Custom Next-Gen Sequencing Oligos

Regardless of next-generation sequencing (NGS) platform, universal and index adapter sequences are required for the proper assembly of sample fragments. Adapters – especially index adapters since they contain the MID (multiplex identifier) or barcode – containing too high a proportion of truncated sequences (unacceptably low purity) or too high a proportion of other adapter sequences (excessive cross contamination) can lead to compromised sequence read integrity (excessive adapter dimerization too) and improper sequence assembly, respectively, during multiplexing experiments. These types of problems are undetectable until the data analysis stage, which make them costly in terms of time and money.

Therefore, the production process (read a technical note about why HPLC will not lower cross contamination to an acceptable level) used for the adapter sequences is critical for a successful sequencing run. Our custom Next-Gen Sequencing Oligos (NGSO) are manufactured under rigorous conditions to ensure suitable purity and low cross contamination to meet research, commercial, and molecular diagnostic needs (read case studies about maximizing read length, minimizing adapter dimer formation, and manufacturing adapter kits with NGSO) by eliminating adapter-quality-at-fault failed sequencing runs. 

Use the table below to decide which of the three options is appropriate to meet your needs. The primary selection criteria should be which cross-contamination ceiling is acceptable along with the quality management system (ISO 9001 for research and commercial needs; ISO 13485 for molecular diagnostic needs). When in doubt, select NGSO-Gold for Illumina instruments and NGSO-Silver for the Thermo Fisher (Ion Torrent™) instrument.

Next-Gen Sequencing Oligos Specifications
Option	NGSO-Bronze	NGSO-Silver	NGSO-Gold
Cross Contamination1	<2%	<1%	<0.25%
Scale & Minimum Yield2	0.05 µmol (1 - 3 OD)  0.2 µmol (3 - 8 OD) 1.0 µmol (9 - 13 OD)  Inquire for larger scales	0.2 µmol (10 nmol or 2 - 6   OD depending on sequence   length) Inquire for larger scales	0.2 µmol (3 - 8 OD) 1.0 µmol (9 - 13 OD) Or, final yields: 25, 50, 100,   250 & 500 nmol Or, inquire for mg quantities
Purity	70-85%	65-80%	85-95%
Sequence Length	10 - 80 bases	10 - 60 bases	10 - 80 bases
Modifications	5’-Phosphate Phosphorothioate 5-Me-dC	5’-Phosphate Phosphorothioate 5-Me-dC	5’-Phosphate Phosphorothioate 5-Me-dC
Format	Supplied dry in tubes	Supplied in solution in   tubes or 96-well plates Standard: 100 µM in water,   TE (10 mM Tris, pH 7.5-8.0,   1 mM EDTA ), or Tris   (10 mM Tris-HCl, pH 8.0) Inquire: 30-150 µM in water   or 30-130 µM in   TE / Tris buffer Mixing Available: Yes Duplexing Available: No	Supplied dry or in water   / buffer in tubes   (pooling & aliquoting also   available)
Platform Compatibility	Illumina, Thermo   Fisher (Ion Torrent™),   & PacBio™	Thermo Fisher   (Ion Torrent™)	Illumina, Thermo Fisher   (Ion Torrent™), & PacBio™
Quality Control	100% Mass Spectrometry Analytical HPLC   available Certificate of Analysis   available	100% Mass Spectrometry Analytical HPLC available Certificate of Analysis   available	100% Mass Spectrometry Analytical HPLC available Certificate of Analysis   available
Quality Management System	ISO 9001	ISO 9001	ISO 13485
Typical Applications	Targeted sequencing   of select genes or   panels	SNP genotyping & variant   detection	Manufacturing of   commercial kits

¹Values determined via independent sources that used adapters manufactured with these processes. Multiplex sequencing was performed using Illumina instruments.
²Expected yields are valid for oligonucleotides 20 bases or longer.

Functional Quality Control

NGSO-Bronze, Silver, and Gold have undergone process validations to ensure that each provides adapters with cross contamination levels below the ceilings in the above table. However, prior to shipment, NGSO adapters as a matter of routine do not undergo functional testing with NGS to provide the actual cross contamination values. If you would like functional quality control of your adapters via NGS, please send requirements. We will perform a feasibility assessment (this includes consulting with you regarding the protocol), and if possible, provide a quotation with pricing and an estimated completion date. Testing will be performed on Illumina instruments.

Other NGS Oligonucleotides

In addition to the adapters, the major sequencing platforms often require other types of oligonucleotides for additional steps in the workflow, e.g. amplification primers (Figure 1). These other oligonucleotides do not have to meet the same low cross-contamination ceiling as the adapters; therefore, they can be ordered as standard Custom DNA Oligos.

Figure 1. A generic next-generation sequencing workflow and the required oligonucleotides.

However, we do recommend certain purifications based on the function of the oligonucleotides as shown in the table.

Recommended Purification for Non-Adapter Next-Gen Oligonucleotides
Platform	Illumina	Thermo Fisher (Ion Torrent™)	PacBio™
Amplification Primers	Cartridge or HPLC	Not available	Not applicable
Target Capture Probes	HPLC	HPLC	HPLC
Blocking Oligos	HPLC	HPLC	HPLC
Sequencing Primers	Cartridge or HPLC	Not applicable	Cartridge or HPLC
Multiplexing Primers	Not applicable	Not applicable	Cartridge or HPLC

Click here to order these additional, custom next-generation sequencing oligonucleotides.