After using your primers for nGS sequencing, I have found out that the sequence corresponding to the primer is not identical to the primer sequence I have ordered. Why?
Base insertions are attributed to a small amount of detritylated
amidite present during coupling, while deletions are probably due to failure
sequences that did not get capped and were subsequently extended.
However, a better explanation for the observation of altered sequences is the incomplete deprotection of the oligo. If an oligo still bears a protecting group in one or more positions, this will be transferred to the subsequent PCR product, which is then transformed into E.coli. Here, the host mismatch repair system will likely attempt to correct the corresponding anomaly with a base, which might be the wrong one. The most likely culprit for incomplete deprotection is the isobutyryl protected dGs. These are the most difficult protection groups to remove.
In general, the longer the oligo, the greater the probability of side reactions during oligo synthesis, along with higher chances to incur into incomplete deprotection. Potential sources of side reactions causing failure products are depurination (which mainly affects the base A) and formation of secondary structure due to the oligos’ sequence. There is no way to completely exclude these effects! However, metabion tries to minimise these failures by continuously optimising synthesis as well as purification protocols!