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™
