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The Recipe for Successful Whole Genome Sequencing

The success of whole genome sequencing (WGS) is shown in the quick and efficient scientific response to the 2011 outbreak of E. coli in Germany and France.1 German and French strains of E. coli were indistinguishable using standard tests.  However, WGS analysis showed 2 single nucleotide polymorphisms (SNPs) in the German strains and 9 SNPs in the French strains. This allowed epidemiologists to trace the origins and track the transmission of the virulent bacterial strains to ensure proper infection control strategies were implemented. The increasing flexibility and scalability of WGS technology allows for the successful and rapid sequencing of human genomes, microbes, livestock and food crops.

Quality control through comprehensive visual feedback at each step of the process is key to obtaining high-quality WGS results and is available exclusively from the Invitrogen™ Collibri™ PS DNA Library Prep Kit and the Invitrogen Collibri PCR-Free PS DNA Library Prep Kit for Illumina Sequencing Systems.

How Is Quality Control Performed?

Quality control is performed at 3 main stages of the WGS workflow: (1) to check the integrity and purity of the DNA sample/input; (2) to use in-process feedback during creation of the library; and (3) to check the robustness and readability of the output data throughout the genome, including challenging GC-rich regions.

Preparing a DNA library from the isolated DNA sample is a multistep process that ensures that the long strands of DNA in the original sample are adequately fragmented, blunted, and ligated to adapters at either end, and finally purified to eliminate any extraneous unwanted DNA such as adapters and adapter dimers. Library prep is a critical step in WGS success and was until recently a time-consuming, labor intensive, and error-prone process. Now, with the use of Invitrogen Collibri PCR-Free PS DNA Library Prep Kits, the hands-on time to prepare a DNA library from physically sheared FFPE samples is about 0.4 hours and requires a total time of about 1.5 hours. This rapid protocol is not only time-efficient, but the in-process visual feedback built into the kit’s workflow prevents costly mixing errors and missteps.

This process not only helps ensure quality but also is lovely to watch. You can see this process here. When the clear and colorless fragmented DNA is successfully added and properly mixed with the End Conversion MastermixTM, which is a shade of aquamarine blue, emergence of an easily detectable visual cue of a light blue solution indicates that the step has been performed optimally. Next, the light blue solution is added to adaptors, labelled yellow. Once again successful adding and mixing with the reagent results in a green DNA-Adaptor mixTM. In the final color-cued reaction, the addition of the green DNA-Adaptor mix to the red Ligation master mixTM results in a purple ligation reaction mix. Post-ligation cleanup step using Magnetic Beads result in pure, clear and colorless DNA Library sample. At any of these three distinct steps, the absence of the light blue, green, or purple solutions, respectively, warns the investigator that either a reagent has not been added or that the reagents have not been mixed thoroughly. This immediate visual cue is easy to detect at a glance even in 96 well plates that are commonly used in WGS workflows.

WGS Applications

WGS is a useful tool for: (1) resequencing; (2) de novo sequence assembly; and (3) population sequencing. The Collibri PS DNA Library Prep Kit for Illumina Systems improves results in these applications by offering consistently even GC coverage, less than 1% contaminating adapter-dimers (even in data from degraded FFPE samples), and high confidence mutation detection from limited input DNA.

References

  1. Grad YH, Lipsitch M, Feldgarden M, et al. Genomic epidemiology of the Escherichia coli O104:H4 outbreaks in Europe, 2011. PNAS. 2012;109(8):3065–3070.
  2. Thirwell C, Eymard M, Feber A, et al. Genome-wide DNA methylation analysis of archival formalin-fixed paraffin-embedded tissue using the Illumina Infinium HumanMethylation27 BeadChip. Methods 2010; 52(3):248-254.
  3. Groisman JI, Hurlimann T, and Goddard B. Parents of a child with epilepsy: Views and expectations on receiving genetic results from Whole Genome Sequencing. Epilepsy Behav. 2019; 90:178-190.

 

 

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