Mitochondria sequence splicing and copy number measuring method based on high-throughput sequencing

A technology of sequence splicing and mitochondria, applied in the field of genomics, can solve the problems of high sample quality requirements, troublesome design, complicated operation, etc., and achieve the effects of low sample quality requirements, low unit cost, and simple experiments.

Active Publication Date: 2018-11-02
沈阳中科赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first method still needs to design multiple pairs of primers and perform amplification, which has the disadvantages of cumbersome design and cumbersome operation
The second method requires higher sample quality, and some specimens preserved in alcohol cannot be applied to this method

Method used

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  • Mitochondria sequence splicing and copy number measuring method based on high-throughput sequencing
  • Mitochondria sequence splicing and copy number measuring method based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The mitochondrial DNA sequencing and assembly of three cases of Eriocheir sinensis included the following steps:

[0037] 1. The total DNA of the muscle tissue of 3 Chinese mitten crabs was ultrasonically interrupted to 500-700bp and used DNA library construction kits construct high-throughput sequencing libraries.

[0038] 2. Construct and use high-throughput sequencing library NextSeq500 high-throughput sequencer performs sequencing, and the sequencing volume of each sample is 2G. And use bcl2fastq software to convert the sequencing results into fastq format.

[0039] 3. Use Trimmomatic software to control the quality of the sequencing data. The specific key parameters are: LEADING:5TRAILING:5SLIDINGWINDOW:4:15MINLEN:30 to obtain high-quality sequencing data.

[0040] 4. Use SPAdes software to splice the above-mentioned high-quality sequencing data. The specific key parameters are: --meta-k 55 (see Table 1).

[0041] 5. The control group used velvet software to splice the ab...

Embodiment 2

[0048] The effect of different sequencing amounts and the proportion of mitochondrial DNA sequences on the splicing effect is as follows:

[0049] 1. Simulate data with different sequencing amounts and mitochondrial DNA sequence ratios. Using the high-quality sequencing data of the sample in Example 1, randomly select 1 / 3, 1 / 6, and 1 / 12 of the data, and use the data without mitochondrial DNA to merge with the aforementioned randomly selected data until the total amount of data reaches 2G, 1G and 0.5G.

[0050] 2. Use SPAdes software to splice the simulated data, and the splicing process refers to step 4 in the above embodiment 1.

[0051] 3. Find the spliced ​​mitochondrial DNA sequence in the splicing result, and the method is the same as step 7 in Example 1.

[0052] The stitching results of the simulation results are as follows:

[0053] sample

[0054] The results show that even if the mitochondrial DNA content in the data is reduced to 1 / 12 of the original data, longer splicin...

Embodiment 3

[0056] To calculate the mitochondrial copy number in the sequencing data, the steps are as follows:

[0057] 1. Sequencing and splicing data are the data and splicing results used in Example 1. Use Reseqtools software to view the total number of bases in high-quality data, denoted as N total .

[0058] 2 Calculate the total length of mitochondrial DNA after splicing and mark it as S mito .

[0059] 3 Use bowtie2 software to compare high-quality data to the sequence of mitochondrial splicing results and generate sam data files. Use the flagstat function of samtools to calculate the above sam file to obtain the total base number of reads that can be compared to the mitochondrial DNA sequence, denoted as N mito .

[0060] 4 Calculate the normalized copy number of the sample M = (N mito / S mito ) / N total . The results are as follows:

[0061] sample

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Abstract

The invention belongs to the technical field of the genomics, and specifically relates to a method for directly measuring eucaryon mitochondria sequence and copy number by using the high-throughput sequencing (the next generation of sequencing, the second generation of sequencing, and the depth sequencing). The method comprises the following steps: performing high-throughput sequencing on the total DNA; performing quality control on the sequencing data; performing sequence splicing based on similar metagenome; and computing the mitochondria content. Through the method disclosed by the invention, the mitochondria assembling is performed by using less sequencing data in the premise of not performing the mitochondria enrichment operation, and the method has the advantages of being simple in experiment, low in sample quality requirement, good in splicing integrity, and capable of measuring the relative content of the mitochondria. And the scientific research and application demand can be satisfied to a certain degree.

Description

Technical field [0001] The invention belongs to the technical field of genomics, and specifically relates to a method for directly determining eukaryotic mitochondrial sequence and copy number by using high-throughput sequencing (next-generation sequencing, second-generation sequencing, deep sequencing) technology. Background technique [0002] Mitochondria are the organelles that provide energy in eukaryotic cells, and occupy a central position in the energy conversion and metabolism processes of organisms. [0003] The mitochondria contains DNA, called mitochondrial DNA (mtDNA). MtDNA forms a genome different from the nucleus, that is, the mitochondrial genome. The size of the mitochondrial genome is much smaller than that of the nuclear genome. Generally, the mitochondrial genome of metazoans is only a dozen kb in size, while the mitochondrial genome of plants is slightly larger, about hundreds of kb. [0004] The number of copies of mitochondrial DNA in different cells varies gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G06F19/18
Inventor 王尧峰薛金会郭海燕王伟伟程恩泽
Owner 沈阳中科赛尔生物科技有限公司
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