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.
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.
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
Figure 2A. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to AT and GC dropout rates. The Watchmaker solution delivered libraries with minimal sequence dropout compared to other solutions, including sonication. See Figure 1 for additional experimental details.
Figure 2B. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 387M read pairs, and assessed with respect to mean coverage of challenging GC-rich promoters. The Watchmaker solution delivered libraries with the highest mean coverage of challenging promoters, even when only 75 ng of DNA was used for library prep. See Figure 1 for additional experimental details.
Figure 2C. Even sequence coverage with minimal bias. All libraries were sequenced on a NovaSeq 6000, subsampled to 1M read pairs, and assessed with respect to start site bias. The Watchmaker solution, along with KAPA HyperPlus and NEBNext Ultra II FS (data not shown), delivered minimal start site bias in comparison to KAPA EvoPlus and Illumina DNA Prep. See Figure 1 for additional experimental details.
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)
Maintaining data quality: Robust variant callingMaintaining 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
Figure 4A. Improve yields (and subsequent sequence coverage) with cfDNA. Final library traces for libraries prepared in triplicate using 1 ng of Isopure cfDNA with the Watchmaker DNA Library Prep Kit, KAPA HyperPrep Kit, or NEBNext Ultra II DNA Library Prep Kit according to each manufacturer’s recommended protocol. Watchmaker library yields were 9-fold higher compared to other solutions enabling better resolution of the disome peak. Additional optimization with increased adapter concentration further improved yields by 20 - 25% with no adapter-dimer formation.
Figure 4B. High-efficiency, high-fidelity amplification. Error rates of the Equinox Amplification Master Mix and KAPA HiFi HotStart ReadyMix were measured after > 9 million bases incorporation events in three separate reactions, using a proprietary NGS-based assay. Base substitution profiles were examined in triplicate over 5.4 million G/C and 4.0 million A/T incorporation events. Equinox displayed an overall polymerase error rate 40% lower than KAPA HiFi.
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Discover more with Watchmaker
Watchmaker DNA Library Prep Kits with Fragmentation
A streamlined single-tube chemistry with reduced bias and artifacts. Ideally suited for PCR-free workflows, ultra-low inputs, and low-quality DNA samples including FFPE
Watchmaker DNA Library Prep Kits
A simple workflow that improves yields and coverage of low-input DNA samples, such as cfDNA.
Equinox Library Amplification Kits
An ultra-high-fidelity DNA polymerase in an optimized hot start master mix format, specifically optimized for high-efficiency, low-bias NGS library amplification