Table 3:

Troubleshooting Advice

Step	Problem	Possible reason	Solution
26	DNA pellet is gooey	DNA is partially dissolved during the washes	Add small amounts of 7.5M Ammonium Acetate (drop by drop) to precipitate the DnA fully again.
93	Excess of dimers	Not enough starting material	Increase the number of starting cells. Cut the agarose gel above the end of the smear and perform a gel purification when preparing libraries.
93	Absence of smear containing Okazaki fragments	Insufficient yield	Perform a second amplification on the same batch of beads. The yield is significantly higher with this second PCR.
93	Absence of smear containing Okazaki fragments	dNTPs are degraded	Use a fresh aliquot of dNTPs and perform a second amplification on the same batch of beads.
93	Heavy banding instead of a smear	Adapters may have degraded	Use fresh aliquot or prepare new adapter on a new sample.
98	Smear on Tapestation is very faint with an excess of signals from the primer dimers	Not enough material	Increase the number of starting cells.
102	High number of reads containing primer-dimer sequences or decreased library complexity	Excess of primers, low amount of produced library	Increase input material. Optimize the number of PCR cycles to prevent excessive PCR duplicates. Test first the libraries on a low throughput machine (e.g. MiSeq) to assess library quality.