RNA sequencing

Next generation RNA sequencing, or transcriptome sequencing, provides a precise measurement of RNA transcript levels, but it can also be used to discover novel transcripts, alternative splicing, or allele-specific expression. We offer several RNA sequencing applications with different outputs:

Bioo Scientific RNAseq (polyA)

With the Bioo Scientific NEXTflex™ Rapid Directional RNA-Seq Kit we can generate NGS libraries from poly(A) tailed RNA. This kit can be used to obtain ‘stranded’ information: it identifies the specific DNA strands a given RNA transcript was derived from with >99% strand specificity. This stranded information improves read alignment and transcript annotation.

Optionally, we can deplete globin RNA transcripts from the RNA sample prior to library preparation. This is recommended for blood RNA samples.

RNA samples are processed with a liquid handler (microplate-format) in sequencing libraries. After library quantification and quality control, samples are loaded on to the Illumina NextSeq500 for single-read or paired-end sequencing.

Illumina RiboZero RNAseq

The Illumina TruSeq Stranded Total Library Preparation kit provides the most complete view of the transcriptome. TruSeq Stranded Total RNA kits couple all of the benefits of standard RNA preparation kits with Ribo-Zero ribosomal RNA reduction chemistry, providing analysis of coding and multiple forms of noncoding RNA with measurement of strand orientation, uniform coverage, and a high- discovery rate of features such as alternative transcripts, gene fusions, and allele-specific expression.

Bioo Scientific Small RNAseq

Small RNA (miRNA) sequencing is used to analyse tissue-specific miRNA expression patterns and disease associations, and to discover previously uncharacterized miRNAs. Bioo Scientific’s line of NEXTflex™ Small RNA Kits for Illumina® sequencing are ideal for the conversion of these small RNA transcripts into barcoded cDNA libraries for next-generation sequencing on Illumina sequencing platforms.

RNA samples are processed manually in microplate-format in sequencing libraries. After library quantification and quality control, samples are loaded on to the Illumina NextSeq500 for single-read sequencing.

Conditions and disclaimer for RNA sequencing

We ask a failure fee for each project to allow for technical problems during the experiment.

To ensure optimal conditions for library preparation and high-quality sequencing data, RNA samples have to be checked by capillary electrophoreses for quantity and quality/integrity. We facilitate this QC measurement on the Fragment Analyzer. Quality criteria of RNA samples will be discussed in advance and described in a contract, which includes our terms of agreement and disclaimers. Under these conditions, we will make a second attempt on library preparation if the first attempt fails. For samples not meeting these criteria, we will only make one attempt at the researcher’s own risk. If this fails, a second attempt will made only at the expense of the researcher.

The number of reads we generate for your NGS library will be determined in advance of the experiment. If this threshold is not obtained because of technical problems, we will re-run the samples on a second run. If the project is cancelled at any point by the researcher, all costs made up to that point will be billed to the researcher.

We can advise you on choosing the most suitable RNA sequencing method from our portfolio to answer your research question. However, downstream analysis is not part of our standard service. Should you need help we can refer you to the Genomics Coordination Centre (email) for more advanced data analysis services, such as mapping reads and variant calling.

Guidelines

For initial information on the RNA quality measurement, please use the guidelines prepared by the facility.

- Download the document on RNA quality measurement using capillary electrophoresis for RNAseq and array-based expression studies. 2014

Laatst gewijzigd:

22 maart 2019 16:07