In NGS applications the use of molecular tags (MTag) to label individual genomic DNA templates prior to PCR amplification provides valuable benefits.
- Original DNA molecules are uniquely identified, counted, and/or analyzed.
- PCR and sequencing induced biases and errors are detected and then removed analytically, reducing false positive rate.
- Variant calling accuracy is increased resulting in improved low allele frequency detection limit.
The MTags are commonly introduced through adapter ligation.
Molecular Tag Design Considerations
- In a Relay-PCR™ specific primers participate only the first two extension reactions. Their participations in amplification reactions are negligible. Therefore, they are the right vehicles to deliver the molecular tags into amplicon seeds.
- Six quaternarily degenerated nucleotides are used in each tag forming 46=4,096 unique tag sequences which would then form 46×2=16,777,216 unique paired tag sequences. This size is sufficient to largely avoid having 2 starting DNA copies carrying the same tag sequence at an input level of 10 ng human genomic DNA (3,000 copies).
- Non-degenerate nucleotides are mixed with the degenerate nucleotides in each MTag to make the tag somewhat rigid. The type and the arrangement of the non-degenerate nucleotides are carefully designed so as to minimize the probabilities of folding formation within individual Omega Primers™ and cross-hybridization between the tag sequences and all primers involved.
Introduction of MTags into amplicon seeds through Omega Primers