NEBNext® Ultra™ Directional RNA Kit Troubleshooting Guide

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  •  Observations  Possible Causes  Effect Suggested
    Solutions
     
    Presence of Bioanalyzer peaks <85 bp
    (Figure 1)
     • Presence of Primers remaining after PCR clean up  Primers cannot cluster or be sequenced, but can bind to flowcell and reduce cluster density  • Clean up PCR again with 1.0X AMPure beads (second clean up may result in reduction of library yield)
    Presence of 127 bp adaptordimer Bioanalyzer peak
    (Figure 1)
     • Addition of non-diluted adaptor
     • RNA input was too low
     • RNA was over fragmented or lost during fragmentation
     • Inefficient Ligation
     Adaptor-dimer will cluster and be sequenced. If ratio is low compared to library, may not be a problem but some reads will be dimers.  • Dilute adaptor (10 fold dilution) before setting up ligation reaction
     • Clean up PCR again with 1.0X AMPure beads (second clean up may result in reduction of library yield).
    Presence of additional Bioanalyzer peak at higher molecular weight than the expected library size
    (~ 1,000 bp)
    (Figure 2)
     • PCR artifact (over-amplification). Represents single-stranded library products that have self-annealed. If the PCR cycle number (or PCR input amount) is too high; in the late cycles of PCR the primers become limiting. Therefore, the adaptor sequences on either end of the fragment anneal to each other. This creates molecules with different insert sizes that run slower in the bioanalyzer.  If ratio is low compared to library, may not be a problem for sequencing  • Reduce number of PCR cycles.
    Broad library size distribution
    (Figure 3)
     • Under-fragmentation of the RNA  Library size will contain longer insert sizes  • Increase RNA fragmentation time

    Figure 1

    Figure 2
    Figure 3