Table 3.

Troubleshooting.

Step	Problem	Possible reason(s)	Solution(s)
10, 13	Signal from negative controls during real time PCR monitoring	Cross contamination from current samples or previous libraries; primer artifact formation	Rigorous cleaning with 10% bleach of all work areas; reprepare library with additional care and maintain separate pre- and post-PCR areas; ensure template concentration is high enough that PCR#1 is run for fewer than 30 cycles and PCR#2 is run for fewer than 20 cycles.
17	Library size smaller than expected	Insufficient template in PCR#1 leading to amplification of a primer artifact instead of target amplicon; contamination	Increase template DNA for PCR#1 or incorporate PCR#0 into the workflow if working with small template amounts. Increase amplification cycles in PCR#1. Assess product size from PCR#1, and if applicable, PCR#0 to confirm amplification of the correct target. Ensure that PCR#1 and PCR#2 reach the early or mid-exponential phase of the amplification. Use negative controls to rule out contamination.
17	Library size larger than expected	Overamplification in PCR#2; contamination	Reduce amplification cycles in PCR#2. Use negative controls to rule out contamination.
18	Large variation in sample coverage	Inaccurate pooling strategy	Prepare sample pooling based on real-time PCR data; quantify separately before pooling for sequencing.
18	Substantially fewer sequencing reads than calculated	Insufficient cycles to add index adapters; inaccurate library quantification	Ensure PCR2 runs for at least 10 cycles; ensure Qubit quantification is not substantially over-estimating library concentration due to non-specific product formation.
18	Large fraction of unidentified reads in Illumina data	contamination by previous library preparations; poor read quality	Rigorous cleaning with 10% bleach of all work areas; reprepare library with additional care and attention to cross contamination.
23	Compilation of Read 1 and Read 2 sequences takes an unreasonably long time	Analysis entails a number of read-write operations	This step entails a large number of read-write operations. Choose a drive with faster I/O capabilities for analysis to shorten run time.
23	Compilation of Read 1 and Read 2 sequences fails	Analysis-provided Blat version may be incorrect	The pipeline includes the version of Blat that is current at the time of this publication. Future versions of Linux and OSX operating systems may require Blat to be updated to the latest version. Check the blat version provided against your operating system, and download the newest version of blat from http://hgdownload.soe.ucsc.edu/admin/exe/. Depending on your operating system, replace blat_linux or blat_osx file in the 1-pair_counting folder of the pipeline with the updated and renamed version.
24	Rscript for filtering identifier fails	Lack of compatible R versions	Check that R and R package version numbers are correct.
26	Rscript for final filtering reports orphan barcodes	Large deletions truncate part of an identifier	Check that the correct founder is selected for this sample. Additional orphan barcodes can be manually corrected in Final-Filtering.R. If the problem is pervasive, this may reflect large errors in the library preparation process or in Illumina sequencing, or cross contamination between samples or previous libraries.