WHOLE GENOME SEQUENCING
METHODOLOGY
Generally, at BRIGHTcore Whole Genome Sequencing (WGS) is performed through 2 main methods : (1) PCR free library preparation for Illumina and (2) PCR free library preparation for NanoPore.
Both methodologies require DNA fragments of a specific size, so fragmentation might be necessary. More on DNA fragmentation can be found here :
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 phosphorylated (polynucleotide kinase) and the 3' ends adenylated (exo-klenow), this 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 needs. More on sequencing can be found here :
For Illumina based sequencing, the read length may vary per project (read length is a parameter inputted on the sequencing device). For NanoPore based sequencing, the read length is determined during the fragmentation process.
Occasionally, a non-PCR free library is generated by e.g. 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.
1. PCR free library preparation for Illumina
Fragmentation of genomic DNA
End repair of fragmented DNA + phosphorylation and adenylation
Adapter ligation
Fragmentation of genomic DNA
2. PCR free library preparation for Oxford NanoPore Technologies (ONT)
Fragmentation of genomic DNA
End repair of fragmented DNA + phosphorylation and adenylation
Adapter ligation
Fragmentation of genomic DNA
INSTRUMENTS
The following devices are used for Whole Genome Sequencing (WGS) in our premises.
Illumina MiSeq
Our lowest capacity short reads sequencer, able to generate reads up to 300 bp in length and this at a throughput of 1M to 30M reads per run.
Illumina NovaSeq 6000
Our highest capacity short read sequencer. This device can generate reads up to 250 bp in length (on the SP flowcell) and generate up to 10B reads per flowcell (S4).
NanoPore GridION x5
Sequencing long reads is no hassle on this device. It can sequence 5 MinION flowcells simultaneously and perform the data analysis on board.
PLAN EXPERIMENT
There is quite some variability on the experimental approaches for Whole Genome Sequencing. Please consult the individual application pages to set up a corresponding experiment.
