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Product Brief

Whole Genome Resequencing ( Re-Sequencing ) It is the sequencing of individuals with known genome sequence information, which can be used to analyze genotype differences of individuals or groups. Genome sequencing is used to assist researchers in finding large single nucleotide polymorphisms. SNP, Copy Number Variation (CNV), insertion deletion (InDel, Insertion/Deletion) and other ectopia sites. It is of great significance to efficiently and accurately obtain the genetic characteristics of biological groups and facilitate genome-wide association analysis (GWAS).


Types of genome alterations that can be detected by next-generation sequencing

Meyerson et al., Nature Reviews Genet, 2010


Our advantages

Accelerated analysis of 1. sequenced analysis: strong ice created a three speed scheduling acceleration framework from task delivery, data segmentation to container multithreading, and eventually achieved a significant speedup of resverfacing analysis, and 4 months to complete a human resampling analysis of a sample, which was more than 10 times faster than the traditional analysis method (68-92 hours).

2. customized analysis strategy: Based on different sequencing species and sequencing programs, customized selection of reference genome version, comparison algorithm and annotation database area information;

3. comprehensive database integration: constantly updating genome database and integrating multi database and multi version to obtain accurate gene information and annotations.

4. powerful combination analysis ability: combining genome sequencing with transcriptome sequencing and methylation sequencing technology to further expand single gene variation data.



Sample requirements

Tissue samples:

One Dry weight of fresh animal tissues More than 0.5g

Two Fresh plant tissue dry weight More than 2G

Three Number of fresh cultured cells More than 4 x 10 Six individual

Four Whole blood (mammal) More than 1ml

Five Whole blood (non mammalian) More than 0.5ml

DNA sample requirements:

1. the total amount of DNA is more than 2 g , concentration It is more than 20ng/ L and the volume is 15-100 L.

2. OD260/280 is between 1.8-2.0.

3. Agilent 2200 quality inspection qualified, DNA sample main peak range is 100-500bp;

Four Electrophoretic detection was not obvious. The genome of RNA is clear and complete without degradation.

Five Please mark the sample number clearly when sending the sample. Parafilm membrane seal;

6. avoid repeated freezing and thawing during preservation.

7. dry ice transportation for sample delivery.



Experiment flow


One Customer samples: number of fresh cultured cells More than 4 x 10 Six One;

Two Genomic extraction The total amount of DNA:DNA is more than 2 g.

3. DNA quality control: Agilent 2200 quality inspection qualified, DNA sample main peak range in 100-500bp;

Four Library Construction: random primers PCR amplification;

Five On board sequencing: sequencing data reached 50X coverage varies from species to species and generally reaches 30X.



Data analysis process

Result example

1. Quality control of raw data
Fastp software was used to conduct low quality sequence, undetected sequence and filtering sequence of the joint sequence. The GC ratio, base mass, length distribution, joint retention and Duplication ratio were analyzed before and after filtration.

Base quality results map

Note: the abscissa of the left picture represents the base site, and the ordinate represents the base mass. The different color curves represent different bases in each. The mass values on the read; the right cross coordinates represent the base sites, and the ordinates represent the ratio of the base content. The different color curves represent the bases of different loci.


2. DNA genome alignment (DNA Mapping) and quality control
The BWA-mem/Bowtie2 algorithm was used to compare the sequencing data with the genome after the quality control, and the BAM files for genome alignment were obtained. Based on the BAM file, the distribution of reads on chromosomes and genomic alignment rate were obtained.

DNA Mapping results

Yamagishi MEB et al., PLoS One. 2017

Note: the map shows different strains. Bull sequencing reads Of Mapping rate


3. Catastrophe analysis
GATK/Samtools and other algorithms were used to analyze genomic comparison files (including SNV and InDel), and the mutation results were obtained.

Call SNP analysis results

Kong HR et al., AJAS. 2018

Note: the graph shows the number of SNP in each chromosome of different groups.



