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Are there any special considerations for designing my NGS library amplification primers?

We recommend including 2 phosphorothiate bonds at the 3′ end of the primer to guard against enzyme mediated primer depletion. Next Generation Sequencing (NGS) library preparation steps tend to rely on high fidelity polymerases for PCR. These polymerases also have varying levels of 3′ exonuclease activity that can degrade primers and affect library amplification efficiency. If library amplification primers are depleted via enzymatic events, heteroduplex tangle structures can result from the continued amplification that occurs with the remaining dNTPs and other library amplification components still present in the reaction. The addition of the 2 phosphorothioate bonds is a simple way to protect the oligonucleotide primers from 3’ exonuclease degradation. Please contact our NGS Specialists at xgen@idtdna.com to discuss further.