Grant and Publication Support Tools

Grant and Publication Support Guide

This guide provides the latest information about our products and technology for microarray, qPCR and Next-Gen Sequencing (NGS) sample preparation to facilitate in the writing of grant applications, scientific publications and abstracts.

I. Description of NuGEN Products

The following text describing NuGEN products may be copied directly into grant applications or scientific submissions.

Next Generation Sequencing

Encore® 384 Multiplex System
(PN 0315)
The Encore 384 Multiplex System consists of a refined set of 384 molecularly "barcoded" library adaptors that enable deep multiplexing of sequencing samples within a library preparation system that is compatible with high-throughput automation.

Encore® Complete Prokaryotic RNA-Seq DR Multiplex Systems
(PN 0327, 0328)
The Encore Complete Prokaryotic RNA-Seq Library Systems provide an end-to-end solution for strand-specific RNA-Seq library construction using as little as 100 ng of total RNA obtained from pure cultures of bacteria or mixed populations. The core technology used in this product enriches for non-rRNA in NGS libraries during cDNA synthesis, and can be applied to transcriptomes extracted from a broad range of prokaryotes. The first strand cDNA synthesis is carried out using proprietary primers to create double-stranded cDNA, which retains RNA strand information. The resulting cDNA is converted to NGS libraries using reagents and barcoded adaptors provided in the same kit.

Encore® Complete RNA-Seq Library Systems
(PN 0311, 0312, 0313, 0333, 0334)
The Encore Complete RNA-Seq Library Systems provide an end-to-end solution for strand-specific RNA-Seq library construction using as little as 100 ng of total RNA. The core technology used in this product enriches for non-rRNA in NGS libraries during cDNA synthesis, and can be applied to transcriptomes extracted from a broad range of higher eukaryotes. The first strand cDNA synthesis is carried out using proprietary primers to create double-stranded cDNA which retains RNA strand information. No dedicated steps are required to reduce rRNA levels. The resulting cDNA is converted to NGS libraries using reagents and adaptors provided in the same kit. The Encore Complete RNA-Seq Multiplex Systems provide optional barcoding for multiplex experiments to further optimize efficiencies and cost savings in transcriptome sequencing.

Encore® NGS Library Systems for Ion Torrent
(PN 0306, 0307)
The Encore NGS Library Systems for Ion Torrent™ provide a simple, fast and scalable solution for producing DNA libraries used in a wide range of sequencing applications with the Ion Personal Genome Machine™ (PGM™) from Life Technologies. The streamlined workflow enables library construction with no need for specialized equipment to perform size selection. Starting from as little as 100 ng DNA, sequencing libraries can be created for genomic DNA/exome sequencing, amplicon sequencing, RNA-Seq, Digital Gene Expression (DGE), ChIP-Seq and more. The efficiency and cost of sequencing projects can be further optimized using either the Encore NGS Multiplex Library System I for Ion Torrent or the System IB for Ion Torrent, to enable up to 16-plex multiplex sequencing on the Ion Chip.

Encore® Rapid Library Systems
(PN 0316, 0317, 0318, 0319, 0320, 0328)
The Encore Rapid Library Systems provide a simple, fast and scalable solution for producing libraries used in next-generation sequencing. These systems enable library construction starting with as little as 100 ng of double-stranded DNA, without PCR amplification. The library construction workflow is suitable for a wide range of sequencing applications including RNA-Seq, Digital Gene Expression (DGE), genomic DNA sequencing, amplicon sequencing and more.

Encore® Target Capture Module
(PN 0332)
The Encore Target Capture Module provides library adaptor-specific blocking reagents to enable target capture workflows with NuGEN® library solutions for the Illumina® NGS platforms. Target or sequence capture methods allow researchers to capture genomic regions of interest and use them for reduced-cost sequencing with simplified data analysis and increased sample throughput as compared to whole genome sequencing. The Encore Target Capture Module has been optimized for the SureSelect Target Enrichment System from Agilent Technologies.

Ovation® 3'-DGE System
(PN 7200)
The Ovation 3'-DGE System provides accurate expression analysis (tag profiling) suitable for quantification of any poly(A)+ transcripts, without the need for prior sequence knowledge. It employs a simple, fast and automatable workflow, powered by the Ribo-SPIA® Single Primer Isothermal Amplification technology and is compatible with all leading NGS platforms. Input for this system is either 10 - 100 ng of purified total RNA or direct cell lysate from 50 - 10,000 cells using the Prelude® Direct Lysis Module (PN 1400).

