Table 1.

Troubleshooting Table

Step	Problem	Possible reason	Solution
Stage I
5	Error creating nucleotide encodings of peptides	Non-standard amino acid letters or characters in the peptide sequences	Look for ‘X’, ‘-’ or other non-standard amino acid characters in peptide sequences and remove them
	Non-unique sequences in first 40 nt of multiple encodings	Similar peptides can end up with identical nucleotide encodings for the first 40 nt, which is problematic if using a 75-cycle sequencing kit	Request multiple (e.g., 10) encodings per peptide and select lower scoring encodings, as necessary, to ensure that all encodings are unique across the initial 40 nt
Stage II
23	MRP is not going into solution	The pellet shape is not allowing for great interaction with the solvent	Break up the pellet by gently pressing it against the tube wall to allow for increased interaction with the solvent
		TE buffer has cooled down	Heat the tube that the MRP and TE buffer is in at 37 °C at 5-min intervals
24	The MRP concentration cannot be read by Qubit Broad Range RNA	The MRP concentration is too high	Dilute the MRP product further before quantification (e.g., 1:800)
		The MRP concentration is too low	Repeating the concentration step with the MRP can often get the product to the >300 ng/μL threshold. Otherwise starting DDP at a higher concentration and synthesizing MRP again is necessary to proceed
55	Bead aggregation	The prepared beads are old	Prepare fresh beads for each capture
73	Faint or absent top band	Capture was unsuccessful	Redo capture steps by using supernatants from Step 67 and rerun the gel
		Translation did not proceed as planned	If the top band is still faint or absent on the second PTC gel (above), repeat the translation step
Stage IV
132	Low raw read counts for specific samples	Loss of PepSeq library during antibody binding assay; uneven pooling of samples before sequencing	Re-run samples that have low raw read counts. For very diverse libraries (e.g., 244,000 unique peptides), we recommend an average of ≥2× reads per peptide. For less diverse libraries (e.g., 15,000 unique peptides), we recommend an average of ≥10× reads per peptide
	A high proportion of unobserved peptides (i.e., zero counts)	Molecular bottleneck of PepSeq library during antibody-binding assay (i.e., loss of most of the PepSeq probes during processing)	Re-run affected samples or replicates
136	Lack of correlation between Z scores of sample replicates	Contamination; samples swapped or misnamed	Re-run the replicates for samples for which Z scores do not correlate between replicates
137	The number of enriched peptides is unexpectedly high or low	Incorrect highest density interval (HDI) setting for PepSIRF zscore module	Adjust the HDI setting to ensure that true enriched peptides are excluded from the calculation of mean and standard deviation for each bin. Lower HDIs are appropriate for library/sample combinations that result in a larger proportion of enriched peptides
		The Z score thresholds for the PepSIRF enrich module are not appropriately set	Run the q2-ps-plot zenrich module to generate Z score enrichment plots to visualize multiple Z score thresholds and pick an appropriate threshold that includes only enriched peptides (i.e., visually distinct from the cluster of non-enriched peptides). For example, a Z score threshold of 10 would be appropriate for the assay shown in Fig. 5