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Ultra-low mRNA Sequencing Service

Send us as little as 100 cells 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 Ultra-low mRNA NGS Services

  • Complete sample-to-answer solution covers everything from initial consultation to advanced data analysis and interpretation
  • Provide as little as 100 cells, 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 mRNA from ultra-low sample amounts – fine needle biopsies, LCM samples, sorted cells and more
  • 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 Ultra-low mRNA 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.

Ultra-low mRNA sequencing is offered human, mouse and rat. However, any species where a reference genome is available may be sequenced. Please contact us for further details.

The workflow of an Exiqon Ultra-low mRNA sequencing services project is summarized in Figure 1. Details of each step in the process are described in the following tab.

Ultra-low mRNA sequencing

Ultra-low mRNA sequencing targets all polyadenylated (poly-A) transcripts of the transcriptome using a library preparation protocol specifically optimized for samples with low RNA content. During the library preparation, poly-A tailed transcripts are enriched from total RNA. The poly-A tailed transcripts include mRNAs (the coding part of the genome), which accounts for 1-4 % of the whole transcriptome as well as some long non-coding RNAs with a poly-A tail. 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. Paired end sequencing increases the mapping percentage to poorly annotated genomes and makes it possible to identify splice variants with much higher confidence.

Table 1: Key specifications for an Ultra-low mRNA sequencing project.

Contact us for more information
Figure 1 Workflow of an Ultra-low mRNA sequencing project
Workflow of an Ultra-low mRNA 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. Sample submission

High quality samples are important for accurate RNA sequencing. At the initial consultation, we offer recommendations on suitable sample collection methods. For Ultra-low mRNA NGS, RNA isolation is included in the project and must be performed by Exiqon. Exiqon has developed an optimized NGS workflow with proprietary steps including unique RNA isolation and QC protocols specifically optimized for challenging samples with low RNA content. Ultra-low mRNA sequencing is ideal for small amounts of cells or tissue including fine needle biopsies, LCM samples and sorted cells. We recommend that you send us minimum 100 cells to get high quality results. See our guidelines for more information.

3. RNA sample quality control (QC)

After RNA isolation, our experts will perform RNA QC using qPCR-based methods specifically optimized for low RNA content samples. RNA input amount is normalized by quantification of GAPDH and ß-actin using qPCR. You will receive a report with the results of these analyses prior to the library preparation and sequencing.

4. Library preparation

Following quality control, we generate libraries using a protocol specifically optimized for samples with low RNA content. according to the type of sequencing to be performed. We enrich poly-A mRNA molecules from total RNA using an Oligo-dT magnetic bead-based system. The resulting RNA is then fragmented and converted into mRNA cDNA NGS libraries. QC of the libraries is performed using the Agilent Bioanalyzer 2100. The libraries are then quantified by qPCR and pooled prior to sequencing.

5. Sequencing

Exiqon Services offer Ultra-low mRNA 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 2 x 50 bp, 60 M paired-end reads per sample.

6. 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 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.

7. 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: 6-8 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 Ultra-low mRNA sequencing project
Workflow of an Ultra-low mRNA 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 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.
 
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