The Spike-In RNA Variant (SIRV) external RNA controls are currently employed in the assessment of RNA sequencing workflows, the comparison of sequencing platforms and protocols, and in the improvement of data evaluation software, among others.1 They have been useful in determining the performance of long read technologies and to assess single-cell setups.2-4 However, Lexogen believes that every RNA-Seq sample should contain a small fraction of spike-in control transcripts to enable not only a technical validation but also the assessment of concordance within and between experiments as well as across platforms.5 Users can then determine whether RNA-Seq experiments are comparable and to which degree. Therefore, the external RNA controls are now available in three distinct SIRV sets, catering for complex validation projects as well as for cost-sensitive applications.
SIRV-Set 1
This set continues the previously available controls set-up; it consists of the 3 SIRV Isoform Mixes E0, E1, and E2 each containing 69 isoform transcripts mapping to 7 genes at concentration differences ranging from equimolar to 128-fold. This Set 1 allows for a thorough validation of RNA-Seq pipelines that rely on the detection and quantification of isoforms; differential gene expression can be assessed even on the isoform level – e.g. by making use of the freely available SIRV Suite software.
SIRV-Set 2
This set contains only the SIRV Isoform Mix E0. Its equimolar setup makes it ideal to assess platforms with high and low read-depth for accuracy and precision of isoform detection. Due to its cost-efficient pricing (starting from EUR 0.27 / USD 0.29 for spiking a 100 ng total RNA sample) SIRV-Set 2 is designed to be broadly applicable for determining concordance between data sets from any RNA-Seq experiment.
SIRV-Set 3
Extending the range of available SIRV modules, SIRV-Set 3 provides the SIRV isoforms mixed with single-isoform ERCC transcripts. For this, the 69 isoforms of the equimolar mix E0 were combined with 92 transcripts of the External RNA Controls Consortium (ERCC) that have unique sequence identity and span a concentration range of 6 orders of magnitude. By using only a single, pre-determined mix, SIRV-Set 3 allows for the efficient evaluation of isoform-specific workflows as well as the assessment of dynamic range, dose response, strandedness, and lower limit of detection. The SIRV Isoform/ERCC Mix is compatible with low and high read-depth setups and can be used to determine experiment concordance by thoroughly assessing the comparability of data sets.
New, dried format
Lexogen has also solved the problem of dry-ice shipment and the degradation-prone storage of RNA standards: the new SIRV-Set 2 and SIRV-Set 3 are now delivered in an innovative, dried format and upon resuspension, the controls can be stored in aliquots at -20 °C for extended periods of time. This enables users to safely spike experiments also at later time-points and to make use of the SIRV sets to the greatest extent.
For more information on the SIRVs please visit its updated product page https://www.lexogen.com/sirvs and consult publications featuring the use of SIRVs at https://www.lexogen.com/publications/sirvs-publications/
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