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Larger than expected fragment sizes during Illumina library preparation

November 17, 2011

A common problem during Illumina library preparation is that after the library is prepared and it is run on the Bioanalyzer, larger than expected fragment sizes are observed. At first thought this makes no sense because the fragments are much larger than the fragments that were used to make the library. There are two reasons I know of that cause this.

1. The library is ‘over amplified’. When primers become limiting during the PCR reaction, fragments with different inserts anneal through the adapter sequences at the ends. This results in DNA molecules with a bubble shape or daisy chains of several DNA molecules. These bands typically start at two times the expected size for your library.
Solution: Since the library is denatured prior to application to the flow cell, there is no need to to anything. If you want to make sure this is the problem, you can add fresh primers, dNTPs and enzyme to your library and do one cycle of PCR.

2. You have Ampure XP beads in your library. This can happen from using a magnet that is too weak, not waiting enough time for the beads to separate or accidentally disturbing the pellet when removing the supernatant. This results in a much higher band around 1,500 bp in your Bioanalyzer readout.
Solution: Put your libraries back in the magnetic separator for a longer period of time.


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One Comment
  1. Alber Michaeel permalink

    Hi Ethan,

    We are having this problem in our lab and decided to go for the PCR step in order to eliminate the adapter ligation. Do you think if running it on Bioanalyzer and the result looks normal, can we run it on the Hiseq, or the extra PCR cycle would produce a bias result? Thanks a lot.


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