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?
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?
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