Support Center

Sequencing Support

Tecan offers NGS library preparation kits for DNA-Seq, RNA-Seq, Methyl-Seq and Targeted Genotyping. General information on sample preparation, library preparation and sequencing are provided below. For more information, please consult the User Guide for each product, or contact Tecan NGS Technical Support at techsupport-gn@tecan.com.




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Tab 01 / DNA-Seq
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DNA-Seq systems

 

Sample isolation and input recommendations

  • ds-cDNA or gDNA
  • 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.

 

Fragmentation

Tecan offers DNA-Seq kits with integrated enzymatic fragmentation as well as kits with mechanical fragmentation.

For standard workflows using the Celero DNA-Seq, Rapid Library System and Ovation® Ultralow V2 DNA-Seq kits, we recommend that cDNA or gDNA be fragmented 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 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 library preparation kits 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.

Note: Low complexity samples and workflows with custom sequencing primers may not be supported by all platforms. Refer to the recommendations for your specific sequencer.




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Tab 02 / RNA-Seq
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RNA-Seq systems

 

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

RNA input recommendations

 

RNA isolation

We recommend a column-based method, including:

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:

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

Sample type Revelo
RNA-Seq
Trio
RNA–Seq
Universal Plus
mRNA–Seq
Universal Plus Total
RNA–Seq
Universal Prokaryotic
RNA–Seq
SoLo
RNA–Seq
Universal
RNA–Seq
Total RNA              
Poly (A) RNA              
Plasma RNA              
Cell free RNA              
Nasal swab derived RNA              
Exosomes              
Whole cells/lysate              
Nuclear/nascent RNA              

 

RNA isolation

We recommend a column-based method, including:

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)

 

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.




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Tab 03 / Target Enrichment
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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:




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Tab 04 / Methyl-Seq
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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

 

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: