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Sample Preparation for NGS


The optimal sample preparation is very critical for NGS library preparation and sequencing. Depending on the definition the sample preparation can also include nucleic acid extraction. Depending on the application and the used protocol, sample prep include cDNA synthesis, target enrichment, depletion (for example ribosomal RNA depletion), and target amplification.


On this website different solutions and products for bisulfite conversion and methylated DNA purification, for target enrichment, for rRNA depletion, as well as for whole genome amplification and whole transcriptome amplification are listed.


 

Bisulfite Conversion

Cells-to-CpG™ Bisulfite Conversion Kit

The bisulfite method is the most commonly used technique for identifying specific methylation patterns within a DNA sample. It consists of treating DNA with bisulfite, which converts unmethylated cytosines to uracil but does not change methylated cytosines. Bisulfite conversion has been utilized in DNA methylation research for the last 20 years with few improvements to the technology until now. With thorough optimization, the Cells-to-CpG™ Bisulfite Conversion Kit provides a quick, streamlined method for bisulfite conversion to reveal methylated cytosines in either loci-specific or genomewide analyses.
Source: Thermo Fisher

Website


Ribosomal RNA Depletion

GeneRead rRNA Depletion Kit

GeneRead rRNA Depletion Kits effectively remove ribosomal RNA, while ensuring complete recovery of mRNA and noncoding RNA from various species, including human, mouse, and rat. By improving the ratio of useful data, decreasing bias, and preserving non-coding RNA types, the kits provide high-quality RNA that is especially suited for next-generation sequencing (NGS) applications. In addition to rRNA, globin mRNA can be effectively depleted from total RNA isolated from human whole blood with GeneRead Globin mRNA Depletion Probes.
Source: Qiagen

Website

NEBNext rRNA Depletion Kit (Human/Mouse/Rat)

The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) employs an RNaseH-based method to deplete both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human total RNA preparations. This product is suitable for both intact and degraded RNA (e.g. FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-Seq, random-primed cDNA synthesis, or other downstream RNA analysis applications.
Source: NEB

Website

RiboGone - Mammalian kit

The RiboGone - Mammalian kit specifically removes ribosomal RNA (rRNA) and mitochondrial RNA (mtRNA) sequences from human, mouse or rat total RNA samples. The RiboGone technology is based on hybridization and RNase H digestion that specifically depletes 5S, 5.8S, 18S, and 28Snuclear rRNA sequences as well as 12S mtRNA sequences. The kit is designed to work with any quality starting RNA, including degraded material like FFPE samples. The RiboGone - Mammalian kit is highly suited for sample preparations prior to random-primed cDNA synthesis and RNA-seq or transcriptome analysis applications. Following the use of RiboGone - Mammalian, and the SMARTer Stranded RNA-Seq Kit or the SMARTer Universal Low Input RNA Library Prep Kit, rRNA reads have been recorded to be between 1-5% of total reads and lower.
Source: Clonetech

Website

RiboMinus™ Technology

RiboMinus™ technology is designed to enrich the whole spectrum of RNA transcripts by selectively depleting ribosomal RNA molecules (rRNA), regardless of their polyadenylation status or the presence of of a 5'-cap structure. The RiboMinus™ method has been shown to remove the vast majority of the most abundant ribosomal RNA molecules (up to 99.9%) to allow for greater interrogation of less abundant transcripts.
Source: Thermo Fisher
Thermo Fisher offers a wide range of different RiboMinus kits for various species.

Website

Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat)

Ribo-Zero (Human/Mouse/Rat) offers the most informative sequencing results by removing unwanted ribosomal RNA prior sequencing. The Ribo-Zero (Human/Mouse/Rat) kit removes cytoplasmic (nuclearencoded) rRNAs and the Ribo-Zero Gold (Human/Mouse/Rat) kit removes both cytoplasmic and mitochondrial rRNAs.
Source: Illumina

Website


Target Enrichment

Target specific sequencing panels are listed under Target Enrichment Panels.

 

Target Enrichment Instruments

Ion OneTouch™ 2

The Ion OneTouch™ 2 System simplifies the workflows for the Ion Proton™ and Ion PGM™ systems by providing an automated solution for scalable and reproducible template preparation.
Source: Thermo Fisher

Website

RainDance ThunderStorm® System

The RainDance ThunderStorm® System is a fully automated, high-throughput NGS content enrichment solution that enables researchers to process more samples per day and generate higher-quality data, faster than ever before. The system features true walkaway capabilities, automatically processing up to 96 samples and accessing up to eight different primer panels. The ThunderStorm System leverages RainDance’s proven single-molecule picodroplet PCR technology that generates millions of unique picodroplet PCR reactions, enabling scientists to target up to 20,000 genomic loci in a single sample. The RainDance ThunderStorm System combines premium performance and a proven technology platform to deliver high daily sample throughput with the most attractive running economics and minimal hands-on time.
Source: RainDance Technologies

