How to remove primer contamination from sequencing libraries? (free primers)

Primer and adapter-dimer contamination in sequencing libraries can lead to serious problems like barcode switching (also called barcode hopping).  Thus, these short molecules should be removed from the libraries as soon as traces of them become visible on the Bioanalyzer or equivalent. Please note that the Bioanalyzer uses double-strand-specific fluorescent dyes which have a very low affinity to single-stranded primers.  Thus, the Bioanalyzer assay severely underestimates the true concentration of free primer molecules.

  • Low concentrations of free primers and adapter-dimers can be removed with a bead cleanup e.g.  adding  1x the original volume in Ampure XP beads (or equivalent).
  • The more stringent option for primer removal is an Exonuclease VII single-strand digest  (e.g. with this  ExoVII) at 37C for 20 minutes using 1 ul enzyme and the accompanying buffer; followed by a bead cleanup with 1.6 x the original volume in Ampure XP beads. This will not remove primer-dimers.

Category: 04 Library Preparation and QC

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