Table 1. Primer sets and their limitations.

Step in protocol	Primer Set	Desired Outcome for Sample	Limitations	Solution
4	Set 1, Initial Primers	Detection	Does not select specifically for expanded arrays. Can only detect expanded arrays when adaptation occurs in at least 10% of the entire bacterial population	Try size selection and re-amplification
17(A)	Set 2, Internal Primers	Detection	Does not specifically bind expanded arrays and thus does not provide an extra level of detection	Try primer set 3 or 4 for more specific binding of expanded arrays
17(A)	Set 2, Internal Primers	Sequencing	Creates a PCR bias in deep sequencing results and could strongly influence the prevalence of certain spacer sequences	Remove duplicate sequences during analysis. Use biological replicates to confirm observed trends
17(B)	Set 3, Degenerate Primers	Sequencing	Creates a PCR bias due to both stronger G-C annealing at the 3’ end and exclusion of the primer ending with the same 3’ nucleotide as the existing leader proximal spacer	Complete a normalization during analysis that accounts for the bias introduced by omitting a primer containing the same 3’ nucleotide as the first spacer21.
17(C)	Set 4, Repeat Primers	Sequencing	Cannot be used for all systems due to the differing repeats sequences. The primers designed need to bind in opposite directions specifically within the repeat sequence. Subsequently these primers may be very short and if the repeat region for a system is AT-rich it may be difficult to design primers with an appropriate annealing temperature.	Try Set 3, degenerate primers
17(C)	Set 4, Repeat Primers	Sequencing	Requires further optimization of annealing temperature to prevent PCR artefacts arising from other sections of the array incorrectly amplified in PCR 1	The degenerate primers (Set 3) require less optimization but increase the PCR bias
17(C)	Set 4 Repeat Primers	Sequencing	Creates PCR bias in deep sequencing results and could strongly influence the prevalence of certain spacer sequences	Remove duplicate sequences during analysis. Use biological replicates to confirm observed trends