Website

ThunderBolts™ System

The ThunderBolts™ System is a fast, accurate and low-cost NGS target enrichment system that features award-winning instrumentation and pre-validated gene panels, as well as flexible open source options for scientists looking for custom solutions. The fast and simple workflow is optimized for Illumina NGS systems and compatible with liquid biopsy, FFPE and tissue samples with as low as 10 ng of starting DNA. Researchers gain rapid, accurate mutation profiles for a low overall cost per sample.
Source: RainDance Technologies

Website


Target Enrichment Kits

GeneRead DNAseq Custom Panels V2

GeneRead DNAseq Custom Panels V2 are the simplest tools for analyzing the genetic variants of a customized panel of genes or genomic regions via next-generation sequencing. Each panel consists of multiplex PCR primer sets, sufficient for 480 samples, to amplify genomic regions of interest tailored to your specific NGS needs. Our primer design and targeted enrichment chemistry provide high design coverage, specificity, and uniformity, which are essential for detecting low-frequency variants in your precious samples.
Qiagen offers different solutions for target enrichment.

Source: Qiagen

Website

HaloPlex custom kits

HaloPlex custom kits provide you with unparalleled flexibility to create panels focused on your genomic regions of interest for any application. Using a simple, library prep-free workflow, complete target enrichment is achieved in less than 6 hours from just 200ng of input DNA. HaloPlex offers superior specificity and coverage that allows you to confidently identify genetic variants, faster.
Source: Agilent

Website

Ion TargetSeq™ Custom Enrichment Kits

Ion TargetSeq™ Custom Enrichment Kits provide a solution-phase DNA probe capture technology that enables selective, specific, and economical enrichment of customer-defined regions of interest in a single tube.
Source: Thermo Fisher

Website

NxSeq® DNA Sample Prep Kit 2

The NxSeq® DNA Sample Prep Kit 2 contain NxSeq Enzyme Mix, NxSeq 2X Buffer, and NxGen™ High Concentration Ligase. Platform-specific adapters are not included and must be supplied by the user. Complete protocols are available on the resources tab.
Source: Lucigen

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Nextera Rapid Capture Custom Enrichment

Nextera Rapid Capture Custom Enrichment provides unparalleled access to genomic regions of interest. This custom assay allows researchers to sequence precious samples faster and more efficiently than ever before, using as little as 50 ng of input DNA. The kit offers add-on functionality to refine content over time, or add regions of unique interest to established panels like Nextera Rapid Capture Exome or TruSight content sets.
Source: Illumina

Website

Ovation cDNA Module For Target Enrichment

The Ovation cDNA Module For Target Enrichment is an optimized system for producing double-stranded cDNA from input total RNA and is used in conjunction with the Ovation Fusion Panel Target Enrichment System and Ovation Custom Gene Fusion Target Enrichment System products.
Source: Nugen

Website

Ovation Fusion Panel Target Enrichment System

The Ovation Fusion Panel Target Enrichment System includes pre-defined content for all exons of 500 genes identified by the Wellcome Trust Sanger Institute's Catalog of Somatic Mutations in Cancer (COSMIC) and ChimerDB 2.0 as being involved in fusion events (Figure 1). This comprehensive fusion panel is used in conjunction with the Ovation cDNA Module to produce double-stranded cDNA from input total RNA, and utilizes NuGEN's proprietary Single Primer Enrichment Technology (Figure 2) to produce multiplexed enriched libraries ready for sequencing.
Source: Nugen

Website

Ovation® Custom Target Enrichment System

Ovation® Custom Target Enrichment System The Ovation Custom Target Enrichment System enables target enrichment studies with researcher-defined genomic or transcriptomic content using NuGEN's proprietary Single Primer Enrichment Technology and is a complete solution for generation of enriched, multiplexed libraries from low-input amounts of genomic DNA or total RNA.
Source: Nugen

Website

REPLI-g UltraFast Kit

The REPLI-g UltraFast Kit includes REPLI-g UltraFast DNA Polymerase, which enables highly uniform and accurate whole genome amplification from small samples in just 60–90 minutes, resulting in typical DNA yields of 7 μg per 20 μl reaction. A variety of starting materials can be used, including genomic DNA, fresh or dried blood, buccal swabs, fresh or frozen tissue, and cells. The DNA generated is suitable for direct use in a range of genetic analyses.
Source: Qiagen

Website

SureSelectXT Reagent Kits

The SureSelect Target Enrichment System is the most proven target enrichment solution today. This technology enables the capture of genomic targets using long 120nt RNA baits which allow for efficient enrichment of regions of interest facilitating confident variant calling. The SureSelectXT Reagent Kit enables post-capture pooling of enriched sequencing-ready libraries. This protocol enables the generation of libraries from either standard 3ug sample input, providing the highest complexity libraries, or lower 200ng input providing compatibility with samples of limited availability. With one library per capture, maximal efficiency and complexity is enabled providing deep coverage of regions of interest facilitating confident variant calling.
Source: Agilent

Website


Human Mitochondrion Enrichment

Ovation® Human Mitochondrion Target Enrichment System

The Ovation Human Mitochondrion Target Enrichment System enables target enrichment studies with the entire human mitochondrial genome using NuGEN's proprietary Single Primer Enrichment Technology and is a complete solution for generation of enriched, multiplexed libraries from low-input amounts of genomic DNA.
Source:Nugen

