SIRVs (Spike-In RNA Variants): external controls for RNA sequencing

[lexogen]

With the invigorated interest in NGS spike-ins we want to focus the posts dealing with Lexogen's SIRVs controls in this thread. While Lexogen will annouce news regarding the SIRVs in this thread, it is meant as a community resource for those interested in RNA spike-ins in general and in SIRVs in particular.

Lukas Paul

[lexogen]

Research Award - "Controlling RNA-Seq Experiments Using SIRVs"

If you plan to perform an RNA-Seq project soon and consider to use spike-ins, apply for Lexogen's Research Award and get a chance to win a set of (Spike-In RNA Variants SIRVs) and sequencing of your samples in an Illumina HiSeq 2500 lane. The splicing and transcription complexity of the SIRVs are key to test, validate and challenge your RNA-Seq workflow. Come up with a clever idea that puts the SIRVs to a good use and apply!

The deadline for your application is November 21st, 2016.

Please also forward this information about the Research Award to your colleague or a friend who would be interested, and get a chance to receive a treat for your lab from Lexogen!

Looking forward to your application,
the Lexogen SIRVs team

[lexogen]

Upcoming Webinar – Controlling RNA-Seq Experiments Using Spike-In RNA Variants

DATE: We, October 19th, 2016
TIME: 9:00 AM PDT, 12:00 PM EDT, 6:00 PM CEST

PRESENTED BY: Lukas Paul, PhD
Head of Services, Lexogen

Hardly anyone would run an RNA gel without a ladder, but transcriptomes are mostly sequenced without the use of external standards. The added layer of transcript isoform complexity in eukaryotes as well as incomplete or incorrect gene annotations further challenge RNA-Seq pipelines to correctly calculate and compare gene expression values. Lexogen, a specialized transcriptomics company, addresses this problem by providing Spike-In RNA Variant Control Mixes (SIRVs) to the RNA-Seq community. These controls are processed together with the RNA sample to allow for an evaluation of the RNA-Seq workflow and, in particular, of transcript isoform detection and gene expression quantification. The mixes contain 69 transcript variants that map to 7 human model genes and mirror the native transcriptome complexity by comprehensively representing splicing isoforms, transcription start-site and end-site variants, overlapping transcripts and antisense transcription. Lukas Paul, Head of Services at Lexogen, describes in this webinar how the SIRVs have been used to estimate absolute accuracy and consistency, as well as concordance in gene expression measurements at the level of workflows, experiments, and samples. A “SIRVs dashboard” is introduced that brings together spike-in derived NGS data, annotations and data evaluation in an easily navigable way, and the webinar will highlight how condensed SIRVs data can function as a “RNA-Seq fingerprint” that enables comparisons across experiments, samples and platforms.

Learning Objective 1: Learn practical considerations on the use of spike-in transcripts in RNA-Seq experiments
Learning Objective 2: Learn how RNA-Seq spike-in reads can be evaluated to assess gene expression quantification

(find out more...)

[lexogen]

Webinar on SIRVs now available on request

The Webinar Controlling RNA-Seq Experiments Using Spike-In RNA Variants is now available on request at LabRoots.

Watch it to find our about:

• The concept of complex RNA spike-ins covering transcription and splicing events
• Design of SIRV mixes to allow for RNA-Seq pipeline quality control and validation
• Assessment of differential gene expression at the transcript level
• The SIRV Suite, a Galaxy-based platform for easy and complete spike-in experiment design, data evaluation and comparison
• The concept of the quality matrix (coverage CoD, accuracy, precision) and concordance
• The SIRV Suite data base to find and research comparable RNA-Seq experiments - on the transcript level
• Upcoming RNA spike-in modules: from transcript length, poly(A) variants to base modifications

UPDATE: The Labroots webinar is now also available on YouTube: https://www.youtube.com/watch?v=uREp3NHWNXA

[lexogen]

Galaxy SIRV Suite now online for the evaluation of transcript isoform spike-in data

The Galaxy SIRV Suite is a set of software tools accompanying Lexogen's SIRVs product. It allows you to design and evaluate your SIRV experiment, and to compare it to other similar experiments. The SIRV Suite streamlines and unifies the data evaluation process. The program package has been embedded in Galaxy, allowing the easy integration of SIRV data evaluation into existing NGS data analyses workflows.

Access the SIRV Suite at: www.sirvsuite.org

The SIRV Suite runs in a CloudMan instance, data processing and reporting can be tested by simply uploading NGS data. The Galaxy installation with the SIRV Suite can be cloned to separate CloudMan instances, and alternatively, the SIRV Suite can be installed into already existing, or new, Galaxy installations.

SIRV resources:

• The SIRV Suite User Guide informs on how to technically operate the SIRV Suite
• https://www.lexogen.com/sirvs for information on molecular biology and statistical aspects of the analysis of SIRV-related experiments
• Webinar Controlling RNA-Seq Experiments Using Spike-In RNA Variants

[lexogen]

SIRV Suite now available as Command Line Interface (CLI) & Amazon Machine Image (AMI)

The SIRV Suite is now also available as CLI (Command Line Interface)
The SIRV Suite tools (currently: Experiment Specifier, Evaluator, and Comparator) can be downloaded and run on your linux system without Galaxy interface at https://github.com/sirvsuite-support...e/master/tools
The software is provided under GNU General Public License v3

Amazon Machine Image configuration
For a less complex yet "private" use of the SIRV Suite tools Lexogen provides an AMI (Amazon Machine Image), which is essentially a preconfigured virtual linux machine hosted by Amazon. Customers with an AWS account can use this AMI / SIRV Suite CLI configuration in a ready-to-go manner. Costs occur when using AWS but are clearly indicated.

If you have any questions, notice any bugs, or want to use an AMI, please contact us at: [email protected].

The Lexogen SIRVs team

[lexogen]

New sets of RNA spike-ins for controlling every sequencing experiment

Lexogen has expanded its line of Spike-In RNA Variant (SIRV) sets to enable cost-effective RNA-Seq experiment concordance determination and to combine isoforms with ERCC controls

The Spike-In RNA Variant external RNA controls (SIRVs) 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. They have been useful in determining the performance of long read technologies and to assess single-cell setups.

However, Lexogen believes that every RNA-Seq sample should contain a small fraction of SIRVs 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. 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 the updated product page https://www.lexogen.com/sirvs and consult publications featuring the use of SIRVs at https://www.lexogen.com/publications...-publications/

[Opplatek]

Dear Lexogen. Looking at the SIRV gtf annotation I have noticed transcript lines often contain different strand than their annotated exons. Why is that? For example, transcripts SIRV108 and SIRV109 have on their transcript line - strand, but their exons (SIRV108_0-SIRV108_2 and SIRV109_0-SIRV109_2) have + strand. According to the Excel information, these transcripts should be on the + strand.

[lauren.sheehan]

Good morning,

My name is Lauren and I am an application scientist with Lexogen. I apologize for the delay in responding to you and would like to ask for your email address so I can contact you directly regarding your question. It may be easier for you to email tech support at [email protected], and you can direct your email to me. Whichever option works best for you.

Thank you and I will talk to you soon,

Lauren

[jobsedu]

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