Custom Next-Gen Sequencing Oligos Overview

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)
• 5.0 µmol (Quote)
• Inquire for larger scales
Purity • 70-85% • 65-80% • 85-95%
Sequence
Length
• 10 - 80 bases • 10 - 80 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 in tubes
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 9001
• ISO 13485
Typical Applications • Targeted sequencing
  of select genes or
  panels
• SNP genotyping & variant
  detection
• Manufacturing of
  commercial kits
Yield, Price, &
Delivery Schedule
Please complete the online request form. Yield, Price, & the expected delivery time will be provided after feasibility assessment.

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 check the box during completion of the online request form. 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.

A generic next-generation sequencing workflow and the required oligonucleotides

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.