Ovation® Prokaryotic RNA-Seq System
(PN 9030)
The Ovation Prokaryotic RNA-Seq System provides a method for the analysis of microbial species and microbiome samples by enabling whole transcriptome profiling. The core technology used in this product enriches for mRNA in NGS libraries and can be applied to transcriptomes extracted from pure and mixed microbial cultures in an easy end-to-end workflow, from total RNA to sequencing. The first and second strand cDNA synthesis are carried out using proprietarily designed primers to create double-stranded cDNA . The resulting cDNA is compatible with NuGEN's Ovation Ultralow Library Systems as well as other library workflows using double-stranded cDNA as input for the creation of sequencing libraries.

Ovation® RNA-Seq FFPE System
(PN 7150)
The Ovation RNA-Seq FFPE System provides a fast and simple method for preparing amplified cDNA from FFPE-derived total RNA for RNA-Seq applications (transcriptome sequencing). Amplification is initiated at the 3' end as well as randomly throughout the transcriptome in the sample, making the Ovation RNA-Seq FFPE System ideal for amplification of the severely degraded and chemically modified RNA typically obtained from FFPE samples. This approach is ideal for processing samples for RNA-Seq on Next Generation Sequencing (NGS) systems, as reads are distributed across the transcript. The Ovation RNA-Seq FFPE System is optimized for the generation of double-stranded cDNA suitable for sequencing library construction for use with a variety of NGS platforms.

Ovation® RNA-Seq System V2
(PN 7102)
The Ovation RNA-Seq System V2 is based on Ribo-SPIA® technology, and provides a fast and simple method for preparing amplified cDNA from total RNA. The Ovation RNA-Seq System V2 is optimized for the generation of double-stranded cDNA suitable for sequencing library construction for use with a variety of NGS platforms.

Ovation® Ultralow Library Systems
(PN 0303, 0304, 0305, 0306, 0330, 0331, 0329)
The Ovation Ultralow Library Systems provide a simple, fast and scalable solution for producing libraries used in next-generation sequencing starting with 1 to 100 ng of DNA. The library construction workflow is suitable for a wide range of sequencing applications, including RNA-Seq, Digital Gene Expression (DGE), genomic DNA sequencing, target capture, amplicon sequencing, ChIP-Seq and more. The Ovation Ultralow Multiplex Systems provide optional barcoding to further optimize efficiencies and cost savings in transcriptome sequencing.

Ovation® WGA FFPE System
(PN 6200)
The Ovation WGA FFPE System provides a fast and simple method for preparing amplified DNA (SPIA®) from genomic DNA prepared from Formalin Fixed Paraffin Embedded (FFPE) tissue. The resulting amplified DNA may be used in a variety of downstream applications, including array CGH (aCGH) and sequencing library construction (requires the Encore® ds-DNA Module).

Microarrays and qPCR

Applause® 3'-Amplification System
(PN 5100)
The Applause 3'-Amp System offers a robust and simple amplification technology based on Ribo-SPIA® technology with sample sizes starting at 50 ng total RNA. The product delivers a single-tube, single purification, add-and-incubate assay that can be completed in a single day.

Applause® WT-Amp ST and WT-Amp Plus ST Systems
(PN 5500 and 5510)
The Applause Systems offer a robust and simple amplification technology based on Ribo-SPIA® technology with sample sizes starting at 50 ng total RNA. The product delivers a single-tube, single purification, add-and-incubate assay that can be completed in a single day.

Encore® Biotin Module
(PN 4200)
Designed for fragmentation and biotin labeling of cDNA, this module is optimized for use with Affymetrix GeneChip® micorarrays. In a simple, robust and automatable add and incubate process, target preparation is completed without the need for any purification steps.

Encore® BiotinIL Module
(PN 4210)
The Encore BiotinIL Module is designed to provide a rapid, simple and easily automatable approach for labeling of cDNA for analysis on the Illumina Whole-Genome Expression BeadChips. It is ideally suited for use with the Ovation® RNA Amplification Systems by NuGEN to enable the analysis of small and compromised RNA samples typically obtained from clinical specimens such as whole blood, LCM, sorted cells, fine needle biopsies and FFPE samples.

Ovation® FFPE WTA System
(PN 3403)
The Ovation FFPE WTA System is a whole transcriptome RNA amplification system based on Ribo-SPIA® technology for gene expression analysis of limited and degraded FFPE RNA samples. Amplification is initiated both at the 3' end and randomly throughout the entire transcriptome, enabling downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

Ovation® Pico WTA System V2
(PN 3302)
The Ovation Pico WTA System V2 is a whole transcriptome RNA amplification system based on Ribo-SPIA® technology, initiating at both the 3' end and randomly throughout the entire transcriptome. This System is intended for use with limited or degraded RNA samples such as LCM, FACS, biopsies, FNA and other limited biological specimens and Enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

Ovation® PicoSL WTA System V2
(PN 3312)
The Ovation PicoSL WTA System V2 is a whole transcriptome amplification solution for preparing cDNA from as little as 500 picograms to 50 nanograms of total RNA. The resulting cDNA can be used for qPCR, sample archiving and microarray gene expression studies.

