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Full-Length mRNA Sequencing-Nanopore

While NGS-based mRNA sequencing is a versatile tool for quantifying gene expression, its reliance on short reads restricts its efficacy in complex transcriptomic analyses. On the other hand, nanopore sequencing 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, a method that relies on nanopore single-molecule real-time electrical signals, provides results in real-time. 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, with immediate results.

Platform: Nanopore PromethION 48

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: Compared to Next-Generation Sequencing (NGS), Nanopore sequencing exhibits lower data requirements, allowing for equivalent levels of gene expression quantification saturation with smaller 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: Our team brings a wealth of experience to every project, having completed over 850 Nanopore full-length transcriptome projects and processed over 8,000 samples.

● 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	Nanopore PromethION 48	6/12 Gb	Average quality score: Q10

Sample Requirements:

Nucleotides:

Conc.(ng/μl)

Amount (μg)

Purity

Integrity

≥ 100

≥ 1.0

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

● 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: 10⁶cells

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

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

Previous: Full-length mRNA sequencing -PacBio

Next: 10x Genomics Visium Spatial Transcriptome

● 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 Alternative Polyadenylation Analysis (APA)

lncRNA prediction

Annotation of novel genes

Clustering of DETs

Protein-Protein Networks in DEGs

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.

Spatially Resolved Transcriptomics