Sequencing Difficult Templates
Various difficulties can arise when attempting to sequence a given template. Beyond any difficulties you may have finding a good set of primers, or subcloning a PCR product, a template itself may provide hindrances in many forms. For most templates, Retrogen’s standard sequencing service is well suited to achieve high quality results. There are, however, a few cases where our proprietary sequencing reactions may be necessary to improve your sequencing. If your templates fit into one of the below categories, consider using one of Retrogen’s Difficult Template Conditions for those sequencing reactions.
GC Rich Templates
There isn’t a defined percentage of GC-content that is used as a cut-off for classification as “difficult,” but if your template contains a GC content over 60% there’s a strong possibility that Retrogen’s GC-Rich conditions will provide increased quality in your sequence.
Templates that contain di- and trinucleotide repeats, inverted repeats, and/or palindromic sequences can also prove difficult under standard conditions. Multiple signals, and low quality base calls can be minimized with Retrogen’s Difficult Template conditions.
Hairpins & Secondary Structures
Hairpins, and other secondary structures can interfere with the sequencing reaction, causing regions to be lost or resulting in the appearance of multiple signals. Retrogen’s Hairpin conditions can stabilize your template and allow sequencing through these difficult regions.
Long Homopolymer Regions
A common source of homopolymers are long stretches of A/T’s resulting from reverse-transcription from mRNA templates. Homoplolymers can cause “slippage” leading to the appearance of multiple signals. Homopolymers can also lead to secondary structures, such as hairpins, which contribute to the difficulty of sequencing these regions. Retrogen’s Difficult Template conditions can stabilize the reaction and allow accurate sequencing through these regions.
Figure 1a shows a difficult template sequenced using Retrogen’s Standard Sequencing conditions. There’s a clear drop in signal intensity around base 470 when a homopolymer and GC-Rich region is reached. Using Retrogen’s Difficult Template Conditions, we were able to sequence through the difficult region, as seen in Figure 1b.