PacBio 2+3 Full-Length mRNA Solution
Features
● Study design:
Pooled sample sequenced with PacBio to identify transcript isoforms
Separate samples (replicates and conditions to be tested) sequenced with NGS to quantify transcript expression
● PacBio 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 includes not only expression at gene and isoform-level but also analysis of lncRNA, gene fusions, poly-adenylation, and gene structure
Advantages
● High Accuracy: HiFi reads with accuracy >99.9% (Q30), comparable to NGS
● Alternative Splicing Analysis: Sequencing all transcripts enables isoform identification and characterization.
● Combination of PacBio and NGS Strengths: It enables quantification of expression at the isoform level, unveiling change that may be masked when analysing the whole gene expression
● Extensive Expertise: We have processed over 2300 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 PacBio Revio |
20/40 Gb 5/10 M CCS |
Q30≥85% |
|
Poly A enriched |
Illumina PE150 |
6-10 Gb |
Q30≥85% |
Nucleotides
|
|
Conc.(ng/μl) |
Amount (μg) |
Purity |
Integrity |
|
Illumina Library |
≥ 10 |
≥ 0.2 |
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≥4.0; For animals: RIN≥4.5; 5.0≥28S/18S≥1.0; limited or no baseline elevation |
|
PacBio library |
≥ 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. |
Plants: RIN≥7.5 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.
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)
- Differentially Expressed Transcripts (DETs) analysis
- Differentially Expressed Genes (DEGs) analysis
- Functional annotation of DEGs and DETs
BUSCO analysis
Alternative Splicing Analysis
Alternative Polyadenylation Analysis (APA)
Differentially Expressed Genes (DEGs) and Transcripts (DETs9 anlaysis
Protein-Protein interaction networks of DETs and DEGs
Explore the advancements facilitated by BMKGene’s PacBio 2+3 full-length mRNA sequencing through a curated collection of publications.
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/FIGURES/7.
