The Xdrop technology

DNA workflow
Capture and/or amplify long DNA fragment (>100kb)

General workflow

Each step of the DNA workflow can be done independently depending on the application.

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Our VP explains ISC & dMDA

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Indirect Sequence Capture

  • Capture long DNA target >100kb
  • No target amplification by PCR
  • No target hybridization

1

ENCAPSULATES single long DNA molecules in droplets with primers for the Detection Sequence.

2

LABELS TARGETS by amplifying a ~150 bp Detection Sequence within or flanking the target.

3

SORTS out droplets containing the target based on the fluorescent signal of the amplified Detection Sequence.

indirect sequence capture ISC

Indirect sequence capture (Xdrop®)


DECOUPLES ENRICHMENT FROM AMPLIFICATION OR HYBRIDIZATION

  • no need to know the target sequence
  • avoids PCR bias
  • works with low sample input

indirect sequence capture aplicon based

Amplicon based

indirect sequence capture Hybridization capture

Hybridization-capture

Where you put the primers opens all kinds of possibilities

WITHIN THE TARGET

  • verification of insertions, gene duplications, and conserved regions where expected and not!
  • gene/pseudogene discrimination
  • haplotype phasing
  • CRISPR editing assessment

Known insertion site

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Unknown insertion site

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Target gene

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Pseudogene

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FLANK THE TARGET

  • gap closing
  • resolving tandem repeats

Known region 1

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Known region 2

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Tandem repeat

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User developed protocol, the Xdrop long-range DNA enrichment workflow

This protocol is a modification of the official Xdrop® DNA enrichment protocol. Relative to the targeted enrichment protocol, the procedure described here does not use droplet MDA to amplify the DNA isolated in the double-emulsion (DE) droplets. Instead of droplet MDA, long-range PCR is applied using primers specific to adapters ligated onto DNA ends of fragmented DNA. The fragmentation and adapter ligation are performed on the input DNA prior to DNA encapsulation in DE droplets.

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Sort the positive droplets

Double-emulsion droplets can easily be sorted.

Sorting can be done with our new instrument Xdrop Sort or by flow cytometry.

The key is that dMDA happens in droplets

Unbiased amplification of single long DNA sequences containing the target Uniform sequence coverage No inter-template chimeras

mdma workflow

Partitioning one single DNA molecule in each droplet extremely reduces amplification bias:

  • no preferred amplification of certain templates
  • no nested products
  • no inter-template chimera

dMDAplot

Amplifying every template in separate droplets (droplet MDA) provides a highly uniform sequence coverage, while bulk MDA preferentially amplifies certain templates.