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Full-length mRNA sequencing -PacBio

While NGS-based mRNA sequencing is a versatile tool for quantifying gene expression, its reliance on short reads restricts its use in complex transcriptomic analyses. PacBio sequencing (Iso-Seq), 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 and poly-adenylation although it is not the primary choice for gene expression quantification, due to the high amount of data required.
PacBio sequencing technology relies on single-molecule, real-time (SMRT) sequencing, providing a distinct advantage in capturing full-length mRNA transcripts. This innovative approach involves the use of zero-mode waveguides (ZMWs), microfabricated wells that enable the real-time observation of DNA polymerase activity during sequencing. Within these ZMWs, PacBio’s DNA polymerase synthesizes a complementary strand of DNA, generating long reads that span the entirety of mRNA transcripts. PacBio operation in Circular Consensus sequencing (CCS) mode enhances accuracy by repeatedly sequencing the same molecule. The generated HiFi reads have an accuracy comparable to NGS, further contributing to a comprehensive and reliable analysis of complex transcriptomic features.

Platform: PacBio Sequel II

Service Details

Bioinformatics

Demo Results

Featured Publications

Features

● cDNA synthesis from poly-A mRNA followed by library preparation

● Sequencing in CCS mode, generating HiFi reads

● Sequencing of the full-length transcripts

● Analysis does not necessitate a reference genome; however, it may be employed

● Bioinformatic analysis enables analysis of transcripts isoform lncRNA, gene fusions, poly-adenylation and gene structure

Service Advantages

● High accuracy: HiFi reads with accuracy >99.9% (Q30), comparable to NGS

● Alternative splicing analysis: sequencing of the entire transcripts enables isoform identification and characterization

● Extensive Expertise: with a track record of completing over 450 PacBio full-length transcriptome projects and processing over 700 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
PolyA enriched mRNA CCS library	PacBio Sequel II	20 Gb	Q30≥85%

Sample Requirements:

Nucleotides:

● 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

Conc.(ng/μl)

Amount (μg)

Purity

Integrity

≥ 100

≥ 0.75

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≥8.0;

5.0≥ 28S/18S≥1.0;

limited or no baseline elevation

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

Experiment design

Sample delivery

RNA extraction

Library construction

Sequencing

Data analysis

After-sale services

Previous: Eukaryotic mRNA Sequencing-Illumina

Next: Full-Length mRNA Sequencing-Nanopore

Includes the following analysis:

● Raw data quality control

● Alternative Polyadenylation Analysis (APA)

● Fusion transcript analysis

● Alternative Splicing Analysis

● Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis

● Novel transcript analysis: prediction of coding sequences (CDS) and functional annotation

● lncRNA analysis: prediction of lncRNA and targets

● MicroSatelite Identification (SSR)

BUSCO analysis

Alternative Splicing Analysis

Alternative Polyadenylation Analysis (APA)

Functional annotation of novel transcripts

Explore the advancements facilitated by BMKGene’s Nanopore full-length mRNA sequencing services in this featured publication.

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.

Chao, Q. et al. (2019) ‘The developmental dynamics of the Populus stem transcriptome’, Plant Biotechnology Journal, 17(1), pp. 206–219. doi: 10.1111/PBI.12958.

Deng, H. et al. (2022) ‘Dynamic Changes in Ascorbic Acid Content during Fruit Development and Ripening of Actinidia latifolia (an Ascorbate-Rich Fruit Crop) and the Associated Molecular Mechanisms’, International Journal of Molecular Sciences, 23(10), p. 5808. doi: 10.3390/IJMS23105808/S1.

Hua, X. et al. (2022) ‘Effective prediction of biosynthetic pathway genes involved in bioactive polyphyllins in Paris polyphylla’, Communications Biology 2022 5:1, 5(1), pp. 1–10. doi: 10.1038/s42003-022-03000-z.

Liu, M. et al. (2023) ‘Combined PacBio Iso-Seq and Illumina RNA-Seq Analysis of the Tuta absoluta (Meyrick) Transcriptome and Cytochrome P450 Genes’, Insects, 14(4), p. 363. doi: 10.3390/INSECTS14040363/S1.

Wang, Lijun et al. (2019) ‘A survey of transcriptome complexity using PacBio single-molecule real-time analysis combined with Illumina RNA sequencing for a better understanding of ricinoleic acid biosynthesis in Ricinus communis’, BMC Genomics, 20(1), pp. 1–17. doi: 10.1186/S12864-019-5832-9.

Spatially Resolved Transcriptomics