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mRNA and Whole Transcriptome Sequencing Service

Send us your samples and let our Next Generation Sequencing (NGS) experts perform your NGS analysis in state-of-the-art automated laboratories with rigorous quality control and fast turn-around times and superior data analysis and interpretation. Exiqon offers a complete sequencing service including RNA isolation, library preparation, sequencing, data analysis and biological interpretation of the results in a comprehensive and easy-to-read format.

Exiqon mRNA and Whole Transcriptome RNA NGS Services

  • Complete sample-to-answer solution covers everything from initial consultation to advanced data analysis and interpretation
  • Provide the sample, and we will take care of RNA isolation, library preparation, sequencing, data analysis and reporting
  • Close consultation with Exiqon’s RNA and NGS experts throughout the process
  • Sequence from as little as 100 ng total RNA
  • Rigorous QC in all steps of the workflow
  • Fast turn-around times – delivery of the final report within 4-6 weeks
  • Combine with downstream validation by market-leading LNA™-enhanced qPCR platform

A sample-to-answer solution for NGS Service

Our NGS service includes every step in the process from initial consultation with our RNA experts through RNA isolation, RNA QC, library preparation and QC, sequencing and QC, data analysis and reporting. All analyses are performed by PhD-level scientists who will ensure that you get the best service throughout the project. You can also track the progress of your project through your own secure web portal.

Each RNA sequencing project can be tailored to your individual needs. At the initial consultation our NGS experts will discuss with you all of the important parameters for an RNA sequencing project (e.g. sequencing depth, read length, number of reads, replicates etc.) to ensure that the project is set up to get the most from your samples.

Exiqon Services provide outstanding and advanced data analysis, interpretation and visualization. Our superior bioinformatics pipeline is based on solid biological expertise in RNA research. The experimental and bioinformatics pipelines are closely integrated, enabling us to provide powerful data analysis and a final report tailored to your specifications.

Data and results from your mRNA or whole transcriptome profiling project is delivered in an easy-to-read report with publication grade illustrations including a data analysis files. The complete encrypted raw data is supplied on a data hard disk. Every project concludes with a scientific follow-up discussion with our NGS experts, to answer any questions you may have about your results.

mRNA and whole transcriptome sequencing is offered for 20 different species including human, mouse and rat (see Table 1). However, any species where a reference genome is available may be sequenced. Please contact us for further details.

The workflow of an Exiqon mRNA or whole transcriptome RNA sequencing services project is summarized in Figure 1. Details of each step in the process are described in the following tab.

mRNA sequencing

mRNA sequencing targets all polyadenylated (poly-A) transcripts of the transcriptome. During the library preparation, poly-A tailed transcripts are enriched from total RNA. The poly-A tailed transcripts include the mRNAs (the coding part of the genome), which only accounts for 1-4 % of the whole transcriptome. By enriching for poly-A tailed transcripts we increase the sequencing depth for coding mRNAs, which improves the sensitivity to mRNAs expressed at low levels. In addition, the library preparation retains information on which of the two DNA strands was used to transcribe a given RNA. This information on strandedness provides increased confidence in transcript annotation and enables the detection of antisense transcript expression.

During data analysis mitochondrial poly-A tailed transcripts are bioinformatically filtered since they are considered to be high abundance sequences. mRNA sequencing is recommended for discovery work and especially differential expression analysis. Paired end sequencing increases the mapping percentage to poorly annotated genomes and makes it possible to identify splice variants with much higher confidence. However, if differential gene expression is the main goal of your project, we recommend single end sequencing.

Table 2 shows a comparison of a typical mRNA and whole transcriptome sequencing project.

Table 2. A comparison of a typical mRNA and whole transcriptome sequencing project A comparison of a typical mRNA and whole transcriptome sequencing project

Whole transcriptome sequencing

Whole transcriptome sequencing enables the characterization of all RNA transcripts for a given organism, including both the coding mRNA and non-coding RNA, such as long non-coding RNAs (lncRNAs), snRNAs and snoRNAs, larger than 170 nucleotides in length, regardless of whether they are polyadenylated or not. However, since ribosomal RNA accounts for vast the majority of the transcriptome, the library preparation is designed to remove the ribosomal RNA prior to sequencing. This increases the depth of sequencing for the biologically relevant part of the transcriptome.

