RNA NGS Service

Our services cover every step in the process from initial consultation through to RNA isolation, RNA QC, library preparation and QC, sequencing and QC, data analysis and reporting. You can also track the progress of your project through your own secure web portal.

Each sequencing project can be tailored to your needs. During the initial consultation, our NGS experts will discuss with you all important parameters for your project to ensure that it is set up in the most effective way.

We also offer advanced data analysis, interpretation and visualization. Our experimental and bioinformatics pipelines are closely integrated, so you get powerful data analysis and a final report tailored to you.

mRNA and whole transcriptome sequencing is offered for 19 different species including human, mouse and rat. Ultra-low mRNA sequencing is offered for human, mouse and rat. However, any species may be sequenced; please contact us for further details.

For miRNA NGS services, libraries are size selected to maximize the number of relevant reads.

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. Our NGS services enrich for poly-A tailed transcripts to 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 provides increased confidence in transcript annotation and enables detection of antisense transcript expression.

During data analysis, mitochondrial poly-A tailed transcripts are bioinformatically filtered, as 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.

Whole transcriptome sequencing
Whole transcriptome sequencing enables the characterization of all RNA transcripts for a given organism, including coding and non-coding RNA (above 170 nt in size), regardless of whether they are polyadenylated or not. However, since ribosomal RNA accounts for the vast majority of the transcriptome, the library preparation is designed to remove ribosomal RNA prior to sequencing. This increases the depth of sequencing for biologically relevant parts of the transcriptome.

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 higher confidence.

miRNA sequencing
miRNA sequencing provides a comprehensive analysis of all miRNAs in the sample. Library size selection (15–40 nt) maximizes the number of miRNA reads. Our service includes novel miRNA discovery as well as isomiR counts, enabling you to see more precisely which miRNA sequences are expressed in your samples. miRNA sequencing is offered for 19 different standard species and a variety of custom species upon request.

Biofluid miRNA sequencing
These services include unique and proprietary protocols for:

• High-quality RNA
• LNA qPCR-based QC to monitor miRNA content, inhibition and hemolysis
• Library preparation protocols optimized for biofluids with limited RNA content
• QC of sequencing data reproducibility and linearity using > 50 RNA spike-ins

Our Biofluid miRNA Sequencing Services provide a comprehensive analysis of all miRNAs in the sample. Library size selection (15–40 nt) maximizes the number of miRNA reads. Our services includes novel miRNA discovery as well as isomiR counts, enabling you to see precisely which miRNA sequences are expressed in your samples. Biofluid miRNA sequencing is offered for human serum and plasma samples and other species or other biofluids (e.g., urine, CSF, saliva, etc.) upon request. The service supports discovery and validation of new non-invasive biomarkers for a range of diseases. Biomarker discovery projects usually begin with a genome-wide screening phase on a limited number of samples, performed either by a hypothesis-free NGS approach or by PCR profiling. From this genome-wide screening dataset, a smaller number of relevant RNAs are identified and the discovery phase can continue. Once a biomarker signature has been identified (including relevant endogenous control RNAs), it is then validated on a large number of individuals using custom Custom PCR panels. Crucially, assays to detect any novel RNAs identified by NGS may also be included in the custom PCR panels.

Consultation

Each project begins with a free consultation with an expert to design an experimental setup that best meets your 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.

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.

mRNA or whole transcriptome
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.

Ultra-low mRNA
For ultra-low mRNA NGS, RNA isolation is included in the project and must be performed by us as we have developed an optimized NGS workflow with proprietary steps including unique RNA isolation and QC protocols specifically optimized for 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 a minimum of 100 cells to get high-quality results. We accept samples from human only.

Biofluid miRNA
For miRNA-seq in serum and plasma, RNA isolation is included in the project and has to be performed by us. Our optimized NGS workflow with proprietary steps, including unique RNA isolation and QC protocols, is specifically optimized for miRNA sequencing from serum/plasma. Although we can get high-quailty results from as little as 250 µl, we recommend sending 500 µl of serum/plasma. We accept human serum/plasma samples, and samples from other species or other biofluids (urine, CSF, saliva etc.) upon request.

Sample preparation

We 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.

Biofluid miRNA
Isolation of RNA from biofluids is performed using QIAGEN kits and protocols developed specifically for this sample type.

RNA sample quality control (QC)
After receiving your RNA samples, we will determine the integrity and quantity of each sample using a Bioanalyzer/TapeStation and Nanodrop instruments and possible contaminations are assessed for each sample. You will receive a report with the results of these analyses prior to the library preparation and sequencing. We also perform the QC prior to any RT-reaction for downstream qPCR profiling.

Ultra-low mRNA
Following RNA isolation, we will perform RNA QC using qPCR-based methods optimized specifically for low RNA content samples. The 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.

Biofluid miRNA 
For biofluid samples, QC of the RNA is performed by qPCR, using a unique combination of RNA spike-ins and endogenous miRNAs to detect outlier samples and monitor RNases, RNA isolation efficiency, hemolysis and presence of inhibitors that may affect library preparation. You will receive a report with the RNA-QC results prior to the library preparation and sequencing.

Library preparation

After QC, we design 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 fragmented and converted into mRNA or whole transcriptome cDNA NGS libraries. A Bioanalyzer/TapeStation is used to perform QC of the libraries.

Biofluid miRNA 
We generate libraries and select size using a bead-based size selection of 15-40 nt. Library QC is performed using a Bioanalyzer/TapeStation.

Sequencing

We 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. For standard projects, the sequencing parameters are 1 x 75 bp, 30 M reads per sample or 2 x 75 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.

miRNA
We offer 2 types of miRNA sequencing using the Illumina platform:

• miRNA sequencing including miRNA discovery
• Biofluid miRNA sequencing including miRNA discovery

Sequencing is performed using Illumina instruments, 1x 75bp reads, 10M reads per sample.

Biofluid miRNA
Biofluid miRNA sequencing is performed using Illumina instruments, 1x 75bp reads, 10M reads per sample.

Analysis and interpretation for mRNA and whole transcriptome NGS projects

Comprehensive data analysis, including statistical analysis is provided using our unique NGS data analysis pipeline:

• 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.
• Statistical analysis is performed, provided there are a sufficient number of samples per group
• Unsupervised analysis: Principal Component Analysis (PCA plot) and heat map   
• 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.

Biofluid miRNA

• Mapping to miRBase and identification of predicted novel miRNAs
• IsomiR counts can be reported
• For biofluids: QC includes analysis of >50 spike-ins which are used to check the reproducibility and linearity of the sequencing data
• Mapping sequences to the appropriate reference genome
• In connection with large service projects, we also offer fully extensive customized bioinformatics analysis
• Normalization
• Differential gene expression analysis to identify known and novel genes and transcripts that are differentially expressed between your sample groups.
• Statistical analysis is performed, provided there are a sufficient number of samples per group
• Unsupervised analysis: Principal Component Analysis (PCA plot) and heat map   
• 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.

Report and final consultation

The final report is delivered by our cloud-solution called XploreRNA 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   
• A 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

Turnaround times
mRNA or whole transcriptome and miRNA: 8–10 weeks

Ultra-low mRNA amounts or biofluids: 8–10 weeks

After you have received your report, we arrange a follow-up discussion to answer any questions you may have about your report and discuss the significance of your results and next steps. We can also help you design appropriate miRCURY LNA or RT2 qPCR assays to validate your NGS data.