Full-Length mRNA Sequencing-Nanopore
Service Advantages
● Low sequence bias
● Revealing full-length cDNA molecules
● Less data required to cover same number of transcripts
● Identification of multiple isoforms per gene
● Expression quantification in isoform level
Service Specifications
|
Library |
Platform |
Recommended data yield (Gb) |
Quality Control |
|
cDNA-PCR(Poly-A enriched) |
Nanopore PromethION P48 |
6 Gb/sample (Depending on species) |
Full-length ratio＞70% Average quality score: Q10
|
Bioinformatics analyses
● Raw data processing
● Transcript identification
● Alternative splicing
● Expression quantification in gene level and isoform level
● Differential expression analysis
● Function annotation and enrichment (DEGs and DETs)
Sample Requirements and Delivery
Sample Requirements:
Nucleotides:
|
Conc.(ng/μl) |
Amount (μg) |
Purity |
Integrity |
|
≥ 100 |
≥ 1.2 |
OD260/280=1.7-2.5 OD260/230=0.5-2.5 Limited or no protein or DNA contamination shown on gel. |
For plants: RIN≥7.0; For animals: RIN≥7.5; 5.0≥28S/18S≥1.0; limited or no baseline elevation |
Tissue: Weight(dry): ≥1 g
*For tissue smaller than 5 mg, we recommend to send flash frozen(in liquid nitrogen) tissue sample.
Cell suspension: Cell count = 3×106 - 1×107
*We recommend to ship frozen cell lysate. In case that cell counts smaller than 5×105, flash frozen in liquid nitrogen is recommended, which is preferable for micro extraction.
Blood samples: Volume≥1 ml
Recommended Sample Delivery
Container: 2 ml centrifuge tube (Tin foil is not recommended)
Sample labeling: Group+replicate e.g. A1, A2, A3; B1, B2, B3... ...
Shipment: 2、Dry-ice: Samples need to be packed in bags and buried in dry-ice.
- RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.
Service Work Flow
Nucleotides:
Sample delivery
Library construction
Sequencing
Data analysis
After-sale services
Service Work Flow
Tissue:
Experiment design
Sample delivery
RNA extraction
Library construction
Sequencing
Data analysis
After-sale services
1.Differential expression analysis -Volcano plot
Differential expression analysis can be processed in both gene level to identify differentially expressed genes (DEGs) and in isoform level to identify differentially
expressed transcripts (DETs)
2.Hierarchical clustering heatmap
3.Alternative splicing identification and classification
Five types of alternative splicing events can be predicted by Astalavista.
4.Alternative poly-adenylation (APA) events identification and Motif at 50 bp upstream of poly-A
BMK Case
Alternative splicing identification and isoform-level quantification by nanopore full-length transcriptome sequencing
Published: Nature Communications,2020
Sequencing strategy:
Grouping: 1. CLL-SF3B1(WT); 2. CLL-SF3B1(K700E mutation); 3. Normal B-cells
Sequencing strategy: MinION 2D library sequencing, PromethION 1D library sequencing; short-read data from same samples
Sequencing platform: Nanopore MinION; Nanopore PromethION;
Key results
1.Isoform-level Alternative Splicing Identification
Long-read sequences empowers identification of mutant SF3B1K700E -altered splice sites at isoform-level. 35 alternative 3′SSs and 10 alternative 5′SSs were found to be significantly differentially spliced between SF3B1K700E and SF3B1WT. 33 of the 35 alterations were newly discovered by long-read sequences.
2.Isoform-level Alternative Splicing quantification
Expression of intron retention(IR) isoforms in SF3B1K700E and SF3B1WT were quantified based on nanopore sequences, revealing a global down-regulation of IR isoforms in SF3B1K700E .
Reference
Tang A D , Soulette C M , Baren M J V , et al. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns[J]. Nature Communications.
