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Nanopore Direct RNA Sequencing Analysis

Nanopore Direct RNA Sequencing Analysis Online Inquiry

CD Genomics is a bioinformatics data analysis provider. Our team is experienced in Nanopore direct RNA sequencing and our high-quality data analysis platform will be used to generate high-quality analysis results in a fast analysis cycle.

Introduction

Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. This approach has many potential advantages over other RNA-seq strategies, and it allows for direct detection of base modifications such as methylation, discovery and characterization of poly(A )RNA molecules and the study of splice variants (Garalde et al., 2018).

Direct RNA-seqFig.1 Direct RNA-seq (Garalde et al., 2018).

Application Field

  • Cancer Research
  • Vaccine and Therapeutic Drug Development
  • Clinical Research

Bioinformatics Analysis Content

  • Clean Data Quality Control
  • Mapping to Reference Genome
  • Quantification of transcript expression
  • The transcriptional structure of genes analysis
  • Differential gene/transcription isoform quantification
  • Functional Annotation, Enrichment Analysis and Protein Interaction Network
  • Methylation Analysis
    Methylation site identification
    Methylation distribution analysis
    Methylation motif analysis
    Methylated gene region annotation
    Analysis of differentially methylated sites (m5C/ m6A)
  • Poly(A) tail length estimation
    Poly(A) length analysis
    Differential Poly(A) length analysis
    Correlation analysis of Poly(A) length and transcript expression

How It Works

CD Genomics is a professional bioinformatics service provider with years of experience in NGS and long read sequencing (Oxford Nanopore platforms) data analysis, integrated analysis services, database construction and other bioinformatics solutions.

Table 1 Partial software and database list

Software or database Versions Uses Link
NanoFilt 2.8.0 TGS data filtering https://github.com/wdecoster/nanofilt
minimap2 2.17 Mapping https://github.com/lh3/minimap2
samtools 1.11 Sorting https://github.com/samtools/samtools
seqkit 0.12.0 FASTA/Q tool https://github.com/shenwei356/seqkit
StringTie 2.1.4 Reconstruct transcripts http://ccb.jhu.edu/software/stringtie/
gffcompare 0.12.1 Discovery of new transcripts http://ccb.jhu.edu/software/stringtie/gffcompare.shtml
SUPPA2 2.3 Variable splicing analysis https://github.com/comprna/SUPPA/
Tapas 2018.5.26 APA analysis https://github.com/arefeen/TAPAS
Nanopollish 0.13.2 Poly(A) tail analysis https://github.com/jts/nanopolish
Tombo 1.5.1 Methylation m5C analysis https://github.com/nanoporetech/tombo

1. What is the minimum starting amount of RNA required for Direct RNA library construction?

Quality qualified total RNA 40-80ug, concentration ≥180 ng/μL.

2. What is the approximate yield of Direct RNA per cell?

Because there is no PCR amplification process for Direct RNA library construction and sequencing, the amount of full-length transcriptome data is relatively low compared with PCR cDNA, and the amount of high-quality total RNA is not less than 1Gb.

References

  1. Garalde, D. R., Snell, E. A., Jachimowicz, D., Sipos, B., Lloyd, J. H., Bruce, M., . . . Turner, D. J. (2018). Highly parallel direct RNA sequencing on an array of nanopores. Nat Methods, 15(3), 201-206. doi:10.1038/nmeth.4577
  2. Workman, R. E., Tang, A. D., Tang, P. S., Jain, M., Tyson, J. R., Razaghi, R., . . . Timp, W. (2019). Nanopore native RNA sequencing of a human poly(A) transcriptome. Nat Methods, 16(12), 1297-1305. doi:10.1038/s41592-019-0617-2
* For Research Use Only. Not for use in diagnostic procedures.
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