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).
Fig.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.