Ovation® RNA Amplification System V2
(PN 3100)
This 3'-focused RNA amplification product based on Ribo-SPIA® technology provides a simple automation-friendly approach for use with intact and non-compromised RNA samples. The add-and-incubate process is amenable to high throughput applications, and enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

Ovation® WGA System
(PN 6100)
The Ovation WGA System offers a robust and simple amplification technology based on SPIA® (Single Primer Isothermal Amplification). This amplification system faithfully replicates genomic DNA, enabling a variety of downstream applications including copy number change analysis using real-time quantitative PCR (qPCR) or microarrays.

Ovation® Whole Blood Solution
(PN 1300)
The Ovation Whole Blood Solution consists of optimized products and protocols for global gene expression profiling of whole blood RNA without the need for globin reduction procedures. The fast, simple, automation-friendly workflow based on Ribo-SPIA® technology, enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

WT-Ovation® Exon Module
(PN 2000)
This module uses whole transcriptome amplified cDNA input to create an ST-cDNA sample for expression analysis on sense-target Affymetrix GeneChip® Gene ST and Exon ST arrays. The method does not require ribosomal RNA removal and is readily automatable.

WT-Ovation® RNA Amplification System
(PN 2210)
This whole transcriptome RNA amplification product based on Ribo-SPIA® technology is specifically designed for pre-amplification approaches prior to real-time quantitative PCR (qPCR) applications. Amplification is initiated at both the 3' end and randomly throughout the transcriptome providing flexibility in qPCR assay design.

WT-Ovation® One-Direct System
(PN 3500)
The WT-Ovation® One-Direct System enables whole-transcriptome gene expression analysis from a single cell. This single-day method prepares targets for microarray or qPCR analysis from lysate of one or more cells, or as little as 10 pg purified total RNA. WT-Ovation® One-Direct is specifically designed to enable the direct interrogation of cell lysate, eliminating the need for RNA purification.

Microfluidic Sample Preparation

Mondrian™ SP System
(PN 8000, 8010)
The Mondrian SP System enables sample preparation using digital microfluidics for liquid handling. The system software includes optimized protocols for a range of sample preparation methods that use pre-configured and quality controlled NuGEN reagents. Sample preparation is performed in sub-microliter volumes within low cost, disposable cartridges. The system provides plug-and-play automation with a simple user interface and load-and-go cartridge operation to significantly reduce the hands-on time for genomic sample preparation.

The Mondrian SP System consists of a benchtop workstation, microfluidic cartridges, system software that includes optimized protocols, and matched reagents.

Encore® SP Rapid Library System
(PN 8040, 8041, 8042)
The Encore SP Rapid Library and Encore SP Rapid DR Multiplex Systems enable fast, automated NGS library construction with no amplification, and are ideal for unbiased sequencing of genomes with unusually high or low GC content. Using 100 - 400 ng of genomic DNA or double-stranded cDNA as input, the protocol results in either non-multiplexed or multiplexed libraries ready for quantitation prior to cluster generation. These libraries are suitable for a wide range of sequencing applications on Illumina sequencing-by-synthesis platforms, including RNA-Seq, Digital Gene Expression (DGE), genomic DNA sequencing, amplicon sequencing, ChIP-Seq and more.

Ovation® SP Ultralow Library System
(PN 8030, 8033, 8034)
The Ovation SP Ultralow Library and Ovation SP Ultralow DR Multiplex Systems 1-8 and 9-16 provide automated, fast and scalable solutions for producing both non-multiplexed and multiplexed libraries starting with as little as 1.0 ng of DNA. The resulting libraries are suitable for a wide range of sequencing applications on Illumina sequencing-by-synthesis platforms, including RNA-Seq, Digital Gene Expression (DGE), genomic DNA/exome sequencing, amplicon sequencing, ChIP-Seq and more.

II. SPIA® and Ribo-SPIA® Technology

The following text summarizes the amplification technology use in NuGEN amplification products and may be copied directly into grant applications or scientific submissions.

