Sequencing quality suddenly drops mid-read (Sanger Sequencing)

Question

Accepted Answer

Early bases read cleanly, but the chromatogram becomes noisy and unreadable from the middle onwards. Common causes: 1) Insert region has high GC content or strong secondary structure 2) Repetitive sequences within the read confuse the polymerase 3) Primer is positioned too far from the target region 4) Template has primary or secondary structures (e.g. hairpins) that stall polymerase Solutions: 1) Walk the read with sequencing primers closer to the difficult region; split into multiple primers 2) Choose primers closer to the target site (≤ 800 bp ideal) 3) Increase template purity or switch to a high-fidelity sequencing polymerase 4) Add sequencing additives (DMSO, betaine) or optimize reaction conditions for difficult templates