only one embryo had a CNV (1/4, 25%). Observed CNVs in both IVF and invivo embryos were segmental duplication or deletion in each chromosome. Hence, to improve pregnancy success in bovine IVF embryos, cytogenetic evaluation may be useful for quality evaluation of embryos that are prone to chromosomal abnormalities, as well as morphological scoring.

scholarly journals Towards Improving Embryo Prioritization: Parallel Next Generation Sequencing of DNA and RNA from a Single Trophectoderm Biopsy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Noga Fuchs Weizman ◽  
Brandon A. Wyse ◽  
Ran Antes ◽  
Zenon Ibarrientos ◽  
Mugundhine Sangaralingam ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Trophectoderm Biopsy ◽  
Next Generation ◽  
Dna And Rna ◽  
Generation Sequencing
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