Sample Submission Guidelines
Why Poly(A) and TAIL Iso-seq Matter
Poly(A) tails are more than simple RNA extensions. They act as key regulators of mRNA lifespan, stability, and translation. Standard RNA-seq methods often discard or overlook this information, missing valuable regulatory signals.
TAIL Iso-seq addresses this gap. It captures full-length cDNA, including intact poly(A) tails, and provides a direct view of poly(A) length, heterogeneity, and associated APA events. Researchers can link 3'UTR changes to miRNA binding, identify isoform-specific APA usage, and explore how tail length affects gene expression.
This makes TAIL Iso-seq a valuable tool for:
- Investigating developmental RNA regulation
- Studying stress responses in plants
- Exploring RNA stability in cancer models
- Supporting RNA therapeutic and vaccine development
Advantages of TAIL Iso-seq with CD Genomics
Full-length precision
TAIL Iso-seq captures the complete transcript, including 5'UTR, coding region, 3'UTR, and poly(A) tail. This eliminates assembly errors common in short-read RNA-seq and provides isoform-level resolution.
Comprehensive poly(A) profiling
Our service quantifies poly(A) tail length distributions across the transcriptome and detects non-A residues (U, G, C) that affect RNA stability and translation efficiency. This allows researchers to study RNA dynamics with unmatched detail.
APA and 3'UTR analysis
TAIL Iso-seq identifies alternative polyadenylation (APA) sites and characterises 3'UTR length changes. This helps link APA to changes in miRNA binding, RNA localisation, and post-transcriptional regulation.
Rich bioinformatics support
We deliver publication-ready reports with tail length distributions, APA maps, isoform catalogues, and pathway enrichment analyses. Visual outputs simplify interpretation and can be integrated into multi-omics projects.
Technology Workflow
CD Genomics provides a complete TAIL Iso-seq workflow, from RNA extraction to bioinformatics reporting. Each step is optimised for accuracy and reproducibility, ensuring reliable insights into poly(A) regulation and isoform diversity.
RNA QC: Input RNA is assessed for integrity (RIN ≥7) and purity.
Full-length cDNA capture: Protocols preserve intact poly(A) tails and barcodes can be added for multiplexed projects.
Nanopore long-read sequencing: Reads cover entire transcripts, including 5'UTR, CDS, 3'UTR, and poly(A) tails.
TAIL Iso-seq workflow: from RNA QC, library preparation, and Nanopore long-read sequencing to comprehensive bioinformatics analysis and publication-ready reports.
Poly(A) Tail Sequencing Methods—When to Use Which
| Method | Platform | What it measures | Non-A residues | Throughput / Cost (relative) | Typical strengths | Typical limitations | Good for |
|---|---|---|---|---|---|---|---|
| TAIL-seq | Illumina (3' end sequencing) | Genome-wide tail length at 3' ends; 3' terminome | Detects terminal U/G additions | High / $$ | Sensitive mapping of 3' ends; links tail length to decay | Raw signal processing and custom analysis required; not full-length isoforms | Deadenylation/uridylation studies; stability assays |
| PAL-seq | Illumina + fluorescence standards | Tail length using calibrated fluorescence; 3' end features | Indirect | High / $$ | Internal standards for calibrated tail length; robust bulk profiling | Added biochemistry; specialised setup; not full-length | Calibrated bulk tail length distributions; method benchmarking |
| Poly(A)-seq | Illumina (direct reading of poly(A) in cDNA) | Global profiling of poly(A) tails; per-gene median tail | Not primary focus | High / $ | Simpler library vs TAIL-seq; scalable cohorts | Isoforms not resolved; homopolymer challenges | Large-cohort screening of tail length shifts |
| FLAM-seq | PacBio (long-read cDNA) | Full-length mRNA including poly(A) tail | Reports non-A residues | Moderate / $$–$$$ | Links isoform structure + tail length per molecule | PacBio access; longer run times | Isoform-resolved tail biology; tail composition per isoform |
| PAIso-seq / PAIso-seq2 | PacBio HiFi | Transcriptome-wide tail length from low input; full-length | Can capture tail composition | Moderate / $$–$$$ | High-accuracy HiFi reads; low-input (e.g., oocytes) | Platform-specific; method complexity | Precious/low-input samples; accurate tail estimates |
| FLEP-seq2 | Nanopore (long-read cDNA) | Full-length RNA with tail length across tissues/species | Detects tail patterns | Moderate / $$ | Depth-friendly; cross-tissue atlases; nuclear vs cytoplasmic comparisons | Nanopore base-level accuracy trade-offs | Tissue atlases; plant and non-model organisms |
| Nano3P-seq | Nanopore (3' end-capture cDNA) | RNA abundance + tail dynamics (poly(A) and non-poly(A)) | Detects non-A residues | Moderate / $$ | Captures polyadenylated and non-polyadenylated RNAs; per-molecule tails | End-capture bias to 3' end; requires toolchain | Developmental time-course; transcriptome-wide tail dyn |
Bioinformatics Analysis
Our in-house pipeline integrates multiple layers of information:
- Isoform identification: Full-length isoform discovery without assembly bias.
