218 RNA-Seq ANALYSIS OF BOVINE OOCYTE TRANSCRIPTOME REVEALS THAT DIFFERENCES BETWEEN HEIFERS AND REPEAT BREEDERS ARE LIMITED TO A FEW KEY TRANSCRIPTS

2013 ◽  
Vol 25 (1) ◽  
pp. 257
Author(s):  
F. Gandolfi ◽  
G. Stradaioli ◽  
V. Gollini ◽  
F. Cattonaro ◽  
C. Galli ◽  
...  
Keyword(s):  
Dna Microarrays ◽  
Transcriptional Profiling ◽  
Rna Extraction ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Oocyte Growth ◽  
Total Rna ◽  
Next Generation Sequencing Technology ◽  
Domestic Species ◽  
Set Up

Maternal transcripts are accumulated during oocyte growth and drive early embryonic development; therefore, their characterisation is a relevant factor for predicting fertility. DNA microarrays have been the method of choice for transcriptional profiling, but this method has some limitations when applied to domestic species because it relies upon existing knowledge about genome sequence and offers a limited quantitative evaluation. These limits are overcome by next-generation sequencing technology. The aim of the work was to define a reference standard for bovine fertility determining the list and the level of transcripts stored in fully grown oocytes collected from heifers (H) and to compare this pattern with that of adult repeat breeders (RB). Oocytes were collected by ovum pick-up (OPU) from 5 Italian Dappled Red heifers of 11 to 15 months of age that became pregnant at the following oestrus and from 4 adult cows of the same breed with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. In both groups, oocytes were aspirated from follicles of 4 to 6 mm in diameter. Each oocyte was carefully denuded and immediately snap frozen in liquid nitrogen. Oocytes from each animal were pooled together (range 4 to 11) and analysed as a single sample. Total RNA extraction was performed by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, from both mRNA and non-polyadenylated transcripts, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (NuGEN Inc., San Carlos, CA, USA). Purified cDNA was ligated directly into an Illumina sequencing library using TruSeq DNA Sample Prep kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on Illumina HiSEqn 2000 in the 50-bp long single-read set-up, at a 4-plex of multiplexing level, producing 30 to 40 million reads per sample. Data were annotated using the cDNA ENSEMBL UMD 3.1.67 database. On average, the number of transcripts present in each sample was 15 438 ± 766 in H and 15 624 ± 768 in RB oocytes. Nineteen thousand one hundred sixty-one transcripts were detected at least in one sample, and 12 174 were detected in all samples. The comparison between H and RB showed that 598 transcripts out of 19 161 (3.12%) and 437 out of 12 174 (3.59%) are expressed at a significantly different level (P < 0.05) in the 2 groups. Taking into consideration only the transcripts detected in all the samples, with an expression rate of at least 10-fold different and a P < 0.05 we identified 39 genes. Seventeen transcripts were more abundant in RB oocytes, whereas 22 were downregulated. This is the first analysis of the oocyte transcriptome performed with deep sequencing technology. The method enabled us to compile a full list of transcripts that are found in highly competent oocytes. A direct comparison with low-quality oocytes indicated that quantitative differences of transcripts level are limited to a small subpopulation of key transcripts. Supported by PRIN 2008.

scholarly journals An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260002
Author(s):  
María José Cárdenas Espinosa ◽  
Tabea Schmidgall ◽  
Georg Wagner ◽  
Uwe Kappelmeyer ◽  
Stephan Schreiber ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Bacterial Degradation ◽  
Rna Seq ◽  
High Quality ◽  
Total Rna ◽  
Aromatic Substrates ◽  
Next Generation Sequencing Ngs ◽  
Total Rna Extraction

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

scholarly journals Novel RNA Extraction Method for Dual RNA-seq Analysis of Pathogen and Host in the Early Stages of Yersinia pestis Pulmonary Infection

2021 ◽  
Vol 9 (10) ◽  
pp. 2166
Author(s):  
Ofir Israeli ◽  
Inbar Cohen-Gihon ◽  
Moshe Aftalion ◽  
David Gur ◽  
Yaron Vagima ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Post Infection ◽  
Rna Extraction ◽  
Early Time ◽  
Transcriptomic Analysis ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Pneumonic Plague ◽  
Bacterial Rna ◽  
Lethal Infection

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.

scholarly journals Rcirc: an R package for circRNA analyses and visualization

2020 ◽  
Author(s):  
Peisen Sun ◽  
Haoming Wang ◽  
Guanglin Li
Keyword(s):  
Closed Loop ◽  
R Package ◽  
Circular Rna ◽  
Loop Structure ◽  
Biological Processes ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
User Friendly ◽  
Generation Sequencing

