Introduction to Whole Genome Sequencing
Whole genome sequencing (WGS) refers to sequencing the entire genetic blueprint of organisms and viruses and has a variety of applications:
- De novo sequencing to establish new reference genomes
- Low pass WGS as a powerful alternative to microarrays for NGS-based genotyping
- Human resequencing to elucidate the genetic basis of inherited, rare, complex, metabolic, and pediatric diseases - especially those associated with large and/or previously uncharacterized structural variation (e.g., deletions, insertions, inversions, duplications, translocations, and copy number variation)
Sonication has long been regarded as the gold standard for unbiased DNA fragmentation and data. However, it can be a bottleneck in high throughput labs and comes with expensive equipment and consumable costs. Enzymatic DNA fragmentation is an attractive alternative that scales easily and minimizes sample loss.
Watch now: Customizing Watchmaker DNA PCR-free Library Prep Kit for Improved NovaSeq X Performance
As presented at ESHG 2024
Key considerations when selecting a WGS workflow
Minimizing bias: Compatibility with PCR-free sequencing
- Library amplification can be a source of both fragment length and sequence bias - where shorter fragments and GC-neutral sequences are preferentially amplified.
- When performing PCR-free sequencing, the core library prep workflow needs to be highly efficient to generate sufficient yields for sequencing without amplification.
- The Watchmaker Library Prep Kit with Fragmentation generates the highest PCR-free yields, enabling the use of lower DNA inputs and broadening the addressable sample range for researchers.
Figure 1. Efficient conversion delivers high ... MORE
Minimizing bias: Compatibility with PCR-free sequencing
Figure 1. Efficient conversion delivers high library yields without amplification. PCR-free WGS libraries were constructed using a variety of commercially available solutions: KAPA HyperPrep (sonication control), Watchmaker DNA Library Prep with Fragmentation, KAPA HyperPlus, KAPA EvoPlus, NEBNext Ultra II FS DNA Library Prep, and Illumina DNA PCR-Free Prep. Either 300 ng (all evaluated kits) or 75 ng (Watchmaker only) of NA12878 human genomic DNA was used as input. Workflows were optimized to deliver similarly sized final libraries by tuning fragmentation and post-ligation cleanup parameters to ensure fair and accurate comparison of key sequencing performance metrics. Watchmaker library prep yields exceeded other sonication and enzymatic fragmentation preps.
Minimizing bias: Maintain a clear picture of the whole genome
- Even in the absence of PCR, sequence bias, including start site bias, can lead to uneven genome coverage and result in certain regions being underrepresented or completely missed.
- The Watchmaker Library Prep Kit with Fragmentation delivers minimal start site bias.
- This results in less AT/GC dropout and improved coverage of challenging GC-rich promoters in comparison to other solutions - including a sonication control.
Minimizing bias: Maintain a clear picture of the whole genome
Maintaining data quality: Robust variant calling
- Many genetic diseases are caused by specific mutations in the genome.
- Sensitive and precise variant calling is essential for identifying disease-causing mutations.
- The Watchmaker DNA Library Prep Kit with Fragmentation delivers excellent F1 scores for SNP and indel variant calling (a combined measure of precision and sensitivity) on-par with sonication.
- Available resource: AGBT 2022 poster (Broad Institute)
Figure 3. Sensitive and precise variant ... MORE
Maintaining data quality: Robust variant calling
Figure 3. Sensitive and precise variant calling. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to SNP and Indel F1-scores. The Watchmaker libraries deliver variant calling performance on-par with the sonication control. See Figure 1 for additional experimental details.
Compatibility with clinically relevant sample types
- WGS of cancer genomes from cell-free DNA (cfDNA) and FFPE samples can be performed to survey somatic mutations at different stages of disease development.
- The Watchmaker DNA Library Prep Kit improves the conversion of low-input cfDNA to final library - translating to improved library complexity and coverage.
- For samples like cfDNA and FFPE where PCR is required, it's critical to use a polymerase system that is highly efficient (to minimize cycle counts), low-bias, and high-fidelity (to minimize PCR errors).
- Equinox® Amplification Master Mix is designed to deliver in all of these areas, limiting bias and significantly reducing base misincorporation events.
Compatibility with clinically relevant sample types
TECHNICAL LITERATURE
Non-invasive prenatal testing: Embracing low-input methodologies
Non-invasive prenatal testing Illumina DNA Plasma/cfDNA DNA NGS DNA Library PrepThis case study highlights how Trisomy Test Ltd improved its NIPT workflow using the Watchmaker DNA Library Prep Kit to generate reliable results from cfDNA inputs as low as 0.5 ng. The streamlined workflow enabled faster turnaround, higher success rates in low fetal fraction samples, and efficient processing of nearly 7,000 clinical specimens.
VIDEOS
Customizing Watchmaker DNA PCR-free Library Prep Kit for improved NovaSeq X performance
PCR-free WGS Whole Genome Sequencing Illumina DNA DNA NGS DNA Library Prep with FragmentationAs presented at ESHG 2024, this video provides recommendations and commentary on whole genome sequencing on the NovaSeq X platform.
Non-invasive pre-natal testing: Embracing low-input methodologies
Non-invasive prenatal testing Whole Genome Sequencing Illumina DNA Blood Plasma/cfDNA DNA NGS DNA Library PrepAs presented at ESHG 2024, this video provides a case study on the use of the Watchmaker DNA Library Prep Kits for non-invasive prenatal testing to improve turnaround times and reduce the percentage of uninformative results.
POSTERS
Benchmarking enzymatic library preparation workflows in a PCR-free whole genome sequencing context
PCR-free WGS Whole Genome Sequencing Illumina DNA Cell Line DNA NGS DNA Library Prep with FragmentationThis poster evaluates eight commercially available library preparation solutions for PCR-free whole genome sequencing (WGS). Results show the Watchmaker DNA Library Prep Kit with Fragmentation achieves high conversion rates with as little as 75 ng of input DNA, delivering superior yields. Sequencing results indicate minimal GC bias and increased coverage across challenging GC-rich regions, highlighting the kit's effectiveness in producing high-quality, uniform, and unbiased WGS data.
An evaluation of enzymatic fragmentation in the library preparation workflow: The Watchmaker Genomics Kit (Broad Institute)
Lab Automation PCR-free WGS SNV/SNP/InDel Detection Variant Calling Whole Genome Sequencing Illumina DNA Cell Line DNA NGS DNA Library Prep with Fragmentation AgilentThis poster from the Broad Institute evaluates the Watchmaker DNA Library Prep Kit with Fragmentation, focusing on its enzymatic fragmentation method as an alternative to traditional mechanical shearing in whole genome sequencing (WGS). The study demonstrates that the enzymatic approach offers comparable sequencing metrics. The workflow is user-friendly, completing high-quality library construction in under two hours, and is suitable for scaling in throughput.