SPIA® Amplification Process
Single Primer Isothermal Amplification (SPIA) is a DNA amplification process that uses a DNA/RNA chimeric primer (SPIA Primer), DNA polymerase and RNase H in a homogeneous isothermal assay providing highly efficient amplification of DNA sequences. (1) Genomic DNA (gDNA) is denatured, and a chimeric DNA/RNA primer mix (WG Primer) hybridizes uniformly across the input gDNA. The RNA portion of the WG Primer includes a unique sequence that serves as the tag for the subsequent SPIA process. DNA polymerase creates the first SPIA template strand by extending from the chimeric WG Primer. The second strand is then synthesized in the same tube without additional reagent addition to form the resulting double-stranded DNA (SPIA Template) with a DNA/RNA heteroduplex at one end that contains the unique tag sequence; (2) The SPIA Template is purified away from the excess primers with magnetic beads; (3) In the SPIA reactions, RNase H is used to degrade the RNA portion of the DNA/RNA heteroduplex introduced during the SPIA Template Synthesis reaction. This process results in exposure of a DNA tag sequence that is now available for binding to a second SPIA Primer. DNA polymerase then initiates replication at the 3' end of the primer, displacing the existing forward strand. The RNA portion at the 5' end of the newly synthesized strand is again removed by RNase H, exposing part of the unique priming site for initiation of the next round of DNA synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of DNA with sequence complementary to the original gDNA.

Ribo-SPIA® 3' Amplification Process
Ribo-SPIA technology is a three-step process that generates micrograms of cDNA from nanograms of total RNA. (1) First Strand Synthesis: Single stranded cDNA is prepared from total RNA using a chimeric DNA/RNA primers and reverse transcriptase. The primer has a DNA portion that hybridizes specifically to poly(A) sequences. The primer has a 3' DNA portion that hybridizes to the mRNA and a unique 5' RNA portion that does not hybridize to the mRNA. The resulting cDNA/mRNA complex includes the unique RNA sequence at the 5' end of the cDNA; (2) Second Strand Synthesis: After partial fragmentation of mRNA in the cDNA/mRNA complex, DNA polymerase generates a second strand, including DNA complementary to the 5' RNA unique sequence incorporated into the first strand, resulting in a double-stranded cDNA with an RNA/DNA heteroduplex of unique sequence at one end; (3) SPIA Amplification: The isothermal amplification step uses an additional DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the unique RNA sequence in the double-stranded cDNA revealing a site for binding the DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3' end of the primer and displacing the existing forward strand. RNA at the 5' end of the newly synthesized strand is again cleaved by RNase H, exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified cDNA products.

Ribo-SPIA® Whole Transcriptome (WT) Amplification Process
Ribo-SPIA® technology is a three-step process that generates micrograms of cDNA from nanograms of total RNA. Amplification occurs across the whole transcript, resulting in successful amplification of mRNA species, even including highly degraded samples. (1) First Strand Synthesis: Single stranded cDNA is prepared from total RNA using a unique mix of chimeric DNA/RNA primers and reverse transcriptase. This mix contains both random as well as poly T primers to achieve comparable representation across the entire transcriptome. The primer has a 3' DNA portion that hybridizes to the mRNA and a unique 5' RNA portion that does not hybridize to the mRNA. The resulting cDNA/mRNA complex includes the unique RNA sequence at the 5' end of the cDNA; (2) Second Strand Synthesis: After partial fragmentation of mRNA in the cDNA/mRNA complex, DNA polymerase generates a second strand, including DNA complementary to the 5' RNA unique sequence incorporated into the first strand, resulting in a double-stranded cDNA with an RNA/DNA heteroduplex of unique sequence at one end; (3) SPIA Amplification: The isothermal amplification step uses an additional DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the unique RNA sequence in the double-stranded cDNA revealing a site for binding the DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3' end of the primer and displacing the existing forward strand. RNA at the 5' end of the newly synthesized strand is again cleaved by RNase H, exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified cDNA products.

III. Publications

A searchable database of scientific publications using NuGEN products:
Publications

IV. NuGEN Patents and Trademarks

©2012 NuGEN Technologies, Inc. All rights reserved. The Ovation® and Applause® families of products and methods are covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251, 7,354,717, 7,771,946 and 8,071,311 and other issued and pending patents in the U.S. and other countries. NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners.

V. General Resources for Grant Writing and Research Funding

National Institutes of Health
Strategy for NIH Funding:
http://www.niaid.nih.gov/researchfunding/grant/strategy/pages/default.aspx

NIH Funding Opportunities:
http://www.niaid.nih.gov/researchfunding/ann/pages/opps.aspx

NIH Grant Programs:
http://grants.nih.gov/grants/guide/index.html

NIH Forms and Applications:
http://grants.nih.gov/grants/forms.htm

National Cancer Institute
NCI Funding Opportunities:
http://deainfo.nci.nih.gov/funding.htm

National Institute for Allergy and Infectious Disease
NIAID Funding Opportunities:
http://www.niaid.nih.gov/ncn/newsletters/default.htm