Whole Genome Sequencing

Human whole genome sequencing enables researchers to catalog genetic constitution of individuals and capture all variants present in a single assay. It is utilized to study cancer and a variety of diseases, as well as human population evolution studies and pharmacogenomics. Equipped with powerful Illumina HiSeq X and NovaSeq 6000 systems, Novogene is capable of sequencing up to 280,000 human genomes per year at the lowest cost per genome. Being one of the first few companies to adopt HiSeq X Ten since early 2014, we have extensive experience providing whole genome sequencing services, having successfully sequenced hundreds of thousands of genomes with high quality results. With the world’s largest sequencing capacity, extensive experience in whole genome sequencing, and advanced bioinformatics capabilities, Novogene is able to expertly meet customer needs for delivering large project results with quick turnaround and the highest quality results.

The Novogene Advantage

• State-of-the-art NGS technologies: Novogene is a world leader in sequencing capacity using state-of-the-art technology, including Illumina HiSeq X and NovaSeq 6000 Systems.
• Highest data quality: We guarantee a Q30 score ≥ 80%, exceeding Illumina’s official guarantee of ≥75%. See our data example.
• Extraordinary informatics expertise: Novogene uses its cutting-edge bioinformatics pipeline and internationally recognized, best-in-class software to provide customers with highly reliable, publication-ready data.

Project Workflow

Sequencing Strategy

• 350 bp insert DNA library
• HiSeq X platform or NovaSeq 6000 platform, paired-end 150 bp

Data Quality Guarantee

• We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, which exceeds Illumina’s official guarantee of ≥ 75%.

Sample Requirements

Sample Type	Amount (Qubit®)	Volume	Concentration	Purity (NanoDrop™/Agarose Gel)
Genomic DNA	≥ 1 μg	≥ 20 μl	≥ 20 ng/μl	No degradation, no contamination
Genomic DNA (PCR-free)	≥ 1.5 μg	≥ 20 μl	≥ 20 ng/μl	No degradation, no contamination
DNA products of single-cell whole genome amplification	≥ 1 μg	≥ 20 μl	≥ 20 ng/μl	Fragments should be longer than 500 bp
FFPE*	≥ 1.5 μg	-	-	Fragments should be longer than 1500 bp

*Formalin-fixed, paraffin-embedded

Turnaround Time

• 18 working days after verification of sample quality without data analysis (depending on sample numbers)
• Additional 8 working days for data analysis

Recommended Sequencing Depth

• For tumor tissues: 50×, adjacent normal tissues and blood 30×
• For rare diseases: 30-50×

Analysis Pipeline

 

Bioinformatics Analysis includes:

• Data quality control: filtering out reads containing adapters or with low quality
• 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 (paired tumor samples)

Advanced Analysis

Monogenic disorders

1. Variant filtering 2. Analysis under dominant/recessive model (Pedigree information is needed) 2.1 Analysis under dominant model 2.2 Analysis under recessive model 3. Functional annotation of candidate genes 4. Pathway enrichment analysis of candidate genes 5. Linkage analysis 6. Regions of homozygosity (ROH) analysis

Complex/multifactorial disorders

1. Variant filtering 2. Analysis under dominant/recessive model (Pedigree information is needed) 2.1 Analysis under dominant model 2.2 Analysis under recessive model 3. Functional annotation of candidate genes 4. Pathway enrichment analysis of candidate genes 5. De novo mutation analysis (Trio/Quartet) 5.1 De novo SNP/InDel detection 5.2 Calculation of de novo mutation rates 5.3 De novo CNV/SV and De novo SV/CNV detection 6. Protein-protein interaction (PPI) analysis 7. Association analysis of candidate genes (at least 20 trios or case/control pairs)

Cancer (for tumor-normal pair samples)

1. Screening for predisposing genes 2. Mutation spectrum & mutation signature analyses 3. Screening for known driver genes 4. Analyses of tumor significantly mutated genes 5. Analysis of copy number variations (CNV) 5.1. Analysis of CNV distribution 5.2.Analysis of CNV recurrence 6. Fusion gene detection (for WGS porject only) 7. Purity & ploidy analyses of tumor samples 8. Tumor heterogeneity analyses 9. Tumor evolution analysis 10. Display of genomic variants with Circos

