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Full-Length mRNA Sequencing-Nanopore

While NGS-based mRNA sequencing serves as a versatile tool for gene expression quantification, its reliance on short reads restricts its efficacy in complex transcriptomic analyses. Nanopore sequencing, on the other hand, employs long-read technology, enabling the sequencing of full-length mRNA transcripts. This approach facilitates a comprehensive exploration of alternative splicing, gene fusions, poly-adenylation, and the quantification of mRNA isoforms.

Nanopore sequencing relies on nanopore single-molecule real-time electrical signals. Guided by motor proteins, double-stranded DNA binds to nanopore proteins embedded in a biofilm, unwinding as it passes through the nanopore channel under a voltage difference. The distinctive electrical signals generated by different bases on the DNA strand are detected and classified in real-time, facilitating accurate and continuous nucleotide sequencing. This innovative approach overcomes short-read limitations and provides a dynamic platform for intricate genomic analysis, including complex transcriptomic studies.

Platform: Nanopore Promethion P48


Service Details

Bioinformatics

Demo Results

Featured Publications

Features

●  Capture of poly-A mRNA followed by cDNA synthesis and library preparation

●  Sequencing of the full-length transcripts

●  Bioinformatic analysis based on alignment to a reference genome

●  Bioinformatic analysis includes not only expression at gene and isoform-level but also analysis of lncRNA, gene fusions, poly-adenylation and gene structure

Service Advantages

 ● Quantification of expression at the isoform level: enabling detailed and accurate expression analysis, unveiling change that may be masked when analysing the whole gene expression

 ● Reduced Data Demands: In comparison to Next-Generation Sequencing (NGS), Nanopore sequencing exhibits lower data requirements, allowing for equivalent levels of gene expression quantification saturation with smaller amounts of data.

 ● Higher accuracy of expression quantification: both at gene and isoform level

 ● Identification of additional transcriptomic information: alternative polyadenylation, fusion genes and lcnRNA and their target genes

 ● Extensive Expertise: with a track record of completing over 550 Nanopore full-length transcriptome projects and processing over 7700 samples, our team brings a wealth of experience to every project.

 ● Post-Sales Support: our commitment extends beyond project completion with a 3-month after-sale service period. During this time, we offer project follow-up, troubleshooting assistance, and Q&A sessions to address any queries related to the results.

Sample Requirements and Delivery

Library

Sequencing strategy

Data recommended

Quality Control

Poly A enriched

Illumina PE150

6 Gb

Average quality score: Q10

 

Sample Requirements:

Nucleotides:

Conc.(ng/μl)

 Amount (μg)

Purity

 Integrity

≥ 100

≥ 0.6

      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≥7.5;

For animals: RIN≥7.0;

5.0≥28S/18S≥1.0;

limited or no baseline elevation

● Plants:

   Root, Stem or Petal: 450 mg

   Leaf or Seed: 300 mg

   Fruit: 1.2 g

● Animal:

   HEart or Intestine: 300 mg

   Viscera or Brain: 240 mg

   Muscle: 450 mg

   Bones, Hair or Skin: 1g

● Arthropods:

   Insects: 6g

   Crustacea: 300 mg

● Whole blood: 1 tube

● Cells: 106 cells

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.

2. 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

Sample delivery

Library Preparation

Library construction

Sequencing

Sequencing

Data analysis

Data analysis

After sale Services

After-sale services

Service Work Flow

Tissue:

Sample QC

Experiment design

sample delivery

Sample delivery

Pilot experiment

RNA extraction

Library Preparation

Library construction

Sequencing

Sequencing

Data analysis

Data analysis

After sale Services

After-sale services


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    ●  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)

     

    Alternative splicing analysis

    图片20 Alternative Polyadenylation Analysis (APA)

     图片21

     

    lncRNA prediction

     图片22

     

    Annotation of novel genes

     图片23

     

     

     Clustering of DETs

     图片24

     

     

    Protein-Protein Networks in DEGs

     图片25 

    Explore the advancements facilitated by BMKGene’s Nanopore full-length mRNA sequencing services through a curated collection of publications.

     

    Gong, B. et al. (2023) ‘Epigenetic and transcriptional activation of the secretory kinase FAM20C as an oncogene in glioma’, Journal of Genetics and Genomics, 50(6), pp. 422–433. doi: 10.1016/J.JGG.2023.01.008.

    He, Z. et al. (2023) ‘Full-length transcriptome sequencing of lymphocytes respond to IFN-γ reveals a Th1-skewed immune response in flounder (Paralichthys olivaceus)’, Fish & Shellfish Immunology, 134, p. 108636. doi: 10.1016/J.FSI.2023.108636.

    Ma, Y. et al. (2023) ‘Comparative analysis of PacBio and ONT RNA sequencing methods for Nemopilema Nomurai venom identification’, Genomics, 115(6), p. 110709. doi: 10.1016/J.YGENO.2023.110709.

    Yu, D. et al. (2023) ‘Nano-seq analysis reveals different functional tendency between exosomes and microvesicles derived from hUMSC’, Stem Cell Research and Therapy, 14(1), pp. 1–13. doi: 10.1186/S13287-023-03491-5/TABLES/6.

     
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