Table 1.

Troubleshooting for DSAGE library construction and data analysis

Problem	Possible Cause	Solution
PCR for no-ligase negative control produced an 88bp band	Contamination from another library	Replace affected reagents. Keep a separate bench area (and materials) for pre- and post- amplification reactions
The 66bp pre- amplification library is not visible	Low yield	Use the marker to excise the 66bp.
No signal for low- expressing genes	Low complexity library or insufficient sequence depth	Check activity of enzymes and purification steps. The amount of material before amplification correlates with the complexity of the library. A total of 10-15 cycles should be sufficient for amplification.
The genes in the gene profiles display mostly null values	Genome assembly used for alignment may not match the annotation tables	Make sure that the genome assembly used for alignment matches the assembly of the reference tables (The provided reference tables with the package are for assemblies hg19, mm9, galGal3)
No signal for a gene that is expressed in the tissue	A small number of mRNAs does not have an NlaIII site	Verify that the gene has an NlaIII site by retrieving the mRNA sequence
The gene profiles of similar biological samples do not strongly correlate	Poor RNA quality	Use RNA with RNA Integrity Number > 8
	Library may not be uniformly amplified.	Keep amplification cycles within the exponential range
	Low complexity library Contamination from another library	Increase yield (see above) See above
Some program commands fail to return an output	Software may be incorrectly set-up. Also, insufficient memory, or other problems with the computing node.	Test-run software using a small dataset.