AbstractCircular RNA (circRNA), which has a closed-loop structure, is a kind of special endogenous RNA and plays important roles in many biological processes. With the improvement of next-generation sequencing technology and bioinformatics methods, some tools have been published for circRNA detection based on RNA-seq. However, only a few tools focus on downstream analyses, and they have poor visualization ability. Here, we developed the R package ‘Rcirc’ for further analyses of circRNA after its detection. Rcirc identifies the coding ability of circRNA and visualize various aspects of this feature. It also provides general visualization for both single circRNAs and meta-features of thousands of circRNAs. Rcirc was designed as a user-friendly tool that covers many highly automatic functions without running many complicated processes by users. It is available on GitHub (https://github.com/PSSUN/Rcirc) under the license GPL 3.0.

139 TRANSCRIPTOME ANALYSIS OF SINGLE BOVINE EMBRYOS BY RNA-Seq

2012 ◽  
Vol 24 (1) ◽  
pp. 182 ◽  
Author(s):  
P. J. Ross ◽  
J. L. Chitwood
Keyword(s):  
Ribosomal Proteins ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Regulation Of Transcription ◽  
Rna Seq ◽  
Specific Expression ◽  
Total Rna ◽  
Structure Information ◽  
Dnase Treatment

Transcriptome sequencing by high-throughput technologies provides global gene expression levels as well as gene structure information. Moreover, analysis of samples from single individuals allows the detection of allele-specific expression. We investigated the possibility of RNA-Seq analysis using single bovine blastocysts. Embryos were in vitro produced using abattoir-derived, in vitro- matured oocytes, TALP-based fertilization and KSOM embryo culture medium. On Day 7 of culture, 5 expanded blastocysts were collected and stored in RNA extraction buffer at –80°C. Total RNA was extracted from each individual embryo using the Arcturus PicoPure RNA isolation kit including DNAse treatment. Approximately 1.5 ng of high-quality total RNA was obtained per embryo. The RNA was amplified using the SPIA-based Ovation RNA-Seq kit (NuGen, San Carlos, CA, USA). After amplification, 6 μg of cDNA was obtained and directly used for library construction with the NuGen Encore NGS Library I kit. Libraries were submitted to the University of California Davis Genome Center for 40-bp single read sequencing on an Illumina GAIIx apparatus. Data analysis was performed using CLC Genomics Workbench. On average, 38 094 173 good-quality reads were produced from each sample. Removing 9 bp from the 5′ end of the sequences greatly improved read alignment. Mapping of trimmed reads to BTAU 4.0 allowing up to 2 mismatches per read resulted in 88.9 ± 0.3% of reads aligning to the genome. Using an SNP discovery algorithm, a total of 31 993 unique SNP were detected with an average of 12 530 ± 496 SNP per sample, 50% of which were heterozygous. Of the total, 45, 21, 14 and 9% were common to at least 2, 3, 4 and 5 samples, respectively. Allelic expression imbalance, defined as 75% of reads corresponding to one allele of a heterozygous SNP with coverage ≥50, was observed in 22% of SNP among those common to at least 2 samples. Mapping the reads to the transcriptome resulted in 71.8 ± 0.4% of reads aligning to genes present in Ensembl. Among those mapping to RefSeq transcripts, 64.4% corresponded to exons, 7.2% to exon-exon boundaries and 0.3% to exon-intron boundaries, with the remainder mapping to introns. An average of 9746 ± 122 genes with RPKM greater than 0.3 were detected in each sample, with 7982 genes expressed commonly among all 5 embryos. The correlation for RPKM between sample pairs was between 0.978 and 0.993. Genes known to be almost exclusively expressed by pre-implantation embryos were present, including OCT4, NANOG and CDX2 among others. Gene ontology analysis of gene groups, divided into quintiles by level of expression, indicated that the most highly expressed genes are enriched in ribosomal proteins and oxidative phosphorylation. Genes with medium-high levels of expression were enriched in structural components including organelles and the cytoskeleton. Genes at the medium level of expression represented nuclear and chromatin proteins, whereas medium-low and low were related to regulation of transcription and DNA metabolism. We conclude that RNA-Seq from a single bovine blastocyst is possible and represents a powerful tool for understanding the biology and pathologies of pre-implantation embryo development.

Whole Transcriptome Sequencing of a Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) with “Next Generation Sequencing” Technology Revealed Novel Point Mutations Associated with Disease-Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3074-3074
Author(s):  
Ilaria Iacobucci ◽  
Alberto Ferrarini ◽  
Marco Sazzini ◽  
Enrico Giacomelli ◽  
Annalisa Lonetti ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Next Generation Sequencing Technology ◽  
Whole Transcriptome ◽  
Generation Sequencing

Abstract Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.

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