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Full-length mRNA sequencing-Nanopore

RNA sequencing has been an invaluable tool for comprehensive transcriptome analysis. Doubtlessly, traditional short-read sequencing achieved numerous important development in here. Nevertheless, it often encounters limitations in full-length isoform identifications, quantification, PCR bias.

Nanopore sequencing distinguishes itself from other sequencing platforms, in that the nucleotides are read directly without DNA synthesis and generates long read at tens of kilobases. This empowers direct read-out crossing full-length transcripts and tackling the challenges in isoform-level studies.

PlatformNanopore PromethION

Library: cDNA-PCR


Service Details

Demo Results

Case Study

Service Advantages

Ø  Low sequence bias

Ø Revealing full-length cDNA molecules

Ø Less data required to cover same number of transcripts

Ø Identification of multiple isoforms per gene

Ø Expression quantification in isoform level

Service Specifications

Library

Platform

Recommended data yield (Gb)

Quality Control

cDNA-PCR(Poly-A enriched)

Nanopore PromethION P48

 4 Gb/sample (Depending on species)

Full-length ratio>70%

Average quality score: Q10

 

Bioinformatics analyses

ü Raw data processing

ü Transcript identification

ü Alternative splicing

ü Expression quantification in gene level and isoform level

ü Differential expression analysis

ü Function annotation and enrichment (DEGs and DETs)

 

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Sample Requirements and Delivery

Sample Requirements:

Nucleotides:   

Purity Integrity Amount
OD260/280≥1.8;OD260/230≥1.0;Clear peak at 260 nmLimited or no protein or DNA contamination shown on gel. For plants: RIN≥7.5;For animals: RIN≥8;28S/18S≥1.0;limited or no baseline elevation Conc. ≥40 ng/μl;Total ≥ 1 μg

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: 1. Dry-ice: Samples need to be packed in bags and buried in dry-ice.

  1. RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.

 

Service Work Flow

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Sample delivery

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Library construction

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Sequencing

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Data analysis

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After-sale services

Service Work Flow

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Experiment design

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Sample delivery

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RNA extraction

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Library construction

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Sequencing

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Data analysis

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After-sale services


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  • 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 

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    expressed transcripts (DETs) 

    2.Hierarchical clustering heatmap

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    3.Alternative splicing identification and classification

    Five types of alternative splicing events can be predicted by Astalavista.

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    4.Alternative poly-adenylation (APA) events identification and Motif at 50 bp upstream of poly-A

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    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.

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