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

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

PROVIDER: PRJNA1086933 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28314000.fastq.gz	Fastqsanger.gz
	SRR28314100.fastq.gz	Fastqsanger.gz
	SRR28314001.fastq.gz	Fastqsanger.gz
	SRR28314101.fastq.gz	Fastqsanger.gz
	SRR28314002.fastq.gz	Fastqsanger.gz

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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.

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Project description:BackgroundGenoLab M is a recently established next-generation sequencing platform from GeneMind Biosciences. Presently, Illumina sequencers are the globally leading sequencing platform in the next-generation sequencing market. Here, we present the first report to compare the transcriptome and LncRNA sequencing data of the GenoLab M sequencer to NovaSeq 6000 platform in various types of analysis.ResultsWe tested 16 libraries in three species using various library kits from different companies. We compared the data quality, genes expression, alternatively spliced (AS) events, single nucleotide polymorphism (SNP), and insertions-deletions (InDel) between two sequencing platforms. The data suggested that platforms have comparable sensitivity and accuracy in terms of quantification of gene expression levels with technical compatibility.ConclusionsGenolab M is a promising next-generation sequencing platform for transcriptomics and LncRNA studies with high performance at low costs.

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