The library preparation also retains information on which of the two DNA strands was used to transcribe a given RNA. This information on strandedness provides increased confidence in transcript annotation and enables the detection of antisense transcript expression.

Whole transcriptome paired end sequencing is recommended for both discovery work and differential expression analysis. By using paired end sequencing, it is also possible to identify splice variants with much higher confidence.

Figure 2. Distribution of RNA species in NGS samples after poly-A enrichment (mRNA) and rRNA depletion (whole transcriptome) sequencing Distribution of RNA species in NGS samples after poly-A enrichment (mRNA) and rRNA depletion (whole transcriptome) sequencing

Contact us for more information
Exiqon mRNA and Whole Transcriptome RNA NGS Services

Table 1 List of species currently supported for mRNA or whole transcriptome RNA sequencing
List of species currently supported for mRNA or whole transcriptome RNA sequencing - for other species, please inquire. (Click to learn more)

Figure 1 Workflow of an mRNA or whole transcriptome sequencing project
Workflow of an mRNA or whole transcriptome sequencing project. (Click to learn more)

1. Consultation

When you engage in a sequencing Service project with Exiqon, you are assured direct communication with the scientists performing your experiments throughout the duration of the project.

Each project begins with a free consultation with an RNA sequencing expert. Together we design an experimental setup that best satisfies your research needs and budget. Following this we will complete a detailed sample-submission form making sure that all experimental details and subsequent analyses are clearly defined and understood by both parties.

2. RNA sample submission

High quality samples are important for accurate RNA sequencing. At the initial consultation, we offer recommendations on suitable extraction and clean-up methods. Alternatively, you can take advantage of our expertise and submit your samples to our RNA isolation service. We recommend that you send us 260 ng total RNA, but we get high quality results with as little as 100 ng total RNA + 60 ng for QC. See our guidelines for more information.

For sequencing from very low amounts of sample as little as 100 cells please see our Ultra-low mRNA sequencing service.

3. Sample preparation

At Exiqon Services we also offer to isolate RNA from your samples for sequencing. We perform high quality RNA isolation from a range of sample types including cells, tissues, FFPE sections, and blood. We also perform RNA isolation from minute amounts of sample as part of our Ultra-low mRNA Sequencing Service (learn more).

4. RNA sample quality control (QC)

After receiving your RNA samples, our experts will determine the integrity and quantity of each sample using Bioanalyzer and Nanodrop™ instruments. Possible contaminations are likewise assessed for each sample. You will receive a report with the results of these analyses prior to the library preparation and sequencing.

5. Library preparation

Following quality control, we generate libraries according to the type of sequencing to be performed.

For mRNA sequencing we enrich poly-A mRNA molecules from total RNA using an Oligo-dT magnetic bead-based system. For whole transcriptome sequencing we perform ribosomal RNA depletion (through the use of a biotin-streptavidin bead-based system).

The resulting RNA is then fragmented and converted into mRNA or whole transcriptome cDNA NGS libraries. QC of the libraries is performed using the Agilent Bioanalyzer 2100.

6. Sequencing

Exiqon Services offer mRNA and whole transcriptome sequencing using the Illumina platform. We use both NextSeq 500 and HiSeq 2500 instruments, enabling us to cater for a range of project sizes from small exploratory pilot studies to very large sequencing projects.

For standard projects, the sequencing parameters are 1 x 50 bp, 30 M reads per sample or 2 x 50 bp, 60 M paired-end reads per sample. However, the sequencing parameters can be tailored to your specific requirements. Please contact us for further information.

