Table 5.

Troubleshooting advice.

Step	Problem	Possible reason	Solution
4	StringTie reports that it cannot open the files in the input mergelist.txt file	The GTF files specified in mergelist.txt do not exist, or you did not run the merge step in the same directory where the GTF files were created.	Either run StringTie in the same directory as the input GTF files or edit the mergelist.txt file to include the full path to each GTF file name description.
8	You get an error message that the file geuvadis_phenodata.csv cannot be opened.	The directory where you started your R session does not contain the phenotype data.	Before proceeding, you need to tell R where all the files are. You can do this with the setwd command. For example if the directory containing the chrX_data is your home directory, then you would use the following R command: > setwd(“~/chrX_data”) You can then run the getwd() command to make sure that the directory has been properly set.
9	The Ballgown function results in an error that the first column of pData does not match the names of the folders containing the ballgown data.	The sample names you have stored in your phenotype file do not match the file names of the samples you ran with StringTie.	To make sure that the file names match the IDs in the phenotype file, run the following R command: > all(pheno_data$ids == list.files("ballgown")) This command should return TRUE.
16	The transcripts and genes you assembled (shown in Table 3 and Table 4 respectively) have different values for the ‘gene id’ and ‘transcript name’ than what you see in the protocol here.	The transcript and gene identifiers assigned by StringTie (by default preceded by the string ‘MSTRG’) are generated in the order that the bundles of reads are processed. This order can differ from one run of StringTie to another when multiple threads are used (with the –p option).	This is not actually an error. If a transcript matches a known, annotated transcript, then the ‘transcript name’ will be consistent from run to run. Otherwise this field can change when you re-run the protocol.