TGS-based Resequencing

Aiming at discovering structural variations and exploring genetic mechanisms of phenotype

Service Overview


Third-generation sequencing (TGS) is prominent for its read length. The leading TGS platforms , including those from PacBio and Oxford nanopore, have gained massive applications in animal/plant genome researches. Taking the advantages of its read length (approx. 10-20 kb), TGS-based resequecing achieves more precise identification of genetic variations compared to reference genome, including structural variation(SV), copy number variations(CNV), etc., which is hardly discovered NGS-based resequencing. The identified SV and CNV can be applied in the genetic analysis at population level. Moreover, it is also able to reveal the relations between these mutations and gene functions, phenotypes, etc. TGS-based resequencing provides a completely new strategy for discovering genetic variations.

Applications

1. Identification of animal/plant structural variation
2. Identification of exogenous gene insertion


Bioinformatic Analysis



Sequencing data quality control
Reference genome mapping
SV identification and annotation
Differential SV analysis among samples
Differential SV annotation and functional enrichment
CIRCOS display of whole genome SV

ONT Resequencing Sample Requirement

DNA sample
Conc. (ng/μl) 
(on Qubit)

Total
(μg)

OD260/280
OD260/230
Agarose gel electrophoresis
Conc. Ratio Nanodrop/Qubit
Appearance
≥50
Data volume related (Normally, 2 μg DNA is required for production of 60 G data
1.8-2.2
1.8-2.5
Size ≥23 kb;
degraded bands>5 kb;
No or very limited contamination observed in loading wells

0.9-2.0
Colourless;
No precipitation observed.



Tissue sample
Species Organ Sampling
Animal
Internal organs, such as liver, spleen, etc. ≥0.35 g
Animal Muscle ≥0.5 g
Animal Mammal blood ≥0.5 ml
Animal Bird or fish blood ≥0.1 ml
Plant Fresh leaves ≥0.1 ml
Plant Flower; stem
≥1.0 g
Plant Root; seed ≥2.0 g
Cells
Tissue culture ≥1e7 cells

Service Advantages

Biomarker Technologies is a leading provider of Third-generation Sequencing services. We keep equipping ourselves with the latest TGS platforms. In 2015, the first PacBio sequencing platform settled. So far, we own PacBio Sequel II, PacBio Sequel and RS II. Nanopore sequencing platform was imported in 2017. To date, we own all ONT platforms including PromethION-48, PromethION-β, GridION and MinION. We are one of the biggest TGS service providers aiming at providing the most comprehensive TGS services.

Case Study

Case 1.

“Title: Mapping and phasing of structural variation in patient genomes using nanopore sequencing
Journal:  Nature Communications
Published: 2017

Main results

  • ONT long reads is superior to Illumina in detection of SVs. de novo chromothripsis rearrangements were identified in two patients with congenital abnormalities.
  • ONT long reads enable efficient phasing of genetic variations (SNV and SV)
  • Large proportion of retrotransposon insertions were missed in short read sequencing

Materials and methods

  • PBMC and lymphoblastoid cells originated from two patients respectively were cultured for DNA extraction.
  • TGS was performed on MinION sequencing platform with an average library length of 10 to 20 kb and average sequencing depth of 16x and 11x.
  • NGS was performed on Illumina HiSeq X platform, with and average library length of 400 to 500 bp and average sequencing depth of approximately 30x. 
Figures quoted from DOI: 10.1038/s41467-017-01343-4

Case 2.

“Title: The population genetics of structural variants in grapevine domestication
Journal: Nature Plants
Published: 2019

Background
Structural variants (SVs) are a largely unexplored feature of plant genomes. Little is known about the type and size of SVs, their distribution among individuals and, especially, their population dynamics. Understanding these dynamics is critical for understanding both the contributions of SVs to phenotypes and the likelihood of identifying them as causal genetic variants in genome-wide associations.

Main results
Here, we identify SVs and study their evolutionary genomics in clonally propagated grapevine cultivars and their outcrossing wild progenitors. To catalogue SVs, we assembled the highly heterozygous Chardonnay genome, for which one in seven genes is hemizygous based on SVs. Using an integrative comparison between Chardonnay and Cabernet Sauvignon genomes by whole-genome, long-read and short-read alignment, we extended SV detection to population samples. We found that strong purifying selection acts against SVs but particularly against inversion and translocation events. SVs nonetheless accrue as recessive heterozygotes in clonally propagated lineages.
Text and figures quoted from DOI: 10.1038/s41477-019-0507-8