- Poly(A) tail analysis: Distribution, median length, and detection of non-A residues (U, G, C).
- APA mapping: Identification of polyadenylation sites (PACs), 3'UTR length variants, and APA switching events.
- Correlation analysis: Linking poly(A) length to transcript abundance, stability, and translational efficiency.
- Functional interpretation: GO and KEGG enrichment for APA-impacted genes, miRNA-binding site gain/loss predictions.
Applications of TAIL Iso-seq
- Decode stress response and developmental biology in plants – profile how poly(A) tail length and APA change under heat, drought or developmental transitions (e.g. Arabidopsis heat shock PAL-seq work)
- Understand disease mechanisms and discover biomarkers in cancer – identify novel isoforms, altered poly(A) tail dynamics, or fusion transcripts that may drive pathology or serve as diagnostic/therapeutic targets (e.g. "Full-length and single-cell Iso-Seq for cancer research")
- Optimise mRNA-based therapeutics and vaccine constructs – ensure transcript integrity, tail length uniformity, and tail composition (including non-A residues) to improve translation and stability
- Crop trait discovery and breeding enhancement – use tail length signatures and APA patterns to link to yield, hybrid vigour, stress tolerance, or tissue-specific expression in crops like rice, maize, soybean
Sample Requirements for TAIL Iso-seq Service
| Sample Type | Recommended Quantity | Minimum Quantity | Concentration / Quality | Notes |
|---|---|---|---|---|
| Total RNA | ≥ 2 µg | 600 ng | ≥ 30 ng/µL | RIN ≥ 8, OD260/280 ≥ 1.8, DNA-free |
| Cells | ≥ 1 × 10⁶ | ≥ 5 × 10⁵ | High viability (>80%) | Snap-freeze pellets in liquid nitrogen |
| Tissue (animal/plant) | ≥ 50 mg | ≥ 10 mg | RNA integrity RIN ≥ 8 | Remove contaminants, freeze immediately |
| Plant tissue (special) | 50–100 mg per aliquot | – | RNA integrity RIN ≥ 8 | Wash with DEPC water, freeze in liquid nitrogen |
| Bacterial culture | ≥ 1 × 10⁷ cells | – | RNA integrity RIN ≥ 8 | Pellet, wash with PBS, freeze in liquid nitrogen |
General Guidelines
- Submit samples in RNase-free water or 10 mM Tris (pH 8.0).
- Avoid repeated freeze–thaw cycles.
- Ship samples on dry ice to maintain RNA quality.
- Clearly label tubes with short codes (≤4 alphanumeric characters).
- Provide QC data (Bioanalyzer/Tapestation traces) along with the submission form.
Deliverables
- Raw and processed sequencing data (FASTQ, BAM).
- Isoform-level transcriptome catalogue.
- Poly(A) tail length distributions and APA site maps.
- Pathway enrichment tables and integrative visualisations.
- Publication-ready figures and a detailed technical report.
Frequently Asked Questions (FAQ)
What is TAIL Iso-seq and how does it differ from regular Iso-seq or RNA-seq?
TAIL Iso-seq is a long-read sequencing service (Nanopore-based) that captures full-length transcripts including the native poly(A) tail, enabling direct measurement of tail length, APA (alternative polyadenylation) sites, and isoform diversity. Regular RNA-seq often loses tail information and requires read assembly, while standard Iso-seq may capture transcript structure but not always the full poly(A) composition and non-A residues.
Can TAIL Iso-seq detect non-A nucleotides inside or at the end of poly(A) tails?
Yes. The service includes detection of non-adenosine residues (such as U, G, C) both at the terminal position and internally in the poly(A) tail. These non-A residues provide insight into transcript stability, degradation, or deadenylation regulation. Tools like tailfindr, nanopolish, and others support this analysis.
How accurate is poly(A) tail length measurement with Nanopore long reads?
TAIL Iso-seq uses state-of-the-art basecalling and signal processing pipelines to estimate tail length per molecule with high resolution, often at single nucleotide precision for shorter tails and good relative accuracy for longer tails. Accuracy also depends on sample quality, sequencing output (.fast5 retention), and tool choice (tailfindr, nanopolish, etc.).
Do I need a reference genome to use TAIL Iso-seq?
No, but having a reference genome or transcript annotation improves mapping, APA site identification, and isoform resolution. In non-model organisms, de novo long-read assembly combined with reference-free transcript annotation is possible, though some analyses (e.g. miRNA-binding prediction) may be more challenging without good annotation.
What are the input sample requirements for TAIL Iso-seq?
