Whole genome sequencing based on short and long reads from GM09237 cell line with and without folate depletion
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ABSTRACT:
PROVIDER: EGAS00001005345 | EGA |
REPOSITORIES: EGA
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Project description:Two DNA samples extracted from GM09237 cell line cultured with either normal medium or medium with no folic acid for 5 days were sequenced using the BGISEQ500 platform (BGI whole genome 100 bp paired-end sequencing 60x) as well as PacBio Sequel long read sequencing.
| EGAD00001007732 | EGA
Project description:Purpose: The aim of this study is to compare different long-read sequencing platforms using reference lung adenocarcinoma cell lines and spike-in controls. Methods - Cell Culture: Lung adenocarcinoma cell lines NCI-H1975 and HCC827 from a range of passages (2-4) were grown on 3 separate occasions in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin. Methods - RNA preparation: mRNA was extracted using a Qiagen RNA miniprep kit and purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490). Purified mRNA spiked with sequins was used for Next Generation Sequencing library preparation using the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) and the cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit (ONT). Completed libraries were sequenced on NextSeq 500 (Illumina) and PromethION (ONT). Iso-Seq libraries were prepared and sequenced by Novogene on Sequel II (PacBio). Reads were mapped to known genomic features of the GRCH38 reference genome and RNA sequin decoy chromosome combined sequences at the gene-level and single reads were then summarized into gene-level counts using featureCounts software (Liao et al. 2014).
2022-07-20 | GSE172421 | GEO
Project description:Summary taken from our paper “Aspergillus terreus Sectorization: A Morphological Phenomenon Shedding Light on Amphotericin B Resistance Mechanism”: WT and ATSec strains were grown in AMM broth for 30 h at 30 °C. Afterwards, the cultures were subjected to either DMSO (control) or 1 µg/ml AmB treatment for 1 h/4 h. Triplicates of each combination of these factors were made, resulting in a total of 24 samples. RNA was extracted with a Promega GmbH kit (Madison, Wisconsin, USA) following the manufacturer’s instructions. The RNA-seq experiment was conducted by BGI, generating a total of around 20 million 100bp paired-end reads with the DNBSEQ platform. BGI employed SOAPnuke (51) to remove rRNA, adaptor-containing sequences, low quality reads, and reads with high unknown nucleotide content, producing "clean reads" datasets that were used in the analysis. On average, clean read datasets contain 45.1 Mb of reads, while the raw read datasets contain 48.7 Mb. For clean reads, the mean Q20 value is 98.3%, and the mean Q30 value is 92.5%.
2024-09-25 | GSE277670 | GEO
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:Single-cell RNA sequencing analysis has recently provided snapshots of gene expression of specific cell types and enabled cell types classification within an heterogenous population. As well as transcriptional changes, alternative splicing events and modifications of components of splicing machinery actively contributes in shaping cellular phenotype as well as ageing process and diseases occurence. Current high-throughput single-cell RNA sequencing methods may lack information on cell-specific isoform expression, missing key aspects of cell biology. In the present work we introduce a novel approach using the 10X Genomics Chromium to generate short-read (Illumina) and long-read (Pacific Biosciences Sequel II) RNA-sequencing libraries from the same single cells. This approach produced single cell parallel transcriptional and splicing profiling that demonstrates for the first time cell-type specific isoform expression and alterations at transcriptional levels associated with ageing in haematopoietic stem and progenitor cells
2022-12-31 | GSE166709 | GEO
Project description:We report the results of performing RNA sequencing using RNA extracted from TC28a2 cells grown in the presence of hypertrophic differentiation medium compared to RNA extracted from TC28a2 cells grown in growth medium. For preparation of samples for RNA sequencing, total RNAs were extracted from TC28a2 cells treated with growth medium (n=2) or hypertrophic medium (n=3) at day 5 of differentiation. RNA was extracted using Trizol (Invitrogen) following the protocols provided by the manufacturer. Isolated RNAs were submitted to the Macrogen Inc. (Macrogen Inc., Seoul, South Korea) for total RNA-sequencing. The overall quality of the extracted total RNAs were validated using spectrophotometry. To remove low quality and adapter sequence, the raw was read by the sequencer before analysis and align the processed reads to the Homo sapiens using HISAT2 v2.1.059. The reference genome sequence of Homo sapiens (hg19) and annotation data were downloaded from the NCBI, and transcript assembly of known transcripts was then processed by StringTie v2.1. Base on the result of that, expression abundance of transcript and gene were calculated as read count or FPKM value (Fragments Per Kilobase of exon per Million fragments mapped) per sample. The expression profiles are used to do additional analysis such as DEG (Differentially Expressed Genes). The relative abundances of gene were measured in read count using StringTie. Genes with one more than zeroed read count values in the samples were excluded. Filtered data were log2-transformed and subjected to RLE Normalization. Statistical significance of the differential expression data was determined using DESeq2 nbinomWaldTest and fold change in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm. For DEG set, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. Enrichment of gene ontology analysis was performed for DEGs using g:Profiler and KEGG pathway analysis was tested based on KEGG pathway (https://www.genome.jp/kegg/) database. We used multidimensional scaling (MDS) method to visualize the similarities among samples. We applied to the Euclidean distance as the measure of the dissimilarity. Hierarchical clustering analysis also was performed using complete linkage and Euclidean distance as a measure of similarity to display the expression patterns of differentially expressed transcripts which are satisfied with fold change ≥2 and raw p <0.05. All data analysis and visualization of differentially expressed genes was conducted using R 3.6.1(www.r-project.org).
2022-04-03 | GSE199847 | GEO
Project description:LocusMasterTE: long-read assisted short-read TE quantification [short]
| PRJNA935371 | ENA
Project description:DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in genome and transcriptome sequencing of various organisms. Nevertheless, challenges such as accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis between the Element Biosciences AVITI platform and its counterpart the Illumina NextSeq platform, testing DNA and RNA short-read sequencing, as well as synthetic long-read sequencing for single cells and RNA transcripts.
2024-08-19 | GSE246832 | GEO
Project description:DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in genome and transcriptome sequencing of various organisms. Nevertheless, challenges such as accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis between the Element Biosciences AVITI platform and its counterpart the Illumina NextSeq platform, testing DNA and RNA short-read sequencing, as well as synthetic long-read sequencing for single cells and RNA transcripts.
2024-08-19 | GSE246830 | GEO
Project description:DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in genome and transcriptome sequencing of various organisms. Nevertheless, challenges such as accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Here, we conduct a comparative analysis between the Element Biosciences AVITI platform and its counterpart the Illumina NextSeq platform, testing DNA and RNA short-read sequencing, as well as synthetic long-read sequencing for single cells and RNA transcripts.
2024-08-19 | GSE269801 | GEO