INVIEW TRANSCRIPTOME ISOFORM DISCOVER
Isoform sequencing with PacBio’s long-read technology to precisely identify new splicing variants
INVIEW TRANSCRIPTOME ISOFORM DISCOVER is a sample-to-data solution which employs remarkably long read lengths to span full-length cDNA. Based on an enhanced ISO-SEQ® protocol, originally developed by Pacific Biosciences, the unique approach enables in-depth profiling of isoform diversity without the need for fragmentation or post-sequencing assembly.
Novel alternatively spliced variants and previously unannotated genes that are difficult or impossible to uncover using short sequencing reads can now be identified.
Highlights of INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- Next-generation sequencing (NGS) of fragments up to 10K to generate high-quality, full-length transcripts without any assembly
- Single-molecule sequencing with one read per transcript
- Unbiased sequencing of difficult genomic regions, such as GC-rich areas and tandem repeats
Service & Specifications
Full-service package offered by INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- Optimised workflows for construction of complex full-length cDNA libraries
- Enhanced protocols based on PacBio’s ISO-SEQ® approach
- Next-generation sequencing of full-length, single-molecule cDNA (four size selections: 1 kb - 2 kb / 2 kb - 3 kb / 3 kb - 5 kb / 5 kb - 10 kb)
- Sequencing using PacBio’s ultra-long read SMRT® technology
- Additional data packages for increased sequencing depth
- Optimised BioIT for unambiguous identification of transcript variants
- Comprehensive GATC Data Report including raw data
- Applicable to any organism with alternatively spliced genes
Accepted starting material for INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- Total RNA (3 µg) with a concentration of 300 ng/µL
- Any number of samples, starting from one sample
Technology used for INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- SMRT® technology on a Pacific Biosciences RS II
Bioinformatics analysis included in INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- Generation of consensus sequences
- Identification of full-length reads
- Detection and removal of artificial chimeras
- Isoform-level clustering
- Consensus polishing
Deliverables of INVIEW TRANSCRIPTOME ISOFORM DISCOVER
- Raw data (fastq, bas.h5, metadata.xml)
- Sorted "Reads of Insert" (fastq, fasta)
- Consensus sequence of isoforms (fastq, fasta)
- Comprehensive Data Analysis Report (pdf)
- 30 days for up to 4 samples, more samples on request
Alternative RNA splicing is a well-known phenomenon, but a complete catalog of isoforms that account for variability in the human transcriptome is still lacking. Significant progress has been made in developing methods to study variability of the transcriptome to provide a full picture of the transcriptome in the near future.
INVIEW TRANSCRIPTOME ISOFORM DISCOVER is our GATC Sequencing Solution for in-depth analysis of transcript isoform diversity. The product combines preparation of complex full-length cDNA libraries, sequencing on the PacBio platform RS II and comprehensive BioIT analysis to enable the systematic characterisation of alternative splicing, alternative promoters and alternative polyadenylation events in eukaryotic cells. Reads that span the entire transcript length make complicated post-sequencing assembly unnecessary. The product instead relies on analysis of single, full-length molecules to provide unambiguous identification of novel gene variants with biological relevance.
Fig. 1. Possible products of alternative splicing of a hypothetical gene
Structure of a three-exon, two-intron gene. Exons are illustrated in dark green and introns in light green. Two alternative promoters, one upstream of the coding region (Pwt) and one in the first exon (Palt), are shown as black arrows. Two alternative polyadenylation sites are shown as green downward arrows and indicated as poly(A)wt for the most frequently used polyadenylation site and poly(A)alt for an alternative polyadenylation site in intron 2.
Some examples of possible mRNAs that could arise from transcription using alternative promoters and using alternative splicing and polyadenylation. The 3’-untranslated region is shown as a black line.
Typical applications for INVIEW TRANSCRIPTOME ISOFORM DISCOVER include:
- Discovery of novel genes, novel splice variants and isoforms
- Analysis of alternative promoters, alternative splicing and alternative polyadenylation events
- Improving genome annotation of eukaryotic organisms
- Investigating mechanisms of post-transcriptional regulation
- Gaining clear insights into protein functional diversity
- Regulation and misregulation of pre-mRNA alternative splicing in cancer and other diseases
1. How is INVIEW TRANSCRIPTOME ISOFORM DISCOVER different from INVIEW TRANSCRIPTOME EXPLORE, ADVANCE and DISCOVER?
INVIEW TRANSCRIPTOME ISOFORM DISCOVER is an RNA-seq standardised service that employs PacBio’s SMRT® technology to profile full-length cDNA transcripts. The product is perfectly suited for thorough investigations of isoform diversity and obtaining a full profile of alternative splicing events in eukaryotic organisms.
INVIEW TRANSCRIPTOME EXPLORE, ADVANCE and DISCOVER are standardised products that use Illumina’s platforms to sequence a variety of library types and facilitate studies on gene expression, gene regulation and identification of rare or novel RNA species. The products are applicable to any organism of interest and provide analysis of coding and noncoding RNA.
2. What starting material will be accepted?
Total RNA extracted from eukaryotic organisms can be used to obtain full-length cDNA sequencing libraries.
3. How much starting material is required?
For exact information on RNA quantity and concentration, please refer to your quote or contact us directly. In general, we require 3 µg of high- quality RNA with an OD 260/280 1.8-2.2 for optimal results.
The quality and quantity of the provided starting material is essential for the success of sample preparation. The use of too little, degraded or contaminated starting material can result in low yields, cause sample preparation to fail, or compromise the quality and amount of the sequencing results.
4. How many data packages do I need to reach my experimental goal?
5. Does GATC Biotech offer RNA isolation?
GATC Biotech can offer RNA isolation based on optimised protocols as an additional service. Please consult us to discuss possible sample preparation methods for your individual project requirements.
6. Should I send my starting material on dry ice or would room temperature be sufficient?
RNA must be shipped on dry ice, unless the RNA is precipitated in ethanol. Tissues / cell cultures must be flash- frozen in liquid nitrogen or dry ice and must be mailed on dry ice. Alternatively, fresh material can be stabilised in “RNAlater” (from companies like Ambion, SIGMA or QIAGEN) and be sent at room temperature. Please check in advance if the couriers that you are using accept dry-ice shipments.
7. What should I do if my sample fails the entry QC?
If the quantity or quality of the starting material does not meet the requirements for further processing, we will contact you directly to discuss how to proceed. If possible, GATC Biotech will recommend additional processing steps that could help improve sample quality.
8. How do I obtain my results and what form will they be in when delivered?
You can download all raw data as well as analysed data in various formats from your secure myGATC online account.
9. Where should I send my samples?
Please mail your samples to:
GATC Biotech AG
European Genome and Diagnostics Centre
Please do not use GATC Collection Points for shipping NextGen samples, because this will delay sample arrival at the appropriate destination.