Generally, Whole Genome Sequencing (WGS) at BRIGHTcore is performed through the creation of PCR free shotgun libraries.

Dependent on the input material, shearing is performed mechanically through the Covaris M220 device (complex genomes) or enzymatically (eg. mitochondrial or bacterial genomes at high concentration).

Following fragmentation, the 3' and 5' overhangs are blunt ended by respectively a 3' exonuclease and a DNA polymerase. After this "end repair" step, the 5' ends are adenylated (polynucleotide kinase) in order to generate a substrate for the adapter ligation.

Finally, the resulting libraries are ready to be sequenced according to the requesting scientist's need (read length may vary per project).

 

Occasionally, a non PCR free library is generated by the use of a tagmentation based library preparation. This strategy is mainly applied to low complexity genomes (bacterial / viral).

 

For more information about WGS, please see our information leaflet or contact us.

 

Whole Genome Sequencing

Methodology

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Minimum requirements

Following are the minimum requirements for us to proceed with WGS. If compliance with any of the following points is not possible, please contact us up front.

 

  • Amount : at least 2 µg DNA (determined fluorometrically, eg. Qubit)

    • Exceptions are possible for precious samples, however we ought to be informed/contacted up front

  • Concentration : should exceed or be equal to 30 ng/µl

  • DNA should not be degraded

  • At least 2 identifiers are required to identify the sample (eg. unique lab number + [surname+name, initials , date of birth,…])

  • A sample list should be provided to us digitally (all delimiters should be included in this list)

    • Please download sample submission form here or see "Downloads" section

  • Tubes should be labelled correctly (with 2 identifiers), preferably with a printed label

  • Per biological sample, only 1 tube should be shipped (no multiple aliquots of the same sample)

Compatible platforms

Dependent on the genome complexity, the most suitable platform (HiSeq/MiSeq) will be selected.

Kits

A total of 3 strategies for the creation of PCR free libraries have been positively evaluated in our setting :

  • Illumina TruSeq PCR free library preparation kit

  • KAPA Hyper prep

  • KAPA HyperPlus

For small genome sequencing (bacteria/viruses), we alternatively offer WGS through tagmentation, using the following kit :

  • Nextera XT DNA Sample Preparation Kit

BRIGHTcore / T +32 (0)2 477 64 79 / F +32 (0)2 477 68 59 / © 2015 by BRIGHTcore

Photography / Bart Moens / http://www.fotobart.be

Content & illustrations / Ben Caljon