How do I size select libraries for the HiSeq 4000 with beads?

The HiSeq 4000 sequencer is the most demanding Illumina sequencer with regards to library insert sizes.  Nevertheless, the majority of existing Illumina sequencing libraries can be sequenced as is on the HiSeq 4000:
The libraries should not have any or no visible adapter dimers and the library fragments should be mostly shorter than 670 bases).
Other sequencing libraries can be made compatible by size-selection (removing both adapter-dimer traces and fragments of more than 670 bases, if the latter are numerous).
We suggest removing library fragments longer than 670 bases libraries for Hiseq 4000 sequencing if there are considerable amounts of these.  The size selection does not need to be “perfect”.
The size selection can be achieved by carrying out a simple “upper cut” with Ampure beads (please see the protocol below), by doing a BluePippin size selection, or by manual gel extraction.   We offer the first two of these options.
If your libraries are homogeneously sized, you could pool the libraries and carry out the size selection only once with the pool.
Ampure XP bead “upper cut” protocol to remove fragments longer than 670 bases:
Please note: It is recommended to verify this protocol first with your batch of Ampure XP beads or similar beads from other manufacturers.   This selection protocol will also remove adapter dimers, if they are not dominating the library.
Bead-based size selection cannot carry out precise “cuts”;  thus, you will also lose some of the library in the size ranges that you intend to keep.
      1. If not mentioned explicitly follow the standard Ampure XP handling instructions from the manufacturer (e.g. equilibrate the beads at to room temperature before use; vortex beads before use, details of the bead washes and elution,…)
      2. If the sample volume is smaller than 50 ul, add EB buffer up to 50 ul to each sample.
      3.  Add 0.55x the sample volume in Ampure beads (e.g. 27.5 ul beads to a 50 ul sample) to your sample, mix, incubate for 5 minutes at RT.
      4. Collect the beads on a magnet.
      5. Transfer the supernatant to a new tube.
      6. Add another 1x original volume Ampure beads to the supernatant; mix; incubate for 5 minutes
      7. Collect the beads on a magnet and remove the supernatant
      8. Carry out the two regular 80% ethanol washes of the beads and elute the samples from the beads according to Agencourt Ampure XP protocol.
      9. Verify the success of the size selection by running an aliquot on a Bioanalyzer or equivalent instrument.
Ampure XP bead “upper & lower cut” protocol to remove fragments longer than 670 bases and shorter than 400 bases:

This protocol is identical to the one above but adds a smaller volume of the beads at step 6 for the final enrichment onto the beads. The reduced bead buffer concentration at this step leads to a removal of longer fragments compared to the protocol above.
Please note: It is recommended to verify this protocol first with your batch of Ampure XP beads or similar beads from other manufacturers.  Bead-based size selection cannot carry out precise “cuts”;  thus, you will also lose some of the library in the size ranges that you intend to keep.

  1. If not mentioned explicitly follow the standard Ampure XP handling instructions from the manufacturer (e.g. equilibrate the beads at to room temperature before use; vortex beads before use, details of the bead washes and elution,…)
  2. If the sample volume is smaller than 50 ul, add EB buffer up to 50 ul to each sample.
  3.  Add 0.55x the sample volume in Ampure beads (e.g. 27.5 ul beads to a 50 ul sample) to your sample, mix, incubate for 5 minutes at RT.
  4. Collect the beads on a magnet.
  5. Transfer the supernatant to a new tube.
  6. Add another 0.25x of the original volume Ampure beads (e.g. 12.5 ul beads for a sample of a 50 ul starting volume sample) to the supernatant; mix; incubate for 5 minutes
  7. Collect the beads on a magnet and remove the supernatant
  8. Carry out the two regular 80% ethanol washes of the beads and elute the samples from the beads according to Agencourt Ampure XP protocol.
  9. Verify the success of the size selection by running an aliquot on a Bioanalyzer or equivalent instrument.

Beckmann/Agencourt also sells beads that are dedicated to size selections named SPRIselect — however, very likely these are actually identical to the AMpure XP beads. The SPRIselect manual provides a lot of additional information and protocols that can be applied to AMpure XP and other beads. Please see here: Beckman SPRIselect Ampure beads

BTW, our favorite magnetic separator for 96-well plates is this one from EdgeBio.


Category: 04 Library Preparation and QC

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