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A method for mitochondrial sequence assembly and copy number determination based on high-throughput sequencing

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

Active Publication Date: 2021-12-17
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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  • A method for mitochondrial sequence assembly and copy number determination based on high-throughput sequencing
  • A method for mitochondrial sequence assembly and copy number determination based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Mitochondrial DNA sequencing and stitching three cases Mitten, comprising the steps of:

[0037] 1. Total DNA 3 Chinese mitten only muscle tissue, sonicated to break 500-700bp, using DNA library construction kits construct a high-throughput sequencing library.

[0038] 2. Construction of high-throughput sequencing and library use NextSeq500 high-throughput sequencing sequencing, sequencing each sample volume of 2G. And use the software bcl2fastq sequencing results into fastq format.

[0039] 3. Trimmomatic software sequencing data for quality control, particularly for the key parameters: LEADING: 5TRAILING: 5SLIDINGWINDOW: 4: 15MINLEN: 30, to give high quality sequencing data.

[0040] 4. SPAdes software sequencing data of the above-described stitching quality, particularly for the key parameters: - meta-k 55 (see Table 1).

[0041] 5. The control group of the above-described velvet software sequencing data quality stitching, specifically the key parameters: -cov_cutoff 3...

Embodiment 2

[0048] The effects of different sequencing amounts and mitochondrial DNA sequence occupy the comparison of splicing effects, the steps are as follows:

[0049] 1. Simulate data of different sequence of sequence and mitochondrial DNA sequence ratio. Using high quality sequencing data in Example 1, the data of 1 / 3, 1 / 6 and 1 / 12, and using data without mitochondria DNA with the aforementioned randomly selected data, to total data 2G, 1G and 0.5g.

[0050] 2. Use the Spades software to splicing the analog data, and the splicing process will be operated in step 4 in the above embodiment 1.

[0051] 3. Find the mitochondrial DNA sequence of splicing in the splicing result, the method is the same as in the first embodiment.

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

[0053] sample Data Size (BP) Sample 1 sequence occupation Splicing length (BP) KMER coverage E1 2G 1 / 3 16096 127.98 E2 1G 1 / 6 16096 64.34 E3 0.5G 1 / 12 16091 ...

Embodiment 3

[0056] The calculation of mitochondrial copy numbers in sequencing data, the steps are as follows:

[0057] 1. Sequencing and splicing data are data and splicing results used in Example 1. Use the ReseqTools software to view the total base base of high quality data, remember to n total .

[0058] 2 calculate the total length of mitochondrial DNA after stitching, remember to mito .

[0059] 3 Use Bowtie2 software to bring high quality data to the mitochondrial splicing result sequence and generate SAM data files. The above SAM file is calculated using the FlagStat function of SamTools to obtain the total base of the total base of READs that can be compared to the mitochondrial DNA sequence. mito .

[0060] 4 Calculate the normalization copy number m = (n mito / S mito ) / N total . The results are as follows:

[0061] sample N total (bp)

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Abstract

The invention belongs to the technical field of genomics, and specifically relates to a method for directly measuring eukaryotic mitochondrial sequences and copy numbers by using high-throughput sequencing (next-generation sequencing, second-generation sequencing, and deep sequencing) technology. The invention includes high-throughput sequencing of total DNA; quality control of sequencing data; sequence splicing based on similar metagenomics; and calculation of mitochondrial content. The method realizes the assembly of mitochondria using less sequencing data without the need for mitochondrial enrichment operations, and has the characteristics of simple experiments, low sample quality requirements, good splicing integrity, and the relative content of mitochondria can be measured. It can meet the needs of scientific research and application to a certain extent.

Description

Technical field [0001] The present invention belongs to the technical field of the genome, particularly relates to a method of using high-throughput sequencing (next generation sequencing, a second-generation sequencing, sequencing depth) techniques and direct determination of the copy number of the mitochondrial sequence eukaryote. Background technique [0002] Mitochondria are eukaryotic cells to provide energy organelle, occupies a central position in the energy conversion organisms and metabolic processes. [0003] Mitochondria containing the DNA, called mitochondrial DNA (mitochondrial DNA, mtDNA), mtDNA and nuclear composition of the different genomes, mitochondrial genomes. Mitochondrial genome size is much smaller than the nuclear genome, usually metazoan mitochondrial genome of only a dozen kb size, plant mitochondrial genome is slightly larger, about several hundred kb. [0004] Number of differences in mitochondrial DNA copy large cells, such as liver cells 1000-2000 c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B30/10
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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