RNA
Sequencing
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What we offer
Our two main approaches
we focus on total RNA or the RNA-exome covering all coding genes
Universal
total RNA Seq includes all types of RNA (>50bp) and therefore delivers a comprehensive picture of the transcriptome capturing both known and novel features
Breadth of results
Identification of fusion transcripts, differentially expressed genes, alternative splicing, expression profiles, but also SNVs and CNVs
Variety of bioinformatic support
depending on the experimental setup different analysis pipelines might be of interest
Our process
RNA sequencing analyzes the transcriptome and is therefore a quantitative determination of the presence and abundance of a transcribed gene in a biological sample at a given moment. The expression of the transcriptome provides the basis for the identity of a cell and the associated functionality and we are happy to share a couple of details about our workflow with you.
1
Library Preparation
We focus on two different approaches for RNA Seq library prep, sequencing of total RNA or RNA enriched for coding genes:
- TruSeq Stranded Total RNA (with Ribo-Zero Globin) (Illumina)
- TruSeq Stranded Total RNA (Illumina)
followed by the xGen hybridization capturing of the xGen Exome Research Panel v2 (IDT) - NEBNext Ultra II Directional RNA (NEB) combined with either
o rRNA depletion (NEB) or
o polyA enrichment (NEB)
2
Sequencing
At MLLSEQ, sequencing is performed using the Illumina sequencing by synthesis method on the latest generation of sequencing devices, the NovaSeq 6000. In contrast to DNA-based assays, the required output for RNA Seq is specified as millions of clusters or millions of reads and depends on the chosen assay and research question.
3
Among other things, in this step we offer different analysis options:
- bcl2fastq (Demultiplexing)
- STAR Aligner (Alignment)
- Arriba, Manta, STAR-Fusion (Variant calling: Fusions)
- Cufflinks 2 (gene expression estimates)
- Kallisto (transcript expression estimates)
- rMats (alternative splicing)
- cnvkit (Variant calling: CNV)