Oligonucleotides for Next Generation Sequencing and Diagnostics
Eurofins Genomics provides better DNA for NGS-based experiments, which mitigates your risk and improves sequencing results.
The expansion of NGS technology over the past decade has empowered scientists to explore a vast new frontier of possibilities. However, NGS projects are typically expensive and highly sensitive. Making a mistake can be extremely costly and even debase the results. Oligonucleotides play a critical role in NGS applications, and although the cost of oligos is relatively small in comparison to other materials, they can make or break your project.
What makes Eurofins Genomics different?
Eurofins Genomics is uniquely positioned to provide several high-quality solutions to ensure that the sequence data generated by NGS technologies is a highly accurate reproduction of the original sequence of the DNA of your sample. Critical to these solutions is the recently developed synthesizer platform developed by the engineers and scientists at Eurofins Genomics. This new technology can synthesize both short and long (up to 200 mers) oligonucleotides of varying scales (intermediate between novel chip-based and traditional synthesis scales) with high-fidelity and shorter synthesis cycle times. When coupled with experience in post-synthesis workflows gathered over three decades, Eurofins Genomics can customize a workflow for you (or in collaboration with you) to address all your custom NGS requirements.
Over the last decade Next-Generation Sequencing, a term that captures many different types of sequencing assays, has become cheaper, faster and more expansive in the types of data it can provide. From Whole Genome Sequencing to Transcriptome sequencing, NGS can provide researchers a wealth of data when compared to more traditional sequencing approaches.
Next Generation Sequencing (NGS) technologies are redefining several aspects of genetic research and discovery. The ability to use NGS data for clinical prognosis and diagnosis coupled with the advancements in high-scale computing and bioinformatics is fueling the growth in healthcare R&D. The AgBio, genealogy, and other applied markets are also using the large quantities of sequence data generated by NGS technologies to make advancements towards a healthier and better tomorrow. The fidelity of the NGS technology-generated sequence data is vital to the success of these advancements.
NGS Adapters and Unique Molecular Indices
Unique Molecular Indices (UMIs) are small unique sequences that can be added to adapter oligos to specifically identify particular samples in a given library. UMIs greatly increase the accuracy of quantifying transcripts that are present in libraries at low levels. They can also accurately identify the presence of PCR duplication artifacts. Usually, libraries need to be amplified in order to increase the material that is generated after the genomic DNA is fragmented. The presence of any inherent biases in the amplification of certain genomic regions may incorrectly under or over represent the true abundance in the library. UMIs can be used to tag these genomic regions prior to their amplification, and have their ratios measured after an NGS sequencing run to much more accurately display an estimate of their concentration in the original sample. UMIs can be used in wide range of applications, such as identifying low-frequency variants, de-duplication of reads, and more.
- Barcoded Adapters
- Random barcodes
- Duel indexing
- Wobbles / degenerate bases
- Adaptive pooling
Target Enrichment and Capture Panels
Initial NGS strategies generated very large amounts of genomic data that while somewhat useful, did not provide deep coverage to specific genomic regions of interest. To eliminate these data, protocols that allow for genomic targeting, or “capture”, of certain regions of interest were developed. These target enrichment strategies have become extremely useful to those who are examining numerous individuals for differences in specific genomic regions, such as single nucleotide polymorphisms or copy number variation. There are a number of protocols that are differentiated based upon the way in which the target region is captured.
- Hybridization panel
- Capture Panels
- Biotin modified EXTREmers
Hybridization-based assay utilize the fragmentation of DNA followed by the ligation of known adapter sequences. The adapters will contain sequence “barcodes” that are unique to each samples which can be searched for and extracted bioinformatically. These ligated sequences are then denatured and subsequently annealed with DNA or RNA single-stranded probes—most of which are biotinylated. Biotinylated allows for the annealed duplex to be captured via streptavidin beads, and after treatment leaves a many fold enrichment of targeted samples compared to the initial starting material. At Eurofins, we can work with you on your Hybridization probe needs. We have the capabilities to synthesize capture panels of probes up to 140 nucleotides in length along with the Biotin modification.
EXTREmers are long oligos up to 200 mer which are synthesized using high-fidelity chemistry on our exclusive new synthesis technology to ensure that the sequence is the most faithful representation of the ordered sequence. Biotin-labeling of these EXTREmers at the 5¢-termini makes these oligonucleotides particularly suited for target enrichment in NGS workflows. Furthermore, we can pool several of these biotin-labeled EXTREmers targeting a region or loci of interest to generate a capture panel. The capture panel can be designed to target genes related to a particular disease, e.g., cancer, inherited diseases, etc., or a specific region, e.g., exome, mitochondria, etc.
Pooling, Annealing, and Multiplexing
Pooling and annealing is a common need for NGS applications. One common example is amplicon sequencing using oligonucleotide pools synthesized for targeted genomic regions. Most often created by combining multiple oligos all at the same concentration, oligo pools can easily contain hundreds of oligos. High specificity assays can require combined oligos to be present at varying concentrations as well. Regardless of the complexity or size of your oligo pool needs, an oligo panel can be prepared utilizing our ISO-certified lab procedures to ensure batch to batch reproducibility.
- Confidence in results with Individually QC’ed oligos: the oligos that are included in your pool are synthesized in an array format and individually QC’ed. This ensures that there is a representation of each of every oligo of the designed oligos in the pools. Furthermore, our experts can assemble pools of any size at the desired concentration. Since there are no primer dropouts, you can be confident that all the regions are effectively represented in your targeted amplicon sequencing project.
- Reduction in Costs with lower synthesis scales and multiplexing: since oligonucleotides can be synthesized at lower synthesis scales, through our proprietary technology, than traditional array-based high-throughput synthesizers, it reduces the internal cost which can be passed along to the customer. Additional price savings can be realized by including molecular barcodes in these primers to allow for sample multiplexing.
These pools can be used for ultra-deep sequencing of PCR products for efficient variant identification and characterization. These pools can be designed to interrogate various genes or regions of the genome, including genealogical studies, carrier screening, noninvasive prenatal testing (NIPT), and much more.
- Multiplex Library Prep solutions with Eurofins Genomics Oligonucleotides: Multiple pairs of individually QC’ed, dual-indexed primers with NGS grade service can be annealed and pooled for use in Multiplex Library preparation. This protocol will ensure lower crosstalk between sequence data obtained from different samples.
- Forward and Reverse PCR Primer mixing: Equal molar concentrations of your primers are prepared separately and then mixed.
Our Production team offers a wide variety of customization options, including specific tubes or plates for delivery. Contact us (below) if you’d like more details and a quote. Our Special Projects representative will contact you with additional details.