Method for constructing human single-cell mitochondrial high-throughput sequencing library and kit for library construction

Abstract

The invention provides a method for constructing a high-throughput sequencing library of human single-cell mitochondria and a kit for constructing the library. The kit includes mitochondrial genome full-circle amplification primers, single-cell lysate, end repair, phosphorylation, and 3' adenylate components for high-throughput library construction, adapters for high-throughput library construction, and forward and reverse Primers for library amplification, etc. This method realizes the high-throughput sequencing library construction of single or several cell mitochondrial genomic DNA in a system based on the complete circle amplification of the mitochondrial genome, and avoids the high off-target rate and high ratio of the conventional single-cell amplification system. The detection rate of false positive sites is high, thereby eliminating a large number of drawbacks in the detection of mitochondrial mutations at the single-cell level, and achieving high sensitivity and accuracy detection; At the same time as the mutation site was identified, large duplications and deletions of mitochondrial DNA were found.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a human single-cell mitochondrial high-throughput sequencing library and a kit for library construction. Background technique [0002] The mitochondrial genome (mtDNA) is the cell's second set of genetic material. The human mitochondrial genome is 16569bp in double-stranded circular shape, the double strands are H chain and L chain, including 37 genes, encoding 13 kinds of proteins and the non-coding region of D ring. mtDNA is easily affected by the external environment, and the mutation rate is 10 to 20 times higher than that of the nuclear genome; because the gene arrangement of mtDNA is compact, the intergenic region is only 87bp, so any mutation may affect an important functional region of the genome. At the same time, mtDNA is a naked molecule without histone protection, and is located near the inner mitochondrial membrane. It is directly exposed to su...

AI Technical Summary

Problems solved by technology

This method is tedious to operate and is extremely dependent on the pretreatment method of sample DNA extraction, and several DNA fragments amplified by multiple pairs of primers are affected by the amplification efficiency of different primers, so it is impossible to analyze the duplication or deletion of large fragments of mtDNA. At the same time, there are mitochondrial pseudogenes in the nuclear genome. If the amplification region of the primers is short, the primers can easily combine with nuclear genomic DNA, resulting in non-specific amplification. The interference of pseudogenes cannot be ruled out in the experimental results; mitochondrial diseases cannot be accurately diagnosed

It is worth mentioning that, based on the PCR method, the base mutation at the binding position of the amplification primer often cannot be detected.

2) Some commercial kits design probes for the human mitochondrial genome, and build a high-throughput sequencing library based on library construction combined with liquid-phase hybridization and capture of mitochondrial probes to find possible pathogenic mtDNA mutations; this method operates, It also depends on the pre-treatment method of sample DNA extraction, whether it is possible to obtain a complete mitochondrial full-circle genome; similarly, since the mitochondrial genome accounts for less than 1% of the nuclear genome, high-throughput libraries that require high-cycle PCR cycles will include mitochondria The inserted fragments are enriched and captured in the high background of nuclear genes. At the same time, the products captured by hybridization need to be amplified with a large number of PCR cycles to meet the requirements of high-throughput sequencing. This method is also unable to perform mtDNA amplification. Analysis of Fragment Duplications or Deletions

[0006] However, the nuclear genome of a single cell or a small number of cells is at the picogram level, and the mitochondrial genome is only at the femtogram level, so DNA extraction pre-treatment cannot be performed, and template enrichment can only be performed based on MALBAC single-cell amplification or SMART single-cell amplification, while These two single-cell amplification techniques have been criticized in practical applications due to the high off-target rate of random primers and the high number of false positive sites caused by high repeatability; A method for amplifying the complete circle of mitochondrial mitochondria and avoiding the disadvantages of routine cell input mitochondrial detection and high-throughput sequencing

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