Skip to content

BLink bead preparation protocol

This page provides the BLink bead protocol in full as a reference. The official PDF is better formatted for printing and using in real time at a laboratory bench.

Official BLink bead Protocol

The BLink bead technology is based on magnetic, barcoded beads loaded with Tn5 protein to generate active transposomes. The preparation of these beads requires a series of barcode ligations (split-pool format) to build out the final combinatorial barcode plus Tn5 mosaic end (ME) sequence. Each bead has many copies of the same barcode; total barcode complexity in a batch of beads is > 7M combinatorial barcodes. A ‘staggered’ barcode segment of variable length ensures the sequencing reads are high complexity, even through the short invariant regions of the barcoded region. The barcode is captured in R1 on Illumina sequencers. See separate BLink-seq Library Prep Protocol to use BLink beads to generate linked-read libraries.

Duration

The preparation of a batch of beads takes ~1 week to complete, ideally with 2-3 people to maximize efficiency of the split-pooling steps.

Day 1

Section titled “Day 1”
  • Recommended Prepare stock buffers and duplex-oligo stocks, if needed
  • Bind biotinylated-duplex oligo to beads (bulk) – overnight incubation

Day 2-3+

Section titled “Day 2-3+”
  • Split beads to one 8-well strip, ligate Stagger-duplex, wash, pool
  • Split beads to 12 x 8-well strips, ligate Segment1-duplex (96 barcodes), wash, pool
  • Split beads to 12 x 8-well strips, ligate Segment2-duplex (96 barcodes), wash, pool
  • Split beads to 12 x 8-well strips, ligate Segment3-duplex (96 barcodes), wash
  • Ligate ME-duplex, wash, final pooling

Day 3+

Section titled “Day 3+”

Test the bead batch

  • Strip one aliquot of beads with NaOH to remove duplex oligos and anneal the ME-REV oligo
  • Test-load small amounts of beads with a Tn5 dilution series
  • Prepare haplotagging libraries with a control DNA sample to determine the optimal Tn5-loading dilution

As needed

Section titled “As needed”
  • Strip aliquots of beads with NaOH and anneal the ME-REV oligo
  • Load beads with Tn5 protein
  • Recommended Pool all beads after stripping and/or loading large batches, and store as a homogeneous stock.

Storage

Section titled “Storage”

BLink beads can be stored in several formats (unstripped, stripped+annealed to ME-REV, loaded with Tn5) at 4°C, in the appropriate buffer as described in the protocol. Beads can be stripped/annealed and loaded with Tn5 as needed to prepare the final transposome-active haplotagging bead stock, as the best long-term storage may be in the unstripped format. The loading seems to be consistent for beads stripped and/or loaded at different times, though test loading can be repeated as needed to confirm the appropriate Tn5 loading concentration.

Materials and Equipment Required: see Appendix A and BLinkBead_oligos.xlsx

Duplex-oligo stocks: see Appendix B to prepare duplex-oligo stocks used in ligation reactions

Technology development: Jennifer Grenier1 designed BLink beads, modified from the original ‘haplotagging beads’ designed by Frank Chan2 and Marek Kucka2. All of the authors of the manuscript BLink- seq delivers population-scale haplotypes without long reads: a scalable framework for non-model genomics contributed to developing BLink-seq.

Footnotes

Section titled “Footnotes”

  1. Genomics Innovation Hub, Biotechnology Resource Center, Institute of Biotechnology, Cornell University, Ithaca, NY. ↩

  2. Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany; https://doi.org/10.1073/pnas.2015005118 ↩ ↩2