Abstract: Embodiments of the present disclosure, pertaining to the technical field of high-throughput sequencing, relate to a method for high-throughput sequencing based on an internal reference with a known index. The method includes: generating a random DNA sequence, adding a single-ended adapter DNA sequence containing the known index at both ends of the random DNA sequence to obtain an internal reference sequence, and synthesizing a sequencing quality control sequence based on the internal reference sequence; performing, based on the sequencing quality control sequence, high-throughput sequencing on a library of samples to be tested to obtain sequencing data; and performing result analysis on the sequencing data to obtain a sample error distribution rate of the library of samples to be tested, and ending the high-throughput sequencing. According to the present disclosure, index hopping in the process of sequencing may be monitored based on the internal reference.

Abstract: Disclosed is a method for constructing a second-generation sequencing library of RNA and DNA. The method includes: performing first-strand synthesis on an RNA nucleic acid and a DNA nucleic acid to obtain a first-strand cDNA; performing second-strand synthesis on the first-strand cDNA to obtain a second-strand cDNA; obtaining an A-tailed product by fragmentation, end repair, phosphorylation, and A-tailing on the second-strand cDNA; performing adapter ligation and a first purification treatment on the A-tailed product to obtain a target RNA fragment and DNA fragment, and recovering the same; and carrying out a PCR amplification reaction on target RNA fragment and DNA fragment to construct and obtain the second-generation sequencing library of RNA and DNA.

Abstract: Disclosed is a preparation method for a DNA next-generation sequencing library, including steps: digestion, end repair, and A-tailing of genomic DNA; adapter ligation of DNA fragments; bead purification of product after adapter ligation; PCR amplification of DNA fragments; and selection and purification of PCR product fragments. The preparation method for the DNA next-generation sequencing library includes steps: fractionating by VVN and T7 through a single-step reaction process with double digestion and end repair, blunting a 5?-overhang under the polymerization action of a Taq DNA polymerase, adding an A (adenine) to a 3?-end, and achieving the preparation of the DNA next-generation sequencing library under an integrated single-step reaction. After the single-step reaction ends, bead purification isn't required, so that the preparation process is simple. In the preparation process, there is no preference for two restriction enzymes, which achieves the sequencing of target fragments.