Human Whole Genome PacBio SMRT Sequencing

Human Whole Genome PacBio SMRT Sequencing

CD Genomics has been providing an accurate and affordable human genome resequencing service for a couple of years. CD Genomics introduces previously hidden PacBio SMRT technology that has great application potential in human genome resequencing. The long single-molecule reads reveal structural variants and produce direct variant phasing information across haplotype blocks and methylation. This is very helpful to broaden the utility of precision medicine efforts to improve human health, and greatly promote the development of human single-gene diseases, complex diseases and tumor genomes.


1. SV detection

SVs represent genomic rearrangements (typically defined as longer than 50 bp), and SVs may play important roles in human disease, evolution and genetic diversity. Many inherited diseases and cancers have been associated with a large number of SVs in recent years. There has been tremendous progress in the detection of single nucleotide variants (SNVs) in the past quarter-century, but intermediate-sized (50 bp to 50 kb) structural variants (SV) remain a challenge with short-read DNA sequencing. The longest read length of the PacBio SMRT sequences is 40~70 K, which can easily cover high repetition and high heterozygosity areas, providing the possibility for SVs detection.

2. Haplotype Genotyping

A haplotype (haploid genotype) is a group of alleles in an organism that is inherited together from a single parent. The correct genotyping of haplotype gene is important; for example, the outcome of unrelated donor marrow transplantation is influenced by donor-recipient matching for HLA. PacBio long reads can span multiple single nucleotides and structural variants, which directly phases the variants into haplotypes.

3. DNA Methylation

DNA methylation is a process by which methyl groups are added to the DNA molecule. It is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging and carcinogenesis. There are many ways to detect DNA methylation, including whole genome bisulfite sequencing (WGBS), methylated DNA Immunoprecipitation Sequencing (MeDIP) and so on. But these methods are difficult to operate experimentally.

While PacBio platform can describe the direct detection of DNA methylation, without bisulfite conversion according to the difference in the fluorescence pulse signal interval. In addition, the long reads sequencing allows for more thorough regional CpG methylation assessment and increases the capacity for studying the relationship between phased single nucleotide variants and allele-specific CpG methylation.

Key Features and Advantages

  • Long-reads. It is beneficial for variation information mining of the whole genome, accurate analysis of chromosomal structural variants (SVs) and fusion genes
  • No PCR amplification. Effectively avoiding the amplification bias, and easily spanning regions with high GC content and high sequence repetition, to ensure the integrity and homogeneity of genome coverage.
  • Directly detects epigenetic modifications by measuring kinetic variation during base incorporation

Project Workflow

Sample Requirements:

1. DNA amount: ≥ 10 μg
2. DNA Purity: OD260/280 =1.8 ~2.0 without degradation or RNA contamination

Sequencing Strategy: 20 kb Library, ≥ 10X genome coverage depth

Data Analysis

In terms of the extensive experience in human genome resequencing service, CD Genomics provides statistical and bioinformatic data analysis services on SV detection, Haplotype Genotyping, DNA Methylation, and customized bioinformatics services are also available upon request.

Analysis pipeline

CD Genomics provides full whole genome resequencing service package including sample standardization, library construction, deep sequencing, raw data quality control, and bioinformatics analysis. We can tailor this pipeline to your research interest. If you have additional requirements or questions, please feel free to contact us.

1. What is the depth of human whole-genome resequencing?

The sequencing depth is determined by research purpose, sample numbers, and your needs. The general depth of human whole-genome resequencing is 30X. For the detection of germline variations, we recommend sequencing depth of 30-50X, such as researches on single-gene disease. For population studies with multiple samples, if you focus on SNP, sequencing depth of 10X is enough. If you focus on structural variations in cancer tissues, we recommend sequencing depth should be more than 50X.

2. What methods can be used to validate the results?

Whole-genome resequencing can detect different types of genetic variations, including SNP, InDel, SV, and CNV.

  • PCR amplification and sequencing or SNP genotyping can be used to validate SNPs.
  • PCR amplification and Sanger sequencing can be used to validate short-fragment InDels.
  • Real-time PCR are useful for validating CNVs.
  • Small scale SVs can be validated by PCR amplification and sequencing, while large scale SVs need to be validated by microscopic observation, such as FISH.
For Research Use Only. Not for use in diagnostic procedures.
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