Construction method of RNA (Ribonucleic Acid) and DNA (Deoxyribose Nucleic Acid) next-generation sequencing library and next-generation sequencing box

Abstract

The invention belongs to the technical field of biology, and discloses a construction method of an RNA (Ribonucleic Acid) and DNA (Deoxyribonucleic Acid) next-generation sequencing library and a next-generation sequencing box. The construction method of the next-generation sequencing library comprises the following steps of carrying out one-chain synthesis on RNA nucleic acid and DNA nucleic acid to obtain one-chain cDNA (Complementary Deoxyribonucleic Acid); carrying out two-chain synthesis on the one-chain cDNA to obtain two-chain cDNA; carrying out fragmentation, terminal repair, phosphorylation and A addition treatment on the two-chain cDNA to obtain an A-added product; carrying out linker connection and first purification treatment on the A-added product to obtain a target RNA fragment and a DNA fragment, and recovering the target RNA fragment and the DNA fragment; and carrying out PCR amplification reaction on the target RNA fragment and DNA fragment to construct and obtain the RNA and DNA next-generation sequencing library. According to the method for constructing the next-generation sequencing library, the overall library construction process is adopted, the process steps are simplified, the operation process is greatly shortened, the experiment time is greatly shortened, the loss of nucleic acid samples can be reduced to the maximum extent, meanwhile, two libraries do not need to be constructed, and the cost of manpower, reagents, time and the like can be greatly saved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing an RNA and DNA next-generation sequencing library and a next-generation sequencing box. Background technique [0002] At present, for samples infected by unknown pathogenic microorganisms on the market, most of the existing solutions use the method of separate construction of RNA library and DNA library, which is time-consuming, laborious and costly. [0003] Such as figure 1 As shown, the existing routine RNA library construction process is: 1. Obtain about 1-10 μg of pure, complete and quality-checked total RNA; 2. Purify mRNA from total RNA; usually, this is done by annealing total RNA to Completed on oligo-dT magnetic beads; two rounds of purification can be performed to remove non-specifically bound ribosomes and other RNA from oligo-dT; then release or isolate mRNA from oligo-dT magnetic beads; 3. Use fragmentation reagent to Purify the mRNA for frag...

AI Technical Summary

Problems solved by technology

It can be seen that the existing RNA library construction steps are cumbersome, the total amount and quality of RNA are high, and it takes a long time to purify multiple magnetic beads. The second-strand synthesis product and the end-repair product adapter ligation product need to be purified, and the end-repair and A steps Unable to be integrated, these processes are not only costly and time-consuming, but also multi-step purification will inevitably increase the loss of RNA samples

However, the RNA virus itself has low content and poor stability, and it is seriously lost during multiple rounds of experimental operations, and it is very easy to miss detection because it cannot reach the detection limit.

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