What are abnormal Bioanalyzer traces for SMARTer stranded kits?
What are abnormal Bioanalyzer traces for SMARTer stranded kits?
All stranded kits:
Few reads from the sequencing run, or few clusters passing filter. SMARTer stranded libraries can have a lower than average pass-filter rate due to low complexity for the first three cycles. Illumina software has problems interpreting low complexity libraries. Decreasing the cDNA library loading concentration and/or spiking in 5–10% PhiX control DNA (Illumina) may correct this issue.
SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian:
Background in the no-RNA control. Background arises due to amplification of environmental contaminants in the reagents and is enhanced by PCR protocols that involve higher amplification cycle numbers. RNA control background will not be visible if performing up to 13–14 cycles in PCR2 of the workflow (typically, no background is detected for up to 18 total PCR cycles). If performing 16 cycles in PCR2, the total number of PCR cycles will be 21.