Total RNA of high integrity is required (recommended RIN ≥ 7), free of contaminants, with sufficient quantity depending on organism / tissue; lower input is possible with optimized methods. The RNA must include intact poly(A) tails and minimal degradation for best results.
How do you handle PCR bias or truncation of poly(A) tails in the pipeline?
We minimise bias by using long-read sequencing (which avoids fragment assembly), capturing full-length transcripts, retaining raw signal data (.fast5) for tail length estimation, and employing QC steps to filter truncated or degraded RNA. Usage of tools that work on raw signal helps ensure accurate tail estimation.
What is the difference between poly(A) tail analysis and APA (alternative polyadenylation) analysis?
Poly(A) tail analysis focuses on the length, composition, and modifications of the tail itself at the transcript-end. APA analysis addresses where polyadenylation occurs—i.e. mapping poly(A) cleavage sites, the varying 3' untranslated region (3'UTR) lengths among isoforms, and how that affects regulation (e.g., miRNA binding). Both are complementary.
Can TAIL Iso-seq be used for comparative studies across conditions or species?
Yes. Because it provides per-isoform tail length distributions and APA site usage per sample, TAIL Iso-seq supports comparisons across tissues, treatments, or species. Statistical pipelines can test for differential tail length or APA usage between groups.
How long will I get the results (bioinformatics + deliverables)?
Typical timelines depend on sample number, complexity, and sequencing depth. Reporting includes raw + processed sequencing data, tail length distributions, APA mapping, isoform catalogues, and visualizations. (Specific turn-around times will be provided based on scope in the project quote.)
How does cost scale with sample number, depth, or target transcript complexity?
Costs generally increase with the number of samples, required read depth, and complexity (e.g. non-model organism or low expression transcripts). CD Genomics offers scalable pricing; clients can choose from standard or premium analysis levels depending on how much resolution (isoform discovery, tail composition, APA precision) they require.
Case Study: "An atlas of plant full-length RNA reveals tissue-specific and evolutionarily-conserved regulation of poly(A) tail length in plants"
Citation: Jia J., Lu W., Liu B., et al. An atlas of plant full-length RNA reveals tissue-specific and monocots–dicots conserved regulation of poly(A) tail length, Nature Plants 2022.
1. Background
Researchers lacked a comprehensive, full-length transcriptome resource in plants that includes poly(A) tail length information across tissues and species. Prior short-read methods often failed to preserve poly(A) tails or did not link tails to full transcript isoforms. This gap limited insights into how poly(A) length, APA (alternative polyadenylation), and mRNA stability vary between tissues or across plant species.
2. Methods
- Used a poly(A)-enrichment-free, Nanopore-based method (FLEP-seq2) to sequence full-length RNAs retaining poly(A) tails.
- Sampled seven different tissues of Arabidopsis thaliana, plus shoot tissue of maize, soybean, and rice. Total >120 million polyadenylated mRNA molecules were sequenced.
- Compared poly(A) tail length distributions between nucleus vs cytoplasm, different tissues, and across orthologous genes in different species. Also assessed relationship between mRNA half-life and tail length.
3. Results
- In most tissues, poly(A) tail lengths peaked around ~20 nt and ~45 nt; in pollen the peaks were different (~55–80 nt).
- Nuclear RNAs had tails almost twice as long as cytoplasmic RNA tails.
- Genes with short mRNA half-lives generally had longer poly(A) tails, whereas stable transcripts had tails peaking near ~45 nt.
- Cross-species comparison showed that orthologous genes tend to maintain similar tail length regulation despite species differences.
Figure: Nuclear poly(A) tails are longer than cytoplasmic tails. This shows the distribution of poly(A) tail lengths in nuclear vs cytoplasmic fractions across plant species/tissues.
4. Conclusions
- Poly(A) tail regulation is gene-specific, tissue-specific, and shows evolutionary conservation among plants.
- Differences in tail length between nucleus vs cytoplasm and among tissues point to dynamic regulation of tail trimming (deadenylation) and APA.
- The dataset provides a resource for studying mRNA stability, translation, APA, and non-coding regulatory features in plants.
- Supports the value of full-length, long-read sequencing for linking tail length to RNA function.
References:
- Jia J, et al. An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of Poly(A) tail length. Nature Plants. 2022
- Wen H, Chen W, Chen Y, Wei G, Ni T. Integrative analysis of Iso-Seq and RNA-seq reveals dynamic changes of alternative promoter, alternative splicing and alternative polyadenylation during Angiotensin II-induced senescence in rat primary aortic endothelial cells. Front Genet. 2023 Jan 19;14:1064624.
- Kuo RI, Cheng Y, Zhang R, Brown JWS, Smith J, Archibald AL, Burt DW. Illuminating the dark side of the human transcriptome with long read transcript sequencing. BMC Genomics. 2020 Oct 30;21(1):751. doi: 10.1186/s12864-020-07123-7. PMID: 33126848; PMCID: PMC7596999.
