Scholarlink

Background <p>Transcriptome sequencing (RNA-seq) is a powerful technology for gene expression profiling. Selection of optimal parameters for cDNA library generation is crucial for acquisition of high-quality data. In this study, we investigate the impact of the amount of RNA and the number of PCR cycles used for sample amplification on the rate of PCR duplication and, in consequence, on the RNA-seq data quality.</p> Results <p>For broader applicability, we sequenced the data on four short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. The native Illumina libraries were converted for sequencing on AVITI and G4 to assess the effect of library conversion, containing additional PCR cycles. We find that the rate of PCR duplicates depends on the combined effect of RNA input material and the number of PCR cycles used for amplification. For input amounts lower than 125&#xa0;ng, 34–96% of reads were discarded via deduplication with the percentage increasing with lower input amount and decreasing with increasing PCR cycles. The reduced read diversity for low input amounts leads to fewer genes detected and increased noise in expression counts.</p> Conclusions <p>Data generated with each of the four sequencing platforms presents similar associations between starting material amount and the number of PCR cycles on PCR duplicates, a similar number of detected genes, and comparable gene expression profiles.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年5月28日

查看全文
  • Natalia Zajac,
  • Ioannis S. Vlachos,
  • Sija Sajibu,
  • Lennart Opitz,
  • Shuoshuo Wang,
  • Sridar V. Chittur,
  • Christopher E. Mason,
  • Kevin L. Knudtson,
  • John M. Ashton,
  • Hubert Rehrauer,
  • Catharine Aquino

摘要

Background

Transcriptome sequencing (RNA-seq) is a powerful technology for gene expression profiling. Selection of optimal parameters for cDNA library generation is crucial for acquisition of high-quality data. In this study, we investigate the impact of the amount of RNA and the number of PCR cycles used for sample amplification on the rate of PCR duplication and, in consequence, on the RNA-seq data quality.

Results

For broader applicability, we sequenced the data on four short-read sequencing platforms: Illumina NovaSeq 6000, Illumina NovaSeq X, Element Biosciences AVITI, and Singular Genomics G4. The native Illumina libraries were converted for sequencing on AVITI and G4 to assess the effect of library conversion, containing additional PCR cycles. We find that the rate of PCR duplicates depends on the combined effect of RNA input material and the number of PCR cycles used for amplification. For input amounts lower than 125 ng, 34–96% of reads were discarded via deduplication with the percentage increasing with lower input amount and decreasing with increasing PCR cycles. The reduced read diversity for low input amounts leads to fewer genes detected and increased noise in expression counts.

Conclusions

Data generated with each of the four sequencing platforms presents similar associations between starting material amount and the number of PCR cycles on PCR duplicates, a similar number of detected genes, and comparable gene expression profiles.