A Beginner’s Guide to Whole Genome Sequencing – Part 1: Introduction to WGS workflow

1.     Introduction

Whole Genome Sequencing (WGS) gives researchers and clinicians the most complete view of an organism’s genetic makeup. By examining the entire genome, WGS captures everything from single nucleotide changes to large structural variations — providing insights that targeted approaches can easily miss. This level of detail has made WGS an essential tool in many areas, from uncovering rare mutations in complex diseases like cancer and hereditary disorders to exploring genetic diversity in large population cohorts.

In clinical settings, WGS helps unravel disease mechanisms by discovering variants relevant to specific patient groups, even within distinct national or ethnic populations. For population studies, it enables researchers to investigate genetic variation at scale and uncover evolutionary patterns in population structure. For example, a study by Degang W. and colleagues in Singapore highlighted the genetic diversity across East, Southeast, and South Asia, reflecting the country’s unique history of immigration.

At Novogene, we’ve designed our WGS solutions to support a wide range of research goals. Whether you’re running a large population study or need high-resolution clinical-grade data, our optimized workflows and experienced team can help you get started quickly and confidently.

2.     Whole Genome Sequencing workflow

The following workflow contains these steps: sample preparation and quality control (QC), library construction and QC, sequencing, data QC and bioinformatics analysis, facilitating detailed dissection of genetic variations, and unraveling the intricate biological aspect of human genome.

Figure 1: The workflow of Whole Genome Sequencing

Step 1: Sample Preparation and Shipment

Your WGS journey begins with preparing high-quality genomic DNA. To ensure the best results, we provide clear instructions for sample handling and require at least 1.2 µg of DNA/sample in a PCR-free approach. Once prepared, samples are strictly shipped following our guidelines to our experimental center for processing.

Figure 2: Sample Guidelines for WGS

Step 2: Sample Quality Control (QC)

When samples arrive, Novogene frequently performs a series of QC steps to assess DNA integrity and concentration:

• Qubit: measure sample concentration.
• 1% agarose gel: assess DNA integrity and contamination.

After completing this step, Novogene will provide you with a Sample QC Report, so you can decide whether to proceed or replace samples if needed.

Step 3: Library Preparation

Qualified DNA samples are processed into sequencing-ready libraries:

• The genomic DNA is randomly fragmented into ~350 bp pieces using sonication.
• Fragments are end-repaired, A-tailed, and further ligated to Illumina-compatible adapters.
• Libraries are size-selected and amplified via PCR unless otherwise specified as PCR-free before being purified with AMPure XP beads.

Figure 3: Workflow of Library construction 

Step 4: Library Quality Control

Before sequencing, each library undergoes rigorous quality checks:

• Labchip is used to check library size distribution. The expected result should show a clean peak in the correct range.
• Qubit and qPCR are used to measure concentration for pooling before being loaded onto sequencers.
• This reduces the risk of wasted sequencing runs and ensures high-quality data output. You can see the illustrated figure below as an example of high adapter dimer contamination.

Figure 4: Lab chip result of high adapter dimer contamination

Step 5: Sequencing

Once libraries pass QC, they are sequenced on the Illumina NovaSeq X Plus or 6000, using a paired-end 150 bp strategy. For more complex genomes or highly polymorphic regions (e.g., HLA typing), we also offer long-read sequencing with PacBio or Nanopore platforms.

With NovaSeq’s ultra-high throughput, even large projects can be completed in under 48 hours.

Step 6: Sequencing Quality Control

Once sequencing is complete, your raw data goes through a robust processing and quality control (QC) pipeline to ensure it meets high standards.

The first step is demultiplexing, where sequencing reads are sorted and assigned to their respective samples based on unique barcodes. Afterward, the data undergoes cleaning to remove any low-quality reads or potential contaminants. This includes:

• Filtering out reads containing more than 10% uncertain bases (N).
• Discarding reads with over 50% low-quality bases.
• Removing adapter sequences to avoid alignment errors in downstream analysis. The expected result after filtering can be observed in figure 4

Next, we evaluate key QC parameters to assess the overall performance of the sequencing run:

• Error Rate: Monitored across all reads, with particular attention to the tail ends where errors may rise slightly due to reagent depletion.
• Quality Score (Q30): A critical indicator of sequencing accuracy, with a target of >80% to meet Illumina’s high standards.
• GC Content: Checked to ensure it aligns with the expected distribution for the organism (typically 40–44% for many genomes).

This careful data processing and QC step ensures that only clean, high-quality reads are passed on for bioinformatics analysis, giving you confidence in the integrity and usability of your dataset.

Figure 5: Different sequencing QC metrics in Whole Genome Sequencing

Step 7: Bioinformatics pipelines

After checking these mentioned QC parameters, we will proceed to alignment and variants detection using a standard workflow from Novogene. Generally, it consists of 3 main steps:

• Alignment with reference genome, statistics of sequencing depth and coverage
• SNP/InDel/SV/CNV calling, annotation and statistics.
• Somatic SNP/InDel/SV/CNV calling, annotation and statistics (tumor-normal paired samples).

The flowchart below depicts the pipeline of data QC and bioinformatics analysis we used. Somatic analysis will be performed only when tumor-normal paired samples are provided.

Figure 6: Whole Genome Sequencing’s bioinformatics analysis pipeline

Novogene provides comprehensive whole genome sequencing with a high-quality, affordable and convenient solution with advanced WGS analysis, making it possible for a wide variety of applications. As Vietnam’s official distributor, GeneSmart is proud to offer comprehensive support to Novogene’s trusted services.

Read more about Novogene’s sequencing services offered by GeneSmart here.

Refer to “Unveiling the Human Genome using cutting-edge Sequencing techniques: WGS & WES” to start your WGS and WES journey.

Stay tuned for our next part where we introduce you to some recognized publications that apply WGS in their research.

References

Novogene. (2021). Unveiling the human genome using cutting-edge sequencing techniques: WGS & WES [Webinar]. https://www.novogene.com/amea-en/resources/onlineevent/unveiling-the-human-genome-using-cutting-edge-sequencing-techniques-wgs-wes/

Novogene Co., Ltd. Whole Genome Sequencing analysis demo report