Human Whole Exome Sequencing

Introduction to Human Whole Exome Sequencing

Whole exome sequencing (WES) employs next-generation sequencing technology (NGS), which provides a cost-efficient alternative to whole genome sequencing (WGS). The human whole exome, composed by about 180,000 exons (protein-coding region of the genome) accounts for only 1-2% of the human genome, but up to 85% of the disease-related mutations associated with Mendelian disorders occur in these regions ^[1]. By targeting these regions, human whole exome sequencing (hWES) provides an in-depth sequencing and analysis approach to indicate genome variants, germline mutations, somatic mutations, and pathogenic mechanisms. hWES service supports a broad range of studies for researchers, including genetic disease-related variants, complex diseases, cancer research, or human population genetics.

Novogene’s hWES service offers a convenient solution with plenty of profits, such as high-quality data, publication-ready results that help you achieve your research goals. Novogene is equipped with clinical-grade sequencing laboratories that are validated and comply with CLIA/CAP/ISO17025 standards, providing precise diagnosis with our clinical whole exome sequencing service.

Applications of Human Whole Exome Sequencing

Human whole exome sequencing has been successfully helping researchers to obtain answers to many different and cutting-edge research and clinical questions:

Genome variant studies through exon detection
Pathogenic mechanism and molecular characterization of samples in research and clinical contexts
Cancer biopsy as a tool

Benefits of Novogene Human Whole Exome Sequencing

hWES focuses on the exome regions which can help achieve higher sequencing depth with significantly fewer data of good quality, compared with WGS.
hWES increases the sensitivity of the analysis which makes the detection of rare mutations easy.
Novogene’s extraordinary professional bioinformatics pipeline and internationally recognized software ensures that our customers always receive reliable and publication-ready data.

hWES Specifications: DNA Sample Requirements

Platform Type	Sample Type	Amount (Qubit^®)	Purity
Illumina NovaSeq 6000	Genomic DNA	≥ 300 ng	A260/280 = 1.8-2.0; no degradation,no contamination
	cfDNA/ctDNA	≥ 30 ng	Fragments should be in multiples of 170 bp, with no genomic contamination
	Genomic DNA from FFPE tissue	≥ 500 ng	Fragments should be ≥ 1000 bp

Note: Values of sample amount are only listed for your reference. Download the Service Specifications and Sample Requirements to learn more. For detailed information, please contact us with your customized requests.

hWES Specifications: Sequencing and Analysis

Sequencing Platform	Illumina NovaSeq 6000
Read Length	Paired-end 150 bp
Sequencing Depth	For Mendelian disorder/rare disease: effective sequencing depth above 50× (6G)
	For tumor sample: effective sequencing depth above 100× (12G)
Data Analysis	Data quality control Alignment to a reference genome SNP and InDel calling Somatic SNP/InDel/CNV mutation detection (tumor-normal paired samples)

Note: Sequencing depths and analysis contents displayed are for your reference only. Download the Service Specifications to learn more. For detailed information, please contact us with your customized requests.

Project Workflow of Novogene hWES Services

From sample preparation, library preparation, DNA sequencing and data quality control, to bioinformatics analysis, Novogene provides high-quality products and professional services. Each step is performed in agreement with a high scientific standard and meticulous design to ensure high-quality research results.

^[1]https://www.nature.com/articles/jhg2013114

Publications of Human Whole Exome Sequencing

Human whole exome sequencing (hWES) enables researchers to cost-effectively unveil protein-coding variants in rare Mendelian disorders, complex disease, cancer, or human population studies. Here we summarized some outstanding academic publications that have used Novogene hWES services.

Association of Rare PTGIS Variants With Susceptibility and Pulmonary Vascular Response in Patients With Idiopathic Pulmonary Arterial Hypertension

JAMA CardiologyIssue Date: April 1, 2020IF: 12.794DOI: 10.1001/jamacardio.2020.0479
- Reference information
  
  Wang, X. J., Xu, X. Q., Sun, K., Liu, K. Q., Li, S. Q., Jiang, X., … & Wu, Y. (2020). Association of Rare PTGIS Variants With Susceptibility and Pulmonary Vascular Response in Patients With Idiopathic Pulmonary Arterial Hypertension. JAMA cardiology, 5(6), 677-684.
Germline genetic patterns underlying familial rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren’s syndrome highlight T cell-initiated autoimmunity

Annals of the Rheumatic DiseasesIssue Date: 2020IF: 16.102DOI: 10.1136/annrheumdis-2019-215533
- Reference information
  
  Wang, Y., Chen, S., Chen, J., Xie, X., Gao, S., Zhang, C., … & Lin, J. (2020). Germline genetic patterns underlying familial rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren’s syndrome highlight T cell-initiated autoimmunity. Annals of the Rheumatic Diseases, 79(2), 268-275.
Integrated multiomic analysis reveals comprehensive tumour heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas

GutIssue Date: 2019IF: 19.819DOI: 10.1136/gutjnl-2019-318912
- Reference information
  
  Zhang, Q., Lou, Y., Yang, J., Wang, J., Feng, J., Zhao, Y., … & Zhang, X. (2019). Integrated multiomic analysis reveals comprehensive tumour heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas. Gut, 68(11), 2019-2031.
Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence

Journal of HepatologyIssue Date: July 23, 2019IF: 20.582DOI: 10.1016/j.jhep.2019.07.014
- Reference information
  
