Jul 07, 2020

Public workspaceBead-based normalization for NGS

This protocol is a draft, published without a DOI.
  • 1W. Szafer Institute of Botany, Polish Academy of Sciences
Tomasz Suchan
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Tomasz Suchan 2020. Bead-based normalization for NGS. protocols.io https://protocols.io/view/bead-based-normalization-for-ngs-q3pdymn
Manuscript citation:
Hosomichi, K., Mitsunaga, S., Nagasaki, H., & Inoue, I. (2014). A Bead-based Normalization for Uniform Sequencing depth (BeNUS) protocol for multi-samples sequencing exemplified by HLA-B. BMC genomics, 15(1), 645.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
We attempted this protocol, but could not get it to work in our group
Created: June 17, 2018
Last Modified: July 07, 2020
Protocol Integer ID: 13135
Abstract
Bead-based normalization protocol for genomic libraries based on Hosomichi et al. (2014). It uses diluted AMPure or other SPRI beads solution in order to bind a small amount of DNA from each sample.
Materials
MATERIALS
ReagentAgencourt AmPure XP beadsCatalog #A63880
ReagentPEG-8000
ReagentIsopropanol
ReagentNaCl
ReagentWater, nuclease free
Reagent1 M Tris-HCl pH 8.0
Reagent80% Ethanol
Reagent0.5 M EDTA pH 8.0
Preparation
Preparation
Prepare 2.5 M solution of NaCl:
  • place a 50 ml Falcon tube on the scale and tare
  • add 14.61 g of NaCl to the tube
  • add water to 40 ml
  • dissolve NaCl
  • fill up the tube with water to 50 ml and mix
Prepare 50% solution of PEG-8000:
  • place a 50 ml Falcon tube on the scale and tare
  • add 12.5 g of PEG-8000 to the tube
  • add 14 ml of nuclease-free water
  • close the tube, and dissolve PEG by mixing vigorously, preferably on an orbital shaker with heating
  • fill up the tube with water to 25 ml and mix gently by flipping the tube to avoid bubbles
Prepare a solution of 20% PEG, 2.5 M NaCl in TE buffer by mixing:
  • 5 ml of 5 M NaCl
  • 0.83 ml of molecular-grade water
  • 100 ul of 1 M Tris
  • 20 ul of EDTA
  • 50 uL of 10% Tween 10
  • 4 ml of 50% PEG-8000
Dilute AMPure or homemade SeraMag beads 20-fold in the above solution (for instance, add 10 ul of AMPure to 190 ul of the solution).
Normalization
Normalization
Add 20 ul of DNA library to the tube.
Add 20 ul of the diluted beads.
Add 20 ul of isopropanol.
Mix well by pipetting.
Incubate for 5 minutes at room temperature.
Duration00:05:00 DNA binding
Place the tube on the magnetic stand and wait until the beads separate.
With the tube still on the magnet, remove and discard the supernatant.
Add 10 ul of 80% ethanol and incubate for 30 sec. at room temperature.
Duration00:00:30 EtOH wash
Carefully remove all the ethanol and discard it.
Repeat steps 13 and 14.
Leave the tube on the magnetic rack and let the beads dry. Do not overdry the beads (the pellet should not be shiny anymore but there should not be cracks on it).
Add 20 ul of molecular-grade water on the beads. Make sure that the beads are wet before removing the tube from the magnet! 
Remove from the magnet and mix the beads with water by pipetting.
Incubate 10 minutes at room temperature.
Duration00:10:00 DNA elution