Troubleshooting Guide for NEBNext® rRNA Depletion Kit (Bacteria) (NEB #E7860)

Presence of Bioanalyzer peaks < 85 bp (Figure 7.1)

• 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 reaction again with 0.9X SPRIselect Beads or NEBNext Sample Purification Beads (second clean up may result in reduction of library yield)

Presence of ~127 bp adaptor-dimer Bioanalyzer peak (Figure 7.1)

• 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 before setting up ligation reaction
• Clean up PCR reaction again with 0.9X SPRIselect Beads or NEBNext Sample Purification 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 7.2)

• 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 heteroduplexes with different insert sequences 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

• Under-fragmentation of the RNA

Library size will contain longer insert sizes

• Increase RNA fragmentation time