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Custom Next-Gen Sequencing Oligos

Regardless of next-generation sequencing 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 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 used for the adapter sequences is critical for a successful sequencing run. Sigma’s custom Next-Gen Sequencing Oligos (NGSO) are manufactured under rigorous conditions to ensure suitable purity and low levels of cross contamination to meet research, commercial, and molecular diagnostic needs.

Use the table below to decide which of the four 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).

 

Next-Gen Sequencing Oligos Specifications
Option NGSO-1 NGSO-2 NGSO-3 NGSO-4
Cross Contamination1 <2% <1% <1% <0.25%
Scale • 0.05, 0.2, & 1.0 µmol
• Inquire for larger
  scales
• 0.05 & 0.2 µmol
• Inquire for larger scales
• 0.2 & 1.0 µmol
• Inquire for larger scales
• 0.2, 1.0, & 5.0 µmol
• Inquire for larger scales
Purity • 70-85% • 65-80% • 75-90% • 85-95%
Sequence Length • 10 - 80 bases • 10 - 50 bases • 10 - 80 bases • 10 - 80 bases
Modifications • 5’-Phosphate
• Phosphorothioate
• 5-Me-dC
• 5-Me-dC • 5’-Phosphate
• Phosphorothioate
• 5-Me-dC
• 5’-Phosphate
• Phosphorothioate
• 5-Me-dC
Format • Supplied dry in
  tubes
• Supplied in solution in 96-well plates
• Supplied dry in tubes • Supplied dry in tubes
Platform Compatibility • Illumina HiSeq™,
  MiSeq®, and
  NextSeq™
• Life Technologies
  Ion Torrent™
• PacBio™ RS II
• Life Technologies Ion Torrent • Illumina HiSeq, MiSeq, and NextSeq
• Life Technologies Ion Torrent
• PacBio RS II
• Illumina HiSeq, MiSeq, and NextSeq
• Life Technologies Ion Torrent
• PacBio RS II
Quality Control • 100% mass spectrometry
• Analytical HPLC available
• Certificate of Analysis available
Quality Management System • ISO 9001 (research applications)
• ISO 9001 (research, commercial applications)
• ISO 9001 (research, commercial applications)
• ISO 13485 (molecular diagnostic applications)
• ISO 9001 (research, commercial applications)
• ISO 13485 (molecular diagnostic applications)
Price2 Lowest     Highest
Turnaround Time2 Shortest   Longest


1Values determined via independent sources that used adapters manufactured with these processes. Multiplex sequencing was performed using Illumina instruments.
2This information along with the minimum yield is provided with the quotation.


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, Sigma does recommend certain purifications based on the function of the oligonucleotides as shown in the table.

Recommended Purification for Additional Next-Gen Oligonucleotides
Platform Illumina HiSeq, MiSeq, and NextSeq Life Technologies Ion Torrent PacBio RS II
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.