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Set4: ERCC bulk Loop-seq long-read RNA-seq by Illumina NovaSeq platform

ABSTRACT: 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.

ORGANISM(S): synthetic construct

PROVIDER: GSE246835 | GEO | 2024/08/19

REPOSITORIES: GEO

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Set2: ERCC bulk short-read RNA-seq by Illumina NextSeq platform

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.

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Set3: ERCC bulk Loop-seq long-read RNA-seq by Element Biosciences AVITI platform

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The impact of PCR duplication on RNAseq data generated using NovaSeq 6000, NovaSeq X, AVITI and G4 sequencers

Project description:Gene expression profiles generated with RNA sequencing can be biased by RNA amount and methods utilised for cDNA library generation. Polymerase chain reaction (PCR) amplification can generate high PCR duplicate proportions and introduce bias in transcript counts. In this study, we investigate the impact of input amount and PCR cycle number on the PCR duplication rate and on the RNA-seq data quality. We used a range of inputs (1 ng - 1,000 ng) and assessed the PCR duplication rate using unique molecular identifiers (UMIs). For broader applicability, we sequenced the data on four different short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. We highlight the limitations of using input amounts below 125 ng and the advantages for using UMIs for deduplication. We contrast the data obtained from different sequencers and discuss the benefits and drawbacks of using Illumina library conversion kits.

2024-03-13 | GSE261432 | GEO

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Multi-omic profiling of pathogen-stimulated primary immune cells

Project description:Objectives: To perform long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors. We aim to discover new transcripts and protein isoforms expressed during immune responses to diverse pathogens. Methods: PBMCs were exposed to four microbial stimuli for 24 hours: the TLR4 ligand lipopolysaccharide (LPS), the TLR3 ligand Poly(I:C), heat-inactivated Staphylococcus aureus, Candida albicans, and RPMI medium as negative controls. Long-read sequencing (PacBio) of one donor and secretome proteomics and short-read sequencing of five donors were performed. IsoQuant was used for transcriptome construction, Metamorpheus/FlashLFQ for proteome analysis, and Illumina short-read 3’-end mRNA sequencing for transcript quantification. Results: Long-read transcriptome profiling reveals the expression of novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. We observe widespread loss of intron retention as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. In general, RNA expression differences did not result in differences in the amounts of secreted proteins. Interindividual differences in the proteome were larger than the differences between stimulated and unstimulated PBMCs. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Conclusion: Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.

2023-09-16 | PXD045237 | Pride

Comparison of methylome data generated with HiSeq X Ten and NovaSeq

Project description:To compare the difference of sequencing platform, a sequencing library was served for sequencing on HiSeq X Ten and NovaSeq 6000.

2019-03-01 | GSE127569 | GEO

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