Website


Methylated DNA Purification

MethylMiner™ Methylated DNA Enrichment Kit

MethylMiner™ Methylated DNA Enrichment Kit enables superior enrichment and differential fractionation of double-stranded DNA based on CpG methylation density, with increased sensitivity over antibody-based methods. Fractionation permits important comparisons between samples and enables researchers to focus analysis on only the methylation densities of interest.
Source: Thermo Fisher

Website

NEBNext Microbiome DNA Enrichment Kit

The NEBNext Microbiome DNA Enrichment Kit facilitates enrichment of microbial DNA from samples containing methylated host DNA (including human), by selective binding and removal of the CpG-methylated host DNA. Importantly, microbial diversity remains intact after enrichment.
Source: NEB

Website


Poly A/ mRNA Purification

GeneRead Pure mRNA Kit

The GeneRead Pure mRNA Kit allows highly specific polyA-oligo-dT–based purification of mRNA ready for next-generation sequencing (NGS) applications. By effectively depleting rRNA and non-adenylated, non-coding, as well as regulatory RNA from a wide variety of eukaryotic species, the kit enriches for mRNA, ensuring optimal use of expensive sequencing capacity and resources. With a preparation time of <45 min and convenient automation options, the GeneRead Pure mRNA Kit offers unparalleled convenience by minimizing the hands-on time required to purify mRNA.
Source: Qiagen

Website

NEBNext Poly(A) mRNA Magnetic Isolation Module

The NEBNext Poly(A) mRNA Magnetic Isolation Module is designed to isolate intact poly(A)+ RNA from previously isolated total RNA. The technology is based on the coupling of Oligo d(T)25 to 1 μm paramagnetic beads which is then used as the solid support for the direct binding of poly(A)+ RNA.
Source: NEB

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NucleoTrap® mRNA Mini

For fast purification of poly(A) mRNA from total RNA

Website

Oligotex spin columns

Small spin columns are suitable for poly A+ mRNA isolation from up to 1 mg total RNA, up to 2 x 107 cells, or up to 100 mg tissue. Oligotex spin columns are provided in Oligotex mRNA and Oligotex Direct mRNA Micro and Mini Kits.
Source: Qiagen

Website

PrepX PolyA mRNA Isolation Kit

The PrepX PolyA mRNA Isolation Kit provides complete walk-away automation of polyA mRNA isolation from total RNA. With 15 minutes of hands-on time and 40 minutes of run time, you can generate 1 to 8 DNA libraries on the Apollo 324 System using our PrepX PolyA protocol. For higher throughput, use the PrepX PolyA 48 protocol to prepare 6 to 48 libraries with 30 minutes of hands-on time and 3 hours, 15 minutes of run time. The output polyA mRNA samples can be then processed in RNA-Seq libraries using one of our PrepX mRNA library protocols. Order the same kit PrepX PolyA mRNA Isolation kit for use with either PrepX PolyA protocol.
Source: Wafergen

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WGA & WTA Kits

REPLI-g Cell WGA & WTA Kit

The REPLI-g Cell WGA & WTA Kit uniquely enables uniform whole genome amplification (WGA) and whole transcriptome amplification (WTA) in parallel reactions, allowing direct correlation of the genome with the transcriptome from very small samples (25–1000 cells). Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to block amplification of contaminating nucleic acids by the REPLI-g method.
Source: Qiagen

Website

REPLI-g Single Cell Kit

For highly uniform whole genome amplification (WGA) from single cells or limited sample material.
The REPLI-g Single Cell Kit is specially designed to uniformly amplify genomic DNA from single cells (1 to <1000 bacterial or tumor cells) or purified genomic DNA with complete genome coverage. Additional protocols are also available for use with fresh or dried blood or fresh or frozen tissue. Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to avoid amplification of contaminating DNA, ensuring highly reliable results every time.
Source: Qiagen

REPLI-g WTA Single Cell Kit

For whole transcriptome amplification of total RNA or mRNA from single cells.
The REPLI-g WTA Single Cell Kit enables reliable investigation of effects on transcription regulation at the single-cell transcriptome level and allows uniform amplification of all transcripts from just single cells (1–1000 cells). Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to block amplification of contaminating nucleic acids by the REPLI-g method. The innovative lysis buffer effectively stabilizes cellular RNA, ensuring the resulting RNA accurately reflects the in vivo gene expression profile.
Source: Qiagen

Whole Transcriptome Amplification (WTA1 & WTA2) Kits

The TransPlex Whole Transcriptome Amplification (WTA1) and the Complete Whole Transcriptome Amplification Kit (WTA2) provide an accurate, fast and simple method of amplifying total RNA from a variety of sources including blood, fixed and frozen tissue, cell culture, FACS sorted cells, plants and microorganisms. The WTA technology accurately amplifies total RNA by the utilization of a unique blend of quasi-random primers to ensure accurate transcriptome coverage and rapid amplification of total RNA. The resulting cDNA is suitable for qPCR, micro array, and traditional cloning.
Source: Sigamaldrich

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cDNA synthesis kits and enzymes for customized target enrichment and sample preparation are listed under Enzymes.