General Sequencing Questions

What is your optional qPCR step for? Can I skip this? Why don’t you recommend a range of library amplification cycles for different inputs?

The optional qPCR step is for estimating the number of cycles needed during Library Amplification. Due to the large variability in sample types and inputs going into the workflow, we recommend that this step be optimized for each specific sample. If this is your first time running the kit, or if a new type of sample is being used, we highly recommend performing this step. For qPCR estimation we recommend 20x EvaGreen (Biotium).

Can I use SYBR green instead of EvaGreen in the optional qPCR step?

We recommend using 20x EvaGreen (Biotium) with the reagents supplied in the kit. EvaGreen binds ds-DNA more specifically than SYBR green, providing a more accurate cycle estimation. In addition, substituting the EvaGreen and reagents in the kit with a 2x SYBR green master mix may provide a different result than the reagents provided with the kit. Please follow the instructions for your specific kit.

For information about input recommendations, fragmentation, sample multiplexing, and stopping points in the protocol, please reference the User Guide and FAQs for your specific product.

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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:

  • 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.
  • Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  • Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN 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?
  • Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  • Ensure that the beads are fully resuspended in solution before adding to the sample.
  • 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.
  • Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.
I have unexpected peaks around 40-80 bp in my BioAnalyzer/Tapestation traces. What are these and how do I get rid of them?

Primer dimers are commonly observed at a peak size between 40-80 bp, depending on the kit. To get rid of these, you may perform a bead purification using the same ratio as the final Library Purification to remove the contaminant. Ensure that the ethanol used is prepared fresh and the beads are allowed to fully dry before eluting to ensure maximum yield and avoid ethanol carryover.

I have unexpected peaks around 120-160 bp in my BioAnalyzer/Tapestation traces. What are these and how do I get rid of them?

Adaptor dimers are commonly observed at a peak size between 120-160 bp, depending on the kit. To get rid of these, you may perform a bead purification using the same ratio as the final Library Purification to remove the contaminant. Ensure that the ethanol used is prepared fresh and the beads are allowed to fully dry before eluting to ensure maximum yield and avoid ethanol carryover.

My library yields are low. How can I increase my library yields?
  • The most common step where sample is lost is during SPRI bead purifications. Ensure that all recommended incubation times are followed, 70% ethanol is prepared the day of the experiment, and all ethanol has evaporated from the beads prior to elution of the sample.
  • Adaptor ligation is another common step where sample is lost. Due to the viscosity of the ligation buffer, we recommend careful pipetting and thorough mixing of both the master mix and the samples with the master mix. Inefficient mixing of the ligation master mix can reduce the ligation efficiency, leading to reduced final yields.
  • Increase the number of PCR cycles used in the library amplification step. Follow the recommended guidelines for library amplification and perform a qPCR optimization of the number of PCR cycles needed during library amplification for each sample where indicated. Please follow the instructions for your specific kit.
What do you recommend for quantitation of final libraries prior to sequencing?

We recommend 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, often significantly overestimate the library concentration.

Do you have a list of barcode sequences?

Please refer to the product page for your specific kit for a list of barcode sequences.

Do you have a sample sheet for sequencing setup?

Please contact NuGEN Technical Support for sample sheets.

What sequencers are compatible with NuGEN library preparation systems?

NuGEN library preparation systems are compatible with Illumina sequencing platforms. Please refer to the User Guide for your specific kit for additional recommendations.

Can you provide me with the adaptor sequences for trimming?

Please contact NuGEN Technical Support for information about adaptor sequences.

DNA-Seq Systems

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 Tapestation may also be useful for optional analytical tests.

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 these systems. Other means of fragmentation, such as sonication 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.

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.

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

We recommend quantifying your final library concentrations using a qPCR-based method. This is the most accurate method of quantification available and is required for PCR-free workflows such as those in the Rapid and Amplicon systems. However, any fluorometric dsDNA quantification method should be acceptable. We have found that spectrophotometric quantification methods, such as NanoDrop, often significantly overestimate the library concentration.

Can I use cDNA as input into NuGEN DNA-Seq Library Systems?

