Table 2:

Troubleshooting

Step	Problem	Possible Reason	Solution
4	Oligo library sequences not generated	Illegal characters in header or protein sequence.	Remove illegal characters
23	Low ELISA signal	Poor binding of capture antibody. TMB expired.	Make sure proper ELISA plates are being used, TMB not expired.
26	ELISA data not within dynamic range	TMB was developed for too long or not long enough.	Increase or reduce the TMB development time.
50, 54	No PCR product	Incorrect primers were used. Incorrect thermocycling conditions were used. dNTPs expired.	Carefully repeat with fresh reagents.
56	Crash or program freeze.	This step essentially rewrites the entire data set and thus may use more disk space than is available.	Ensure your filesystem has enough disk space.
57	Too much of library is missing	Library was bottlenecked during construction or became too skewed during expansion.	Reconstruct library
58	Extreme, unreproducible enrichments	Contamination by host bacterial cells	More stringent centrifugation to remove cells, addition of antibiotic to prevent growth
58	Unreproducible enrichments	Sample cross-contamination. This can usually be determined by examining overlapping hits between samples.	Avoid antibody cross-contamination, PCR1 cross-contamination, PCR2 barcode cross-contamination

Timing

Steps 1–6, peptide library design: 1 day

Steps 7–8, construction and expansion of the phage screening library: 3 weeks

Steps 9–26, IgG quantification by ELISA: 1 day

Steps 27–41, antibody binding and immunoprecipitation: 2 days

Steps 42–55, DNA sequencing library preparation: 1 day

Steps 56–60, PhIP-Seq data processing: several hours