MiSeq errors on scFv linker

[superpyrin]

Hi, we used MiSeq 250 to sequence amplicon library of scFv antibody variants. Heavy and light chains are connected by a peptide linker (HeavyChain-GGGGSGGGGSGGGGS-LightChain). We saw dramatic increase in quality of forward reads in the region of the linker from which it never recovered (see figure). We think it is due to high G content and repetitive occurrence of GGC/GGT motifs that are known to cause sequencing errors.

What can be done to maintain a good quality throughout the read?
Can this be resolved by suitable sample preparation?
Can you recommend alternative scFv linkers that are known to work well with MiSeq?

[GenoMax]

Was there a spike-in added (phiX or some other high diversity DNA)? If not that would be one thing to start looking at.

[superpyrin]

yes, there was around 60% of PhiX

[GenoMax]

Have you talked with Illumina tech support to see if they have any recommendations? What was the cluster density for this run? Since you had plenty of phiX you are not going to see any additional improvement by that strategy.

You need to start thinking about changing your experimental strategy by inclusion of some spacers (padding) in your primers etc. Someone with experimental expertise will need to add the specifics for how you could do that.