SIRVs: RNA-seq controls from @Lexogen

This article was commissioned by Lexogen GmbH.

My lab has been performing RNA-seq for many years, and is currently building new services around single-cell RNA-seq. Fluidigm’s C1, academic efforts such as Drop-seq and inDrop, and commercial platforms from 10X Genomics, Dolomite Bio, Wafergen, Illumina/BioRad, RainDance and others makes establishing the technology in your lab relatively simple. However the data being generated can be difficult to analyse and so we’ve been looking carefully at the controls we use, or should be using, for single-cell, and standard, RNA-seq experiments. The three platforms I’m considering are the Lexogen SIRVs (Spike-In RNA Variants), or SEQUINs, or ERCC 2.0 (External RNA Controls Consortium) controls. All are based on synthetically produced RNAs that aim to mimic complexities of the transcriptome: Lexogen’s SIRVs are the only controls that are currently available commercially; ERCC 2.0 is a developing standard (Lexogen is one of the groups contributing to the discussion), and SEQUINs for RNA and DNA were only recently published in Nature Methods.

You can win a free lane of HiSeq 2500 sequencing of your own RNA-seq libraries (with SIRVs of course) by applying for the Lexogen Research Award

Lexogen’s SIRVs are probably the most complex controls available on the market today as they are designed to assess alternative splicing, alternative transcription start and end sites, overlapping genes, and antisense transcription. They consist of seven artificial genes in-vitro transcribed as multiple (6-18) isoforms to generate a total of 69 transcripts. Each has a 5’triphosphate and a 30nt poly(A)-tail, enabling both mRNA-Seq and TotalRNA-seq methods. Transcripts vary from 191 to 2528nt long and have variable (30-50%) GC-content.

Want to know more: Lexogen are hosting a webinar to describe SIRVs in more detail on October 19th: Controlling RNA-seq experiments using spike-in RNA variants. They have also uploaded a manuscript to BioRxiv that describes the evaluation of SIRVs and provides links to the underlying RNA-Seq data. As a Bioinformatician you might want to download this data set and evaluate the SIRV reads yourself. Or read about how SIRVs are being used in single-cell RNA seq in the latest paper from Sarah Teichmann’s group at EBI/Sanger.

Before diving into a more in-depth description of the Lexogen SIRVs, and how we might be using them in our standard and/or single-cell RNA-seq studies, I thought I’d start with a bit of a historical overview of how RNA controls came about...and that means going back to the days when microarrays were the tool of choice and NGS had yet to be invented!

RNA-seq advice from Illumina

This article was commissioned by Illumina Inc.

The most common NGS method we discuss in our weekly experimental design meeting is RNA-seq. Nearly all projects will use it at some point to delve deeply into hypothesis driven questions, or simply as a tool to go fishing for new biological insights. It is amazing how far a project can progress in just 30 minutes of discussion, methodology, replication, controls, analysis, and all sorts of bias get covered as we try to come up with an optimal design. However many users don't have the luxury of in-house Bioinformatics and/or Genomics core facilities so they have to work out the right sort of experiment to do for themselves. Fortunately people have been hard at work creating resources that can really help and most recently Illumina released an RNA-seq "Buyer’s Guide" with lots of helpful information....including how to keep costs down.