Step	Section	Problem	Possible reason	Solution
3	Trinity assembly	‘bad_alloc’ error	Insufficient computing resources resulting in a fatal out-of-memory error.	Ensure you have ~1G of RAM per ~1 M PE reads to be assembled. See Box 2 for computing requirements and services available.
3	Trinity assembly	Large numbers of fusion transcripts	Not using strand- specific RNA-Seq, or applying assembly to a transcriptome derived from a compact genome having (minimally) overlapping transcripts.	If PE reads are being used, try running Trinity.pl with the ‘--jaccard_clip’ parameter, which uses PE reads to separate minimally overlapping transcripts.
3	Trinity assembly	Retained introns are prevalent	Unprocessed RNA is captured and assembled, or contaminating genomic DNA contributes to the assembly.	Setting Trinity.pl ‘--min_kmer_cov’ to 2 or higher should reduce the number of retained introns, but will also reduce sensitivity for transcript reconstruction. Alternatively, lowly expressed transcripts (often enriched for retained introns) can be filtered from a given component post- abundance estimation.
5-11	Quality assessment and abundance estimation	Cannot find makeblastdb, blastn, or Bowtie	The additional required software tools were not installed or available via the Unix PATH setting.	See EQUIPMENT section, be sure software tools are installed as required and that the software utilities are accessible via your PATH setting. Check with a systems administrator as necessary.
13-15	Differential expression analysis	Few or no transcripts identified as differentially expressed	Assuming transcripts are truly differentially expressed, increased sensitivity is required to detect them.	Adjust the sensitivity thresholds of ‘analyze_diff_expr.pl’, increasing the allowed FDR and lowering the fold- change requirements. Try running Bioconductor tools directly and examine the available options for data exploration. Increase your depth of sequencing to improve upon the detection of lowly expressed transcripts.