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Product Brief

Exons only account for the human genome. 2.5%, but contains most of the information related to gene expression. The variation in this region is closely related to cancer, monogenic diseases, and some complex diseases. Exon capture sequencing only sequenced the sequence of exon regions. Compared with genome sequencing, the cost of genome sequencing is low, and a large amount of useful information can be obtained with very small amount of data. It has great advantages in studying SNP and InDel of genes.


Aglient SureSelect exon capture system ( Www.Agilent.com )

Our advantages

1. when integrating existing databases, new mutation sites can be found.

2. adopt the authoritative exon data analysis process in bioinformatics field, and support the high score literature.

3. integrate the latest annotation database to ensure the accuracy and high veracity of the results.

4. design multiple sets of perfect alternatives for specific research subjects and recommend the best one.



Sample requirements

Tissue samples

One Dry weight of fresh animal tissues More than 0.5g

Two Fresh plant tissue dry weight More than 1G

Three Number of fresh cultured cells More than 8 x 10 Six individual

Four Whole blood (mammal) More than 2ml

Five Whole blood (non mammalian) More than 0.5ml

6. formalin fixed paraffin embedded tissue (FFPE) > 10 slides or 50mm. Two 5-10 slices of 10 m thick slices.

DNA sample

One Please provide the total amount. More than 3 mu g/ Sample concentration DNA greater than 50ng/ mu L;

2. OD260/280 is between 1.8-2.0.

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

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

5. avoid repeated freezing and thawing during preservation.

6. dry ice transportation for sample delivery.

Note: there are differences between different samples. Please refer to the ice cream for more details.



Experiment flow


1. DNA extraction and quality control: gel electrophoresis quality control > Nanodrop quality control > Agilent 2200 quality control;

2. target acquisition: exon acquisition of Agilent Sureselect V6 , Complete the crosses within 90 minutes.

3. primer sequence degradation: removal of primer sequences;

Four Library construction and quality control: PCR amplification;

5. on board sequencing: Strong ice recommended choice NovaSeq, dual terminal sequencing, has high flux, high base precision, low cost and fast speed. Sequencing data: 10Gb.




Data analysis process

Result example

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

Quality control results of raw data

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
BWA-mem/Bowtie2 algorithm was used to compare the sequencing data after quality control to the genome and get the BAM files for genome alignment. Based on the BAM file, the distribution of reads on chromosome and genomic alignment rate were obtained.

DNA Mapping results

Note: left picture is Reads The distribution on the chromosome; the right picture is Comparison of reads and reference genome sequences



3. DNA genome alignment optimization
Samtools and GATK tools were used to analyze the BAM files of genome alignment by Remove Duplicate and Recalibrate, and the Bam files optimized after each sample were obtained.

Genome alignment optimization

Note: the graph shows. GATK tools before correction Later Reads base mass



4. Somatic mutation analysis (SNP Calling)
Mutect2 and other algorithms were used to carry out SNP Calling analysis.

SNP Calling

Jiang et al., Hepatology, 2014

Note: IGV confirm candidate somatic mutation site map



5. Somatic mutation notes
VEP software was used to annotate the loci of mutation sites, and the display of mutation types and mutation sites in various databases were also presented.

Somatic mutation gene annotation and mutation type

Coco S et al., Cancers, 2019

Note: the above is a mutation gene in the tumor tissue samples. The following is a mutation frequency of the mutant type, a transverse axis is a mutation type, and a mutation frequency with a longitudinal axis is a specific mutation type.



6, mutation filtering
Using the industry standard tumor somatic mutation filtering strategy, all SNP annotations were mutated and filtered to obtain meaningful SNP loci and their annotation information.

SNP/SNV screening

Jiang et al., Hepatology, 2014

Note: the map shows tumor somatic cells. Filtering strategy for SNP/SNV mutation



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

All differential genes GO analysis results

Note: the map shows significant pre concentration. Fifteen individual GO entries, abscissa coordinates -log10 (P-value). Ordinate representation The name of the GO entry.



8. Mutation gene signaling pathway analysis (Pathway Analysis)
The KEGG database was used to analyze the signal transduction pathways of mutant genes and obtain the signal pathway entries that were significantly enriched in mutant genes.

Pathway enrichment scatter plot

Note: the map shows significant enrichment. Twenty strip Pathway entries. Abscissa represents Rich factor corresponding to pathway. Ordinate representation Pathway annotation information, the color of the dot represents the size of P-value, and the size of the dot represents the number of differential genes contained in pathway.



Advanced data analysis
1. Constructing phylogenetic tree.
The homologous proteins of several species were selected to construct phylogenetic tree to further understand the conservatism and evolution history of the protein gene in different species.

Phylogenetic tree

Jiang et al., Hepatology, 2014

Note: the phylogenetic tree contains HECT domain homologues. Scale bar The number of amino acids per locus is expressed, and the value at each node represents the corresponding number. Bootstrap value.



2. Build a protein model.
Mutations occurring at protein active sites or modified sites will greatly affect the function of proteins. Therefore, mutations in the protein active center can be found through the 3D pattern of proteins.

Protein model

Jiang et al., Hepatology, 2014

Note: bioinformatics is used to simulate protein structure and establish a model for analysis and display. The Glu959 mutation is located on the alpha helix on the surface of HECT domain, which may be related to the protein active site.



3. Evolutionary analysis
Amino acid conservation was predicted by homologous genes of other genes and other species, and whether the mutation sites were conserved amino acid sites. If conserved, the mutation might affect the protein function.

Evolutionary analysis

Jiang et al., Hepatology, 2014

Note: the map shows different species. UBE3Cparalogs Multiple sequence alignment results. Among them, The mutation sites in UBE3C are marked with red arrows.


Examples of literature

[1] Xu LX, He MH, Dai ZH, et al. Genomic and, al. 2019, 27.,


[2] Ricciuti B, Kravets S, Dahlberg SE, et al. Use of, al., B, Kravets, S, and S.


[3] Coco S, Bonfiglio S, Cittaro D, et al. Integrated Somatic, al. 2019, 28, 11 (4).


[4] Cho SY, Chae J, Na D, et al. Unstable Genome, al. 2019, 22.,


[5] Jiang J, et al. Clinical Significance of the Ubiquitin Ligase Ubiquitin 2014 J; 59 (6):

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