  Zhou, S. L., Zhou, Z. J., Hu, Z. Q., Song, C. L., Luo, Y. J., Luo, C. B., … & Huang, X. W. (2019). Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence. Journal of hepatology, 71(6), 1152-1163.
Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension

European Respiratory JournalIssue Date: 2019IF: 12.339DOI: 10.1183/13993003.01609-2018
- Reference information
  
  Wang, X. J., Lian, T. Y., Jiang, X., Liu, S. F., Li, S. Q., Jiang, R., … & Xu, X. Q. (2019). Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension. European Respiratory Journal, 53(3).
Multiregion Sequencing Reveals the Genetic Heterogeneity and Evolutionary History of Osteosarcoma and Matched

Cancer ResearchIssue Date: 2019IF: 9.727DOI: 10.1158/0008-5472.CAN-18-1086
- Reference information
  
  Wang, D., Niu, X., Wang, Z., Song, C. L., Huang, Z., Chen, K. N., … & Wang, Y. (2019). Multiregion sequencing reveals the genetic heterogeneity and evolutionary history of osteosarcoma and matched pulmonary metastases. Cancer research, 79(1), 7-20.
Mutational landscape of secondary glioblastoma guides MET-targeted trial in brain tumor

CellIssue Date: October 18, 2018IF: 38.637DOI: 10.1016/j.cell.2018.09.038
- Reference information
  
  Hu, H., Mu, Q., Bao, Z., Chen, Y., Liu, Y., Chen, J., … & Sa, J. K. (2018). Mutational landscape of secondary glioblastoma guides MET-targeted trial in brain tumor. Cell, 175(6), 1665-1678.
Whole-exome sequencing reveals the origin and evolution of hepato-cholangiocarcinoma

Nature CommunicationsIssue Date: 01 March 2018IF: 12.121DOI: 10.1038/s41467-018-03276-y
- Reference information
  
  Wang, A., Wu, L., Lin, J., Han, L., Bian, J., Wu, Y., … & Wang, W. (2018). Whole-exome sequencing reveals the origin and evolution of hepato-cholangiocarcinoma. Nature communications, 9(1), 1-10.

Data Quality Control

Sequencing Error Rate Distribution

The sequencing error rate is the major confounding factor of precise detection of low-frequency variations by deep sequencing. It determines the quality of the sequencing data. The sequencing error rate is highly associated with the sequencing cycle, escalating towards the end of each read because of the consumption of chemical reagents, which is a common feature of the Illumina high throughput sequencing platform.

Novogene hWES Error Rate Distribution

Note: The x-axis represents the position in reads, and the y-axis indicates the average error rate of bases of all reads at a position.

GC Content Distribution

GC content distribution aims to check the potential of AT/GC separation. Sample contamination, sequencing bias, and errors during library preparation can impact on the sequencing results.

Note: The x-axis represents the position in reads, and the y-axis indicates the percentage of each type of bases (A, T, G, C); different bases can be distinguished by different colors.

Alignment to Reference Genome

Sequencing Depth & Coverage Distribution

Sequencing depth and coverage illustrate the average number of pair-end clean reads which are aligned to the known reference nucleotides. The sequencing coverage distribution determines whether the identification of variations can be done with a certain degree of confidence at specific base positions.

Novogene hWES Sequencing Depth & Coverage Distribution

Average sequencing depth (bar plot) and coverage (dot-line plot) in each chromosome

Note: The x-axis represents the chromosome; the left y-axis indicates the average depth; the right y-axis shows the coverage (proportion of covered bases).

SNP and InDel Calling, Annotation and Statistics

Single nucleotide polymorphisms (SNPs), also known as single nucleotide variants (SNVs), constitute the largest class of genetic variants in the genome. Another class of genetic variations includes small insertions and deletions (InDels) which are <50 bp in length. The InDels present in the coding region or splicing sites may cause changes in mRNA transcripts and proteins.

Novogene hWES SNP Detection

The number of SNPs/inDels in various genomic regions

Novogene hWES SNP Detection

The number of different types of SNPs/InDels in the coding region

Advanced Analysis

Driver Gene Analysis

Heatmap of significantly mutated genes

Only a few mutations related to cancer can drive tumorigenesis by affecting genes. Significantly mutated genes (SMGs) refer to those mutations that show a significantly higher mutation rate than the background mutation rate (BMR), thus indicating a positive selection during tumorigenesis. Analysis of SMGs helps us pinpoint the key genes that are critical for cancer initiation and progression.

Novogene hWES Heatmap of Significantly Mutated Genes

Heatmap of significantly mutated genes (SMGs) across samples

Note:
The bar plot at the top shows the mutation rate of each sample (Mutations/Mb). The heatmap in the center shows the mutation types of each SMG across samples. The horizontal axis represents samples, and the vertical axis represents SMGs. Different mutation types are distinguished by different colors. The bar plot on the left side of the heatmap shows the percentage of samples affected by mutations in each SMG, and the plot on the right side shows p values of SMGs.

Tumor Heterogeneity Analysis

Intra-tumor Heterogeneity Analysis

Intra-tumor heterogeneity refers to the heterogeneous composition of tumor cells. Deciphering the intra-tumor heterogeneity and clonal architecture may contribute towards the understanding of therapeutic resistance.
The number and content (subclonal somatic mutations) of tumor subclones were identified by analyzing the variant allele frequencies of somatic mutations.