Sequencing Support

RNA-Seq systems

cDNA generation (Ovation RNA-Seq System V2, Part no. 7102)

RNA input recommendations

• Total or poly A selected RNA
• High quality, with an A260:A280 ratio of 1.8 or above, an A260:A230 of 2.0 or above and a RIN score of 7 or above.
  Note: While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
• DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include:
• Qiagen RNase-free DNase (included with the RNeasy Mini Kit and RNeasy Micro Kit.
• Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Cleanup Kit.
• Column-based methods are not recommended as they may not provide sufficient removal of DNA.

RNA isolation

We recommend a column-based method, including:

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

Ensure that the elution volume/final RNA concentration is appropriate for the cDNA kit used. Tecan cDNA kits use input volumes of 5-10 µL.

Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.

cDNA clean-up

cDNA generated with the Ovation RNA-Seq System V2 should be cleaned up using a column-based method such as:

• QIAGEN MinElute Reaction Cleanup Kit
• QIAGEN QIAquick PCR Purification Kit
• Zymo Research DNA Clean & Concentrator TM -25

Alternatively, an SPRI bead-based system such as Agencourt® RNAClean® XP or AMPure XP are suitable. If choosing a bead-based method, we recommend using a magnet compatible with 0.2 mL tubes, tube strips, or plates.

Note: Columns or beads for final cDNA cleanup must be purchased separately.

Library preparation

cDNA prepared by the Ovation RNA-Seq System V2 is compatible with any library preparation kit that accepts ds-cDNA as input. Please contact Tecan NGS Technical Support for input recommendations.

Note: cDNA generated by the Ovation RNA-Seq System V2 must be fragmented prior to library prep. We recommend use of Tecan’s Celero, Celero EZ, Ultralow V2 or Rapid EZ library systems.

RNA-Seq library preparation kits

RNA input recommendations

• Total or poly A selected RNA
• High quality, with an A260:A280 ratio of 1.8 or above, an A260:A230 of 2.0 or above and a RIN score of 7 or above.
  Note: While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
• DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include:
• Qiagen RNase-free DNase (included with the RNeasy Mini Kit and RNeasy Micro Kit.
• Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Cleanup Kit.
  Note: Column-based methods may not provide sufficient removal of DNA.

RNA isolation

We recommend a column-based method, including:

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

Do not use carriers during RNA isolation.

Ensure that the elution volume/final RNA concentration is appropriate for the RNA-Seq kit used. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.

Fragmentation (optional; Universal and Universal Prokaryotic only)

• For standard workflows, we recommend that cDNA be fragmented to an average of 200-300 bp using a Covaris Focused-ultrasonicator. Refer to the DNA Shearing guide for your specific ultrasonicator. Follow the settings for the desired fragment size.
• Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Tecan NGS Technical Support for more information on these workflows.

Library preparation

Follow all library preparation steps as written, including bead ratios and incubation times. We recommend performing a qPCR step prior to library amplification of any new sample or input into our kits to determine the optimal number of library amplification cycles. Recommendations are provided in each User Guide, or contact Tecan NGS Technical Support for additional information.

Sequencing guidelines

Tecan’s RNA-Seq library systems are compatible with Illumina sequencing platforms. The length and number of reads should be chosen based on the needs of the experiment. General guidelines for library pooling can be found here. Please note that low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.

Target Enrichment systems (Allegro Targeted Genotyping)

Sample isolation and input recommendations

• gDNA
• Samples should be of high quality, with an A260:A280 ratio of 1.8 or above and an A260:A230 of 2.0 or above.
• Degraded DNA is not recommended for Allegro Targeted Genotyping kits.

Library preparation

Follow all library preparation steps as written, including bead ratios and incubation times. We strongly recommend optimization of library amplification cycle number when using any new panel, sample or input. A protocol for qPCR optimization is provided in each User Guide, or contact Tecan NGS Technical Support for additional information.

Sequencing guidelines

Tecan’s Target Enrichment kits are compatible with Illumina sequencing platforms.

Important: Allegro Targeted Genotyping kits require a custom R1 sequencing primer. Certain workflows may also require a custom index 2 sequencing primer. Custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer:

• NovaSeq Series Custom Primers Guide
• NextSeq System Custom Primers Guide
• MiSeq System Custom Primers Guide
• MiniSeq System Custom Primers Guide
• HiSeq System Custom Primers Guide

Methyl-Seq systems

Sample isolation and input recommendations

• gDNA
• Samples should be of high quality, with an A260:A280 ratio of 1.8 or above and an A260:A230 of 2.0 or above.
  Note: While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples. Degraded DNA is not recommended for oxidative bisulfite sequencing (oxBS) workflows.

Fragmentation

• The Ultralow Methyl-Seq library kit requires fragmentation of DNA prior to library preparation. For intact gDNA, we recommend using a Covaris Focused-ultrasonicator. Refer to the DNA Shearing guide for your specific ultrasonicator. Follow the settings for the desired fragment size.
• Alternatively, a Diagenode Bioruptor® ultrasonicator or enzymatic fragmentation such as NEBNext® dsDNA Fragmentase® may be used, but optimization and sample clean-up prior to library preparation may be required. Please contact Tecan NGS Technical Support for more information on these workflows.

Library preparation

Tecan’s Ultralow Methyl-Seq and RRBS Methyl-Seq library preparation kits require bisulfite conversion of DNA for successful library preparation. Both kits include the include TrueMethyl oxBS Module for bisulfite (BS) and oxidative bisulfite (oxBS) sequencing.

Sequencing guidelines

The Ultralow Methyl-Seq and RRBS Methyl-Seq library preparation kits are compatible with Illumina sequencing platforms. When preparing the library pool, addition of PhiX is recommended to add diversity to the BS and oxBS libraries. Please consult the product User Guide or email techsupport-gn@tecan.com for current recommendations.

Important:

• A custom sequencing primer, MetSeq1, is required for the forward read and is provided with both kits. Custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer:
• NovaSeq Series Custom Primers Guide
• NextSeq System Custom Primers Guide
• MiSeq System Custom Primers Guide
• MiniSeq System Custom Primers Guide
• HiSeq System Custom Primers Guide
• Refer to Illumina’s recommendations on sequencing low complexity samples, or contact Tecan NGS Technical Support for more information.