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Ovation® SoLo® RNA-Seq library preparation kit

Complete end-to-end sample to library prep solution for stranded, whole transcriptome RNA-Seq analysis of ultra-low inputs from exosomes, FFPE samples and cell-free RNA.
Overview
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Tab 01 / Overview
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Ultra-low input RNA-Seq library preparation kit

The Ovation® SoLo RNA-Seq System for ultra-low input RNA sequencing is a flexible end-to-end library preparation solution producing strand-specific, rRNA depleted libraries from 10 pg to 10 ng of total RNA.

Utilizing the highly flexible and customizable AnyDeplete technology, this ultra-low input RNA-Seq kit offers depletion of rRNA and other high-abundant transcripts to increase the dynamic range, reduce sequencing costs, and simplify data analysis.

Sample types include purified RNA or lysates from cell lines including fresh or FFPE tissue, liquid biopsy, cell-free RNA and other ultra-low input RNA samples.

 

Selection guide

Key benefits of SoLo RNA-Seq

  • Fully integrated workflow for whole transcriptome analysis of as little as 10 pg of precious samples, providing access to complete biological information.
  • Customizable, targeted transcript depletion with AnyDeplete, allowing elimination of unwanted reads after library preparation, even from ultra low inputs.
  • Direct integration with cell lysis protocols, providing an easy-to-use workflow for ultra-low RNA-Seq.




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Tab 02 / Highlights
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Fully integrated workflow for whole transcriptome analysis

Ovation SoLo RNA-Seq is a complete workflow enabling whole transcriptome analysis from ultra-low inputs.

Fully integrated workflow for whole transcriptome analysis

FSuperior whole transcriptome RNA-seq

Superior whole transcriptome RNA-seq

  • Assess whole transcriptome for complete biological information.
  • Consistent coverage across transcripts.

Libraries generated from 5 ng of cell-free plasma RNA with a DV200 score of 10-20 %. Analysis of the whole transcriptome provides complete information from coding, non-coding and regulatory transcripts.

Reproducible sequencing results across a wide range of inputs

  • Consistent results from 10 pg to 10 ng total RNA inputs.
  • Excellent reproducibility with FFPE samples.
Show more

Excellent reproducibility with FFPE samples

Ovation SoLo RNA Seq provides highly correlated data between fresh frozen and FFPE adenocarcinoma samples.

High correlation from low quality samples

High correlation between technical replicates from inputs as low as 10 pg of total RNA to 1ng, giving you confidence in your data.

Deplete unwanted transcripts even at ultra low-inputs

  • Detect more transcripts across a greater dynamic range.
  • Customizable depletion to eliminate any uninformative transcripts.

AnyDeplete dramatically eliminates rRNA reads from your library.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina HiSeq, MiSeq, NextSeq, MiniSeq

Library Type

Library Type

RNA

Starting Material

Starting Material

Total RNA, FFPE tissue, Cell-free RNA

Input Amounts

Input Amounts

10 pg – 10 ng




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with the Ovation SoLo RNA-Seq System?

The Ovation SoLo RNA-Seq System provides all necessary buffers, primers and enzymes necessary for library construction from cell lysates or isolated RNA. The kit also provides DNA Resuspension Buffer for purification elution steps. This kit does not provide EvaGreen used for Library Amplification qPCR nor the Agencourt RNAClean XP or AMPure XP Beads required for the purification steps. AnyDeplete probes used for targeted transcript depletion are available separately or in a bundle with the Core Kit. For custom designs Tecan will provide AnyDeplete probes with the Core kit.

Can this system be used with other library preparation workflows?

There is no need to combine the workflow with other library preparation workflows. The Ovation SoLo RNA-Seq System is an end-to-end solution designed to generate Illumina-compatible libraries starting from cells or total RNA.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a magnetic plate for 0.2 mL tubes, strips, or plates and a spectrophotometer or fluorometer. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A comprehensive list of required and recommended equipment can be found in Section II.B.

Input recommendations

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

  • Norgen Biotek Total RNA Purification Kit
  • Zymo Research Quick-RNA™ Kits
  • Arcturus PicoPure® RNA Isolation Kit
  • Ambion PureLink® RNA Mini Kit
  • Qiagen RNeasy Kits

For FFPE RNA isolation, we recommend a kit designed for FFPE samples, including:

  • Norgen Biotek FFPE RNA Purification Kit
  • Zymo Research Quick-RNA™ FFPE Kit
  • Arcturus® Paradise® PLUS FFPE RNA Isolation Kit
  • PureLink™ FFPE RNA Isolation Kit
  • Qiagen RNeasy FFPE Kit

Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method. Isolation methods that capture RNAs shorter than 200 nt may result in increased 5S and 5.8S rRNA. Do not use carrier RNA during isolation.

Can I use TRIzol® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.

Can I use Ovation SoLo RNA-Seq System with RNA from any organism?

The Ovation SoLo RNA-Seq System should be suitable for total RNA input from any organism. For information on organism specific rRNA or targeted transcript depletion contact Tecan NGS Technical Support.

Do I need to use high-quality total RNA?

When using with the Ovation SoLo RNA-Seq System, samples should be of high molecular weight with little or no evidence of degradation. While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of somewhat degraded samples. With such samples, users may experience lower yields and may encounter affected sequencing metrics.

Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Ovation SoLo RNA-Seq System?

No. The system is designed to use total RNA as input. rRNA depletion or poly(A) enrichment are not necessary. rRNA, as well as other transcript types, can be targeted for depletion using the AnyDeplete technology embedded in the workflow.

Can I sort my cells into PBS or another buffer prior to beginning the cell lysis protocol?

For best results we recommend sorting cells directly into the Lysis Buffer Master Mix prepared with the kit, or resuspending cell pellets in the Lysis Buffer Master Mix. Other buffers (e.g. sheath fluid) may inhibit reactions in the kit and yield variable sequencing results. It is recommended for other buffers not to exceed 1 ul. Contact Tecan NGS Technical Support for additional information.

What cell types are compatible with the lysis buffer provided with the kit?

The lysis buffer has been tested with mammalian cell inputs up to 500 cells and should be compatible with most dissociated cell types. Some direct cell inputs may need to be optimized depending on cell type. Decreased performance may be observed with cell types containing excessive lipids, minerals, collagen, etc. or a cell wall, or with very large cells. Non-dissociated cells, tissue, or LCM samples may require additional optimization of the workflow.

Can contaminating genomic DNA interfere with Ovation SoLo RNA-Seq System performance?

Yes, contaminating genomic DNA may be incorporated into libraries. For this reason DNase treatment is incorporated into both the Cell Lysis and Total RNA Input workflows.

General workflow

Does cDNA generated with the Ovation SoLo RNA-Seq System require fragmentation?

No, fragmentation of cDNA occurs during the cDNA Processing steps in the protocol.

Does this system contain a SPIA®-based amplification?

No. The cDNA is generated with random and poly(T) primers, but no SPIA-based amplification is used.

Can I combine the barcoded libraries prior to the PCR amplification step?

This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantified independently before being pooled for use on the sequencer.

Where can I safely stop in the protocol?

Samples may be stored at –80 °C after Cell Lysis. Samples may also be placed in short-term storage at –20 °C after First Strand cDNA Synthesis, End Repair, Adaptor Ligation, Library Amplification, or after any purification step.

Custom AnyDeplete

Custom depletion designs can be tailored to any transcript, any organism. Please contact Tecan NGS Technical Support at techserv-gn@tecan.com for more information on available existing designs or to develop a new custom design.

SPRI bead purifications

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.

What magnetic separation devices do you recommend for the SPRI bead purifications?

Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:

  1. Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the Tecan workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
  4. Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

Library quantification and qualification

How do I measure my final library yield? Can I use an Agilent Bioanalyzer to evaluate the product?

Please refer to section V.Q of the User Guide for guidelines on quantitative and qualitative assessment. A qPCR based-method is required in combination with the Agilent Bioanalyzer or Fragment Analyzer.

Are irregular Bioanalyzer traces normal for libraries generated with the Ovation SoLo RNA-Seq System?

Yes, irregular Bioanalyzer traces are normal for libraries generated with the Ovation SoLo RNA-Seq System.

What is the average size of the library generated by the Ovation SoLo RNA-Seq System?

Ovation SoLo RNA-Seq libraries are 300–350 bp in length for high-quality human samples and may be shorter for FFPE and other degraded samples. Libraries generated from model organism samples may also be longer or shorter than human libraries. Take care not to overestimate library size for loading on the sequencer.

How many bases do Ovation SoLo RNA-Seq System adaptors add to the library?

The adaptors add 132 bp to the Ovation SoLo RNA-Seq System library.

Sequencing recommendations

Is the Ovation SoLo RNA-Seq System compatible with all Illumina sequencing platforms?

Special sequencing parameters may be required depending on the Illumina sequencing platform used. Contact Tecan NGS Technical Support for recommendations.

How much material should I load into the sequencer?

Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.

Can the Ovation SoLo RNA-Seq System be used with paired-end sequencing?

Yes, it can be used for both single- and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The workflow generates library fragments with an average size of 330 bases.

What kind of sequencing primers can I use with your library?

Ovation SoLo RNA-Seq libraries are designed to use standard Illumina sequencing primers for both single- and paired-end sequencing when multiplexed with standard Illumina libraries (see User Guide section Sequencing Recommendations and Guidelines for more information). For dedicated Ovation SoLo RNA-Seq sequencing runs, contact Tecan NGS Technical Support for recommendations.

Data analysis

What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?

Each barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.

Do you recommend trimming sequences before downstream analysis?

Yes. We recommend trimming the 5’ end of the sequence as recommended in the Data Analysis section of the User Guide. This portion of the read corresponds to the overhang of the forward adaptor and may not accurately reflect the biological sequence.

Can I use standard alignment algorithms to analyze strand-specific sequencing data?

Yes. Strand-specific reads can be processed and mapped to reference sequences using the same methods used for other RNA-Seq libraries. Note that in libraries generated by the Ovation SoLo RNA-Seq System, the forward read corresponds to the sense strand.

What percentage of rRNA reads can I expect in my data?

The number of rRNA reads present in the sequencing results is dependent on the abundance of rRNA transcripts in the starting material. For a sample containing 10% mRNA and 90% rRNA, a 90% depletion of rRNA transcripts results in a sample containing 53% mRNA and 47% rRNA (i.e. 10% and 9% of the original pool of RNA, respectively).




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Tab 05 / Literature
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