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