Table 1.

Troubleshooting

Step	Problem	Possible Reason	Solution
6	Small colony sizes and slow growth for some library strains on target plates	Insufficient amount of cells transferred from the source plate to the target plate at step 6.	Replicate in 1:2 format instead of 1:4 (i.e. use 1 source plate for 2 target plates instead of for 4). Plates should still be pinned in two cycles, and on the second cycle, the plates should switch red/blue positions, as one would normally do for 1:4 replication. For particularly difficult scenarios, library plates can be grown for an additional 12 hours prior to mating to increase colony sizes.
15	Poor growth on all 3 replicates	Library array plates for mating were taken directly from 4 °C storage.	Grow both query and library plates for 24 hours (from fresh pinnings) prior to mating (step 7) to ensure efficient mating.
15	Spatial artifacts on all 3 replicates	Uneven plates or insufficient colony transfer at either library array (step 6) or mating (step 7).	Ensure that library array plates are even with consistent growth across plates. Ensure that mating plates are even with consistent colony transfer from both query and library plates. For library array troubleshooting, see the entry for step 6 in this table.
15	Spatial artifacts or poor growth on one replicate	Uneven plates, damaged pads, or insufficient colony transfer at SPO (step 9) or HS1 (step 10).	Ensure that SPO and HS1 plates are even, and that colony transfer at steps 9 and 10 is maximized (see ‘Critical step’ flags). If colonies on diploid plates are very small after 48 hours of growth (step 8), they can be grown an additional day prior to pinning to SPO (step 9). For general tips regarding plate pouring, see Pouring agar-based media into plates in the Reagent setup section. Recycling of pads (cleaning and re-use) can result in damages, so check any recycled pads for damaged pins prior to pinning.
18	Incorrect alignment of colony measuring grid	May occur for plates with particularly poor growth.	The colony measure program provides a manual mode for re-processing plates that experience this issue.