Double-stranded cDNA, including the SPIA cDNA generated by the Ovation RNA-Seq System V2 and Ovation FFPE RNA-Seq System may be used as input into the Ovation Rapid Library System and Ovation Ultralow System V2.

Can I use FFPE or other degraded DNA as input into NuGEN DNA-Seq Library Systems?

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.

RNA-Seq Systems

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 carry over of organics 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 Technical Support for more information.

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, we recommend a kit specific 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.

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 NuGEN 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 RNA-Seq library systems, including Universal, SoLo, and Complete Prokaryotic RNA-Seq, the Covaris fragmentation step can be omitted with little or no impact on genes detected, FPKM concordance, or differential expression concordance. With some samples NuGEN has observed a modest elevation in the percentage of sequencing reads mapping to rRNA when using AnyDeplete primers targeting these transcripts. In addition, omitting Covaris fragmentation leads to a small increase in reads mapping to the 5’ end of transcripts.

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 these systems. Other means of fragmentation, such as sonication 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, as well as information about your organism’s reference genome to check for probe specificity.

What if I my organism does not have an annotated/reference genome?

While we do not require an annotated/reference genome for custom depletion designs, we recommend that you submit a FASTA file of the draft genome or sequences available in order to check for probe specificity and minimize any off-target effects.

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 require the sequence for both strands.

For more information about custom AnyDeplete, please see our page on custom AnyDeplete designs.

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

We recommend 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, often 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).

Can contaminating genomic DNA interfere with the Ovation RNA‑Seq System V2 and RNA-Seq FFPE System performance?

Yes. These systems are 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 and a magnetic plate for 0.2 mL tubes, strips, or plates. An Agilent Bioanalyzer or Tapestation 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.

Do your Ovation RNA-Seq System V2 and Ovation FFPE RNA-Seq System deplete ribosomal RNA?

Our Ovation RNA-Seq System V2 and Ovation FFPE RNA-Seq System do 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.

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 Tapestation may also be useful for optional analytical tests.

Can genomic DNA interfere with the Ovation RNA-Seq FFPE System?

Yes. When using purified total RNA from FFPE samples, genomic DNA may be amplified as well. For this reason we recommend DNase treatment during RNA purification.

Target Enrichment

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 Tapestation may also be useful for optional analytical tests.

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 these systems. Other means of fragmentation, such as sonication 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.

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.

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

We recommend 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, often significantly overestimate the library concentration.

Where can I get the scripts for duplicate determination?

For duplicate determination, you can download NuDup at https://github.com/nugentechnologies/nudup

Can target enrichment be customized to my fusion/gene/SNP/ region of interest?

Our target enrichment kits can be customized for a variety of targeted sequencing workflows including DNA targets, gene fusions, SNP genotyping and viral integration, as well as for FFPE samples. Please see our product pages for custom Gene Fusion Target Enrichment and custom Target Enrichment, or contact Technical Support for more information.

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 Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
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 carry over of organics 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 FFPE or other degraded DNA as input into Ovation Target Enrichment Systems?

We recommend using high quality DNA with an A260:A280 ratio of 1.8 or above. While high quality DNA will guarantee the best results, many of our kits will perform with degraded samples.

Methyl-Seq

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 Tapestation may also be useful for optional analytical tests.

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

The bisulfite conversion reagents are included with the purchase of the Ovation Ultralow Methyl-Seq System with TrueMethyl oxBS (P/N 0535/0536-32). For workflows with the Ovation Ultralow Methyl-Seq core kit (P/N 0335/0336-32), NuGEN does not provide the reagents used in the bisulfite conversion steps. The QIAGEN EpiTect Fast DNA Bisulfite Kit (QIAGEN Cat. #59824) is a suitable substitute. Other commercial bisulfite conversion kits may be suitable as well, but these have not been validated.

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

We recommend 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, often significantly overestimate the library concentration.

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

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Microarray & qPCR Products

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 Tapestation may also be useful for optional analytical tests.

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 carry over 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 Technical Support for more information.

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.

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:

  • 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.
  • Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  • Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN 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?
  • Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  • Ensure that the beads are fully resuspended in solution before adding to the sample.
  • 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.
  • Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

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