Table. Representative data of Novogene's human whole genome sequencing service.  ¹  Original sequencing data (in gigabases).  ²  Percentage of clean reads from all raw reads.  ³  Average error rate of all bases in read1 and read2.  ⁴  Percentage of reads with an average quality greater than Q20.  ⁵  Percentage of reads with an average quality greater than Q30.  ⁶  Percentage of G and C bases from total bases.  ⁷  Percentage of total reads that mapped to the reference genome (UCSC hg19).  ⁸  Average sequencing depth.  ⁹  Percentage of genome covered by sequencing. ¹⁰  Percentage of bases in genome with a sequencing depth ≥ 4x. ¹¹  Percentage of bases in genome with a sequencing depth ≥ 10x. ¹²  Percentage of bases in genome with a sequencing depth ≥ 20x.

Project Example

The following studies utilized Novogene's expertise in whole genome sequencing on HiSeq X Ten. Genetic alterations in esophageal tissues from squamous dysplasia to carcinoma Gastroenterology 153:166-177 (2017) Esophageal cancer is a major worldwide health threat, and intraepithelial neoplasia (IEN) is considered to be a precancerous lesion of esophageal squamous cell carcinoma (ESCC), one subtype of esophageal cancer. This study incorporated Novogene’s whole genome sequencing, together with whole exome sequencing and targeted sequencing, to track genetic changes in patients during development of ESCC. The study revealed significant similarities in the types and frequency of mutations between IEN and ESCC, including similarity in the DNA damage mutation signature. Mutations in the CCND1, CDKN2A, and FGFR1 genes were also revealed as the early driver events from phylogenetic and clonal analysis. However, the number of non-overlapping SNVs in tissues taken from the same individuals indicated that various lesions formed independently and that there was independent clonal expansion of mutations. As shown in this study, using multiple NGS applications provides novel approaches for exploring early diagnostics and treatments for cancer. Figure. The mutation variation landscape of ESCC, IEN, and simple hyperplasia (ESSH) from whole genome sequencing and whole exome sequencing Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions Oncogene, 36: 746-755 (2017) Genome instability, caused by DNA repair failure and DNA response system damage, is a hallmark of cancer. BRCA1 and BRCA2 play important roles in homologous recombination repair. Here Novogene’s HiSeq X Ten was incorporated on chicken DT40 cell clones whole genome sequencing to compare the consequences of loss of BRCA1/2 on genomic mutagenesis, under normal cell culture or under a MMS treatment that designed to accelerate one class of endogenous mutagenic processes. Loss of BRCA1 or BRCA2 increased seven- to eightfold higher level of spontaneous base mutation rate, and this increased mutation is strongly correlated with a BRCA1/2 mutant signature. Loss of BRCA1 or BRCA2 also induced more insertion/deletion mutations and large rearrangements under the endogenous damage induction condition. The high rate of base substitution mutagenesis demonstrated in this study indicates significant oncogenic effect of the inactivation of BRCA1/2, with distinct roles of BRCA1/2 in the DNA lesions processing. Figure. Number and spectrum of SNVs

Examples of Publications Using Novogene’s Services

Journal	Title
Gastroenterology, 153(1), 166-177 (2017)	Genetic alterations in esophageal tissues from squamous dysplasia to carcinoma.
Oncogene, 36: 746-755 (2017)	Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.
Nature Communications, 8:823 (2017)	Simultaneous evolutionary expansion and constraint of genomic heterogeneity in multifocal lung cancer.
Cancer Research, 77(23) (2017)	Clonality, heterogeneity, and evolution of synchronous bilateral ovarian cancer.
Molecular Psychiatry, doi: 10.1038/s41380-018-0020-x (2018)	A set of regulatory genes co-expressed in embryonic human brain is implicated in disrupted speech development.