Support FAQ's

Do I need to fragment my gDNA/cDNA prior to End Repair and Adaptor Ligation?

Please see the recommendations for your specific kit for information on fragmentation.

I don’t have access to a Covaris instrument, can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of Ovation Ultralow System V2, Celero DNA-Seq, and Ovation Rapid Library Systems. Other sonication instruments or enzymatic fragmentation may be suitable as long as the method generates a tight size distribution of DNA fragments. Please be aware that alternative methods require optimization by the end user. We also offer Celero EZ and Rapid EZ DNA-Seq which feature enzymatic fragmentation.

What do you recommend for quantification of final libraries prior to sequencing?

We recommend, and in certain cases require, quantifying your final library concentrations using a qPCR-based method. This is the most accurate method of quantification available. However, for most of our kits any fluorometric dsDNA quantification method should be acceptable. We have found that spectrophotometric quantification methods, such as NanoDrop, may significantly overestimate the library concentration.

Can I use cDNA as input into Tecan DNA-Seq kits?

Yes. Contact Tecan's NGS Technical Support for input recommendations.

Can I use FFPE or other degraded DNA as input into Tecan DNA-Seq kits?

We recommend using high quality DNA with an A260:A280 ratio of 1.7-2.0. While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples.

Do I need to fragment my cDNA prior to End Repair and Adaptor Ligation?

Many of our kits do not require fragmentation, or the fragmentation is built into the workflow of the kit with no need for additional reagents or equipment. Please see the recommendations for your specific kit for additional information.

Does Tecan provide reagents for performing the fragmentation step of the protocol?

Some of our kits include fragmentation during library preparation with no need for additional reagents or equipment; others do not provide reagents or do not require fragmentation. Please see the recommendations for your specific kit for additional information.

Are there other options besides Covaris fragmentation?

Yes. For our Universal RNA-Seq with NuQuant and Universal Prokaryotic RNA-Seq kits, the Covaris fragmentation step can be omitted with little or no impact on genes detected, FPKM concordance, or differential expression concordance. However please consult with Tecan NGS Tech Support for guidelines on library quantification and sequencing. With some samples Tecan has observed a modest elevation in the percentage of sequencing reads mapping to rRNA when using AnyDeplete probes targeting these transcripts. In addition, omitting Covaris fragmentation leads to a small increase in reads mapping to the 5’ end of transcripts. We also offer Universal Plus Total RNA-Seq with NuQuant which features integrated chemical fragmentation.

I don’t have access to a Covaris instrument, can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of Universal RNA-Seq with NuQuant and Universal Prokaryotic RNA-Seq. Other sonication instruments or enzymatic fragmentation may be suitable as long as the method generates a tight size distribution of cDNA fragments. Please be aware that alternative methods require optimization by the end user.

Can I customize AnyDeplete for transcripts other than rRNA? Can I customize AnyDeplete for other organisms?

Custom depletion designs can be tailored to any transcript, any organism.

What information do I need to provide for a custom AnyDeplete design?

We require a FASTA file with the sense strand of the sequences that you want targeted for depletion. You may optionally provide information about your organism’s reference genome to check for probe specificity.

What if I do not know which strand my transcript maps to?

For non-annotated genomes or sequences where the strand/sequence location is uncertain, we will design probes to both strands.

For more information about custom AnyDeplete, please contact Tecan Genomics technical support at techserv-gn@tecan.com.

What do you recommend for quantification of final libraries prior to sequencing?

We recommend, and in certain cases require, quantifying your final library concentrations using a qPCR-based method. This is the most accurate method of quantification available. However, for most of our kits any fluorometric dsDNA quantification method should be acceptable. We have found that spectrophotometric quantification methods, such as NanoDrop, may significantly overestimate the library concentration.

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).

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a spectrophotometer and a magnetic plate for 0.2 mL tubes, strips, or plates. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests. A system for fragmentation, such as a Covaris system, is required for use with most downstream library preparations.

Do you have a kit that can be used for qPCR, archiving and NGS?

The Ovation RNA-Seq System V2 generates ds-cDNA that can be used for all three purposes.

Does Ovation RNA-Seq System V2 deplete ribosomal RNA?

Ovation RNA-Seq System V2 does not selectively deplete ribosomal RNA. You may, however, observe a reduction in the amount of ribosomal RNA content with this system when used on mammalian samples.

Can contaminating genomic DNA interfere with Ovation RNA‑Seq System V2 performance?

Yes. This kit is designed to amplify RNA, but contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a spectrophotometer, a fluorometer and a magnetic plate for 0.2 mL tubes, strips, or plates. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests.

What do you recommend for quantification of final libraries prior to sequencing?

We recommend, and in certain cases require, quantifying your final library concentrations using a qPCR-based method. This is the most accurate method of quantification available. However, for most of our kits any fluorometric dsDNA quantification method should be acceptable. We have found that spectrophotometric quantification methods, such as NanoDrop, may significantly overestimate the library concentration.

Does Tecan provide reagents for performing the bisulfite conversion step of the protocol?

The bisulfite conversion reagents are included with the purchase of the Ultralow Methyl-Seq System with TrueMethyl oxBS (Part No. 0541-32 and 9513-A01) and the Ovation RRBS Methyl-Seq System 1-16 with TrueMethyl oxBS (Part No. 0553-32). Other commercially available bisulfite conversion kits may be suitable as well, but these have not been validated.

What do you recommend for quantification of final libraries prior to sequencing?

We recommend, and in certain cases require, quantifying your final library concentrations using a qPCR-based method. This is the most accurate method of quantification available. However, for most of our kits any fluorometric dsDNA quantification method should be acceptable. We have found that spectrophotometric quantification methods, such as NanoDrop, may significantly overestimate the library concentration.

For information on Methyl-Seq data analysis, please refer to the User Guide for your specific kit.

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.

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.