Methods to do bulk sequencing of protein coding, non-coding or small RNA, with short or long reads. Select an application to learn more about its advantages and sample requirements. We also offer different options for low input samples or degraded RNA within each application.
NGI offers several types of transcriptome sequencing approaches. Are you interested in analysing protein coding or non-coding RNAs, or small RNAs such as miRNAs? Get in touch with us to discuss the best approach for your project!
Please consult the flowchart for an overview of the applications that might best suit your project. You can also read more about each application further down in the applications section.
For projects focused on transcriptome annotation, please refer to our de novo page.
The term “coverage” is often used when sequencing DNA samples, referring to the average number of reads that align to, or "cover", known reference bases. When sequencing RNA samples, we are instead referring to “number of reads”, as transcripts are present at different levels.
For high quality RNA sequencing data, we ask for high-quality RNA samples in adequate amounts. Degraded or low amounts of starting material can yield low complexity libraries, meaning that the number of unique fragments present in a given library will be very low. This could complicate downstream analyses. It is therefore of utmost importance that our sample requirement guidelines are followed, and high quality RNA material is submitted to us.
Each application has its own requirements regarding concentration and volume, as different starting amounts are needed. We strongly encourage our users to check sample amounts and quality before submitting them to us. Making sure you have good quality samples from the start will improve the success rate and facilitate handling of your project. Note that the quality control performed at NGI is mainly for confirmation purposes.
Concentration measurements are most accurately performed using fluorometric measurements (Qubit, Quant-it). For quality scoring we recommend a capillary electrophoresis method (Fragment Analyzer, Bioanalyzer, TapeStation) that can measure RNA integrity by providing a RNA integrity Number (RIN) value. RIN values range from 1–10, with lower values indicating poor quality and higher values indicating good quality RNA (RIN > 8). Please note that RIN values can be a bit tricky to obtain on certain types of RNA, such as insect RNA. Read more about RIN scores here and here. In cases where your project involves FFPE samples, we also recommend using a capillary electrophoresis method to determine the DV200 value.
The RNA should be free from any contaminating DNA to minimize the contribution of sequence reads derived from residual genomic DNA in the sample. NGI does not perform DNase treatment prior to library preparation. In particular for library preparation based on ribosomal depletion, failure to treat RNA samples with DNase or inefficient DNase treatment can result in a significant fraction of intergenic reads in the sequence data.
PacBio SMRT sequencing generates reads tens of kilobases in length enabling high quality genome assembly, structural variant analysis, amplicon resequencing, full-length transcript isoform sequencing, full-length 16S rRNA sequencing and amplification free epigenetic characterization.sequel hifi clr de novo iso seq sv smrt assembly pacbio methylation amplicon