Full-length mRNA sequencing -PacBio
Service Advantages
● Direct read-out of full-length cDNA molecule from 3'- end to 5'- end
● Iso-form level resolution in sequence structure
● Transcripts with high accuracy and integrity
● Highly compatible to vaiours species
● Large sequencing capacity with 4 PacBio Sequel II sequencing platforms equipped
● Highly experienced with over 700 Pacbio-based RNA sequencing projects
● BMKCloud-based result delivery: Customized data-mining available on platform.
● After-sale services valid for 3 months upon project completion
Service Specifications
Platform: PacBio Sequel II
Sequencing library: Poly A- enriched mRNA library
Recommended data yield: 20 Gb/sample (Depending on species)
FLNC（%）： ≥75%
*FLNC: Full-length non-chimeric transcipts
Bioinformatics analyses
● Raw data processing
● Transcript identification
● Sequence structure
● Expression Quantification
● Function Annotation
Sample Requirements and Delivery
Sample Requirements:
Nucleotides:
|
Conc.(ng/μl) |
Amount (μg) |
Purity |
Integrity |
|
≥ 120 |
≥ 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≥8.0; 5.0≥ 28S/18S≥1.0; limited or no baseline elevation |
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
Microorganism: Mass ≥ 1 g
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
1.FLNC length distribution
Length of full-length non-chimeric read(FLNC) indicates length of cDNA in library construction. FLNC length distribution is a crucial indicator in evaluating quality of library construction.
FLNC read length distribution
2.Complete ORF region length distribution
We use TransDecoder to predict protein coding regions and corresponding amino acid sequences to generate unigene sets, which contains complete non-redundant transcript information in all samples.
Complete ORF region length distribution
3.KEGG pathway enrichment analys
Differentially expressed transcripts(DETs) can be identified by aligning NGS-based RNA sequencing data on full-length transcript sets generated by PacBio sequencing data. These DETs can further processed for various functional analysis, e.g. KEGG pathway enrichment analysis.
DET KEGG pathway enrichment -Dot plot
BMK Case
The developmental dynamics of the Populus stem transcriptome
Published: Plant Biotechnology Journal, 2019
Sequencing strategy:
Sample collection: stem regions: apex, first internode(IN1), second internode(IN2), third internode(IN3), internode(IN4) and internode(IN5) from Nanlin895
NGS-sequence: RNA of 15 individuals were pooled as one biological sample. Three biological replicates of each points were processed for NGS sequence
TGS-sequence: Stem regions were divided into three regions, i.e. apex, IN1-IN3 and IN4-IN5. Each region was processed for PacBio sequencing with four types of libraries: 0-1 kb, 1-2 kb, 2-3 kb and 3-10 kb.
Key results
1.A total of 87150 full-length transcripts were identified, in which, 2081 novel isoforms and 62058 novel alternative spliced isoforms were identified.
2.1187 lncRNA and 356 fusion genes were identified.
3.From primary growth to secondary growth, 15838 differentially expressed transcripts from 995 differentially expressed genes were identified. In all DEGs, 1216 were transcription factors, most of which has not yet been reported.
4.GO enrichment analysis revealed importance of cell division and oxidation-reduction process in primary and secondary growth.
Alternative splicing events and different isoforms
WGCNA analysis on transcription factors
Reference
Chao Q, Gao ZF, Zhang D, et al. The developmental dynamics of the Populus stem transcriptome. Plant Biotechnol J. 2019;17(1):206-219. doi:10.1111/pbi.12958