4. Copy number variation (CNV) analysis
Using CNVKit algorithm, copy number variation analysis of genome alignment files is carried out, and copy number variation results are obtained.

Genome wide distribution of CNV region

Khatri B et al., PLoS One. 2019

Note: the map shows different strains. Japanese quail CNV of HS and LS) The distribution of the region on the whole genome. Outer ring HS strain, inner circle is LS strain.


5. Database annotation
The results of the mutation analysis were annotated by VEP, and the annotations of the corresponding genes and their mutation loci were obtained.

Mutation annotation result

Note: the map shows the type of mutation based on database identification and the proportion of different types of mutations.

6. Site selection
All mutation annotation results were taken as research objects, and mutation filtering was carried out to obtain mutation sites with research significance and annotation information.

Multiple criteria selection

Note: the map is also shown in the sample population. Variation frequency of CNV and SNV



7. Functional analysis of mutant genes (GO Analysis)
NCBI/UNIPROT/SWISSPROT/AMIGO and other GO databases were used to carry out functional analysis of the mutant genes and obtain GO entries that were significantly enriched in mutant genes.

GO analysis results

Note: the graph is from biological process. BP, molecular function (MF) and cell component (CC) showed three significant GO entries (Top 15).



 


8. Mutation gene signaling pathway analysis (Pathway Analysis)
The KEGG database was used to analyze the signal transduction pathway of the mutant gene, and Pathway items with significant enrichment were obtained.

chart 9 Pathway analysis results

Note: the map shows mutant genes. Pathway analysis results (Top 15), red is a significant item, and blue is a non significant item.



Advanced data analysis
1. Conservation analysis of mutation sites
According to the mutation type of missense, the pathogenicity of loci was judged by SIFT and Polyphen algorithm based on the conservatism of loci between species and the role of locus in protein structure.

Conservativeness analysis of mutation sites

Guo Q et al., Dna & Cell Biology, 2014

Note: Multi species MYH7 amino acid sequence alignment results The results showed that the mutant site was highly conserved and predicted the protein before and after the mutation. 3D structure


2. Wayne analysis (Venn Analysis)
The mutation sites identified by each sample were analyzed by Wayne analysis, and the unique or common mutation sites among the samples were obtained by Wayne mapping analysis. The corresponding genes were further obtained by mutation annotation, and GO and Pathway analysis were performed.


SNVs and InDels Wayne diagrams

Yamagishi MEB et al., PLos ONE. 2017

Note: the graph shows. SNVs and InDels identified in 4 strains. Of Venn analysis results



Examples of literature

[1] Khatri B, Kang S, Shouse S, et al. Copy number Copy, B 2019, 14 (3):


[2] Yu Y, Fu J, Xu Y et al. Genome re-sequencing Genome, Y 2018, 9 (1): 5404.


[3] Reimer C, Rubin CJ, Sharifi AR, et al. Analysis of Analysis, C 2018, 19 (1): 687.


[4] Kong H R, Anthony N B, Rowland K C, et C, H 2018, 31 (1):


[5] Yamagishi MEB, Chud TCS, CaetanoAR, et Al.Single Nucleotide variants and InDels identified from whole-genomere-sequencing of Guzerat, Gyr, Girolando, Guzerat 2017, 12 (3):


[6] Linn e a Smeds, Mugal C F, Anna Qvarnstr m m, et m, 2016, 12 (5):


[7] He Y, Wang C, Higgins J, et al. MEIOTIC F-BOX, al. 2016, 28 (8):


[8] Wei F, Jie Z, Zhijing L, et al. Development of, al. 2016, 11 (1):


[9] Torkamaneh D, Laroche, J e r r me, Belzile, Fran C OIS, et OIS, D 2016, 11 (8).


[10] Guo Q, Xu Y, Wang X, et al. Exome Sequencing Exome, Q 2014, 33 (10):


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