7. Analysis & interpretation

Comprehensive data analysis including statistical analysis is provided using Exiqon’s unique NGS data analysis pipeline (see Figure 2):
  • Comprehensive QC of raw data
  • Mapping: Alignment of sequence to the appropriate reference genome
  • QC of mapped data
  • Quantification of known transcripts (both Ensembl and UCSC are supported)
  • Prediction and quantification of novel transcripts
  • Tests for significant changes in promotor usage
  • Identification of splice-junctions with list of exon positions associated with each transcript
  • Identification of alternative splicing and splice variants with list of exons associated with each isoform
  • Identification of antisense transcripts
  • Normalization
  • Differential gene expression analysis to identify known and novel genes and transcripts that are differentially expressed between your sample groups (unlimited number of group comparisons). Statistical analysis is performed, provided there are a sufficient number of samples per group.
  • Unsupervised analysis: Principal Component Analysis (PCA plot) and heatmap
  • Supervised analysis: For each specific group comparison we generate a Volcano plot to display genes which are differentially expressed between the groups, list differentially expressed genes as well as identify statistically significant signals
  • Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within differentially expressed genes. This gives a picture of the impact of the experiment on biological pathways and processes.
In connection with large service projects we also offer fully extensive customized bioinformatics analysis.

8. Report and final consultation

The final report is delivered as a link to our secure webserver and contains:
  • An easy-to-read data report containing a description of the project, assessments of sample and data quality and an overview of the results of the data analysis with publication-grade illustrations.
  • Data analysis file which is sufficient for publication, or performing your own analysis of the data if needed.
  • A materials and methods section ready to use for publication purposes.
The complete encrypted raw data (including FASTQ and BAM files) are provided.

Expected turnaround time: 4-6 weeks.

After you have received your report, we arrange a scientific follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. Exiqon scientists can also help you design appropriate LNA™ qPCR assays to validate your NGS data, or Antisense LNA™ GapmeRs to study the function of the candidate genes of interest identified in the study.

Contact us for more information
Figure 1 Workflow of an mRNA or whole transcriptome sequencing project
Workflow of an mRNA or whole transcriptome sequencing project. (Click to learn more)

Figure 2 Overview of the NGS data analysis workflow
Overview of the NGS data analysis workflow. (Click to learn more)

Comprehensive report including biological interpretation of your data

Based on our extensive bioinformatics expertise we have developed a unique pipeline for collecting, analyzing and visualizing NGS results.

At the end of each project, you will receive a comprehensive and easy-to-read final report.

Download a report example

Comprehensive data analysis including statistical analysis is provided:
  • Comprehensive QC of raw data
  • Mapping: Alignment of sequence to the appropriate reference genome
  • QC of mapped data
  • Quantification of known transcripts (both Ensembl and UCSC are supported)
  • Prediction and quantification of novel transcripts
  • Tests for significant changes in promotor usage
  • Identification of splice-junctions with list of exon positions associated with each transcript
  • Identification of alternative splicing and splice variants with list of exons associated with each isoform
  • Identification of antisense transcripts
  • Normalization
  • Differential gene expression analysis to identify known and novel genes and transcripts that are differentially expressed between your sample groups (unlimited number of group comparisons). Statistical analysis is performed, provided there are a sufficient number of samples per group.
  • Unsupervised analysis: Principal Component Analysis (PCA plot) and heatmap
  • Supervised analysis: For each specific group comparison we generate a Volcano plot to display genes which are differentially expressed between the groups, list differentially expressed genes as well as identify statistically significant signals
  • Gene Ontology (GO) enrichment analysis is performed to identify GO terms that are over-represented within differentially expressed genes. This gives a picture of the impact of the experiment on biological pathways and processes.
In connection with large service projects we also offer fully extensive customized bioinformatics analysis.

After you have received your report, we arrange a scientific follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. Exiqon scientists can also help you design appropriate LNA™ qPCR assays to validate your NGS data, or Antisense LNA™ GapmeRs to study the function of the candidate genes of interest identified in the study.

Contact us to discuss your project.
Hanna Taipaleenmäki

NGS identifies therapeutic microRNA targets for skeletal disorders


"Exiqon Services performed microRNA and mRNA sequencing from the exact same RNA samples....We have been combining the NGS results with functional studies using In vivo LNA™ microRNA inhibitors in mouse models."

Dr. Hanna Taipaleenmäki is a research scientist within the group of Prof. Dr. Dr. Eric Hesse, in the Molecular Skeletal Biology Laboratory at the University Medical Center Hamburg-Eppendorf in Germany.

Read full story...

 
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