Table 6.

ShapeMapper Troubleshooting

Problem	Possible cause	Solution
No output files produced.	Missing pipeline binaries, file permissions not set correctly, missing files, incorrectly-formatted configuration file, or out of memory.	Ensure executable binary files trimPhred, parseAlignment, and countMutations are present in the ShapeMapper directory (see section on installing ShapeMapper). Check the log file (log.txt) in the run directory for details of the error. If running in a load sharing facility-enabled cluster computing environment, also check the output of the submitted job, which will display errors due to file permissions or memory issues that cannot be captured in the ShapeMapper log file.
Noisy reactivity profiles.	Incorrectly labeled samples, low read depths, poor signal above background (low RNA modification rate or cDNA mutation rate in SHAPE-modified sample), or DNA contamination.	Check mutation rate and depth histograms in RUN/output/reactivity_profiles to determine cause.
Missing samples.	Unbalanced sequencing library sample loading or failed PCR.	Quantify missing samples with a high-sensitivity assay and resequence. Redesign directed primers if necessary. Increase the amount of RNA in reverse transcription and increase the amount of first-strand cDNA in PCR.
Uneven read depth or regions of low depth.	RNA degradation, poor primer binding, or low RNA concentration.	Sequence using longer reads; use Nextera kits (if using the Illumina platform); use more RNA in reverse transcription; use a battery of paired PCR primers instead of random primers. For RNAs with regions of high AU-content, consider using the LNA+ random primers (Fig. 5).
No reactivity profiles produced, even though alignment files (./output/aligned_reads/*.sam) are present.	Sample name(s) in the “[profiles]” section of the configuration file do not exactly match the name(s) given in the FASTA reference sequence files (.fa).	Fix errors in the configuration file and rerun the generateReactivityProfiles stage.
Pipeline run incomplete, but no error message or unhelpful error message in the log file.	Out-of-memory error or other error that ShapeMapper does not currently identify.	Check the output from the ShapeMapper.py script itself, in addition to the log file (log.txt). Check the contents of the subfolders in the ./output/directory in order of module execution (Table 3). The first folder in which some or all of the expected output files are missing, blank, or empty indicates the stage that failed.