A position-anchored barcoding system for nanopore sequencing library construction

Abstract

The present application relates to a position-anchored barcode system for nanopore sequencing library construction, a preparation method and an application thereof. The position-anchored barcode system described in this application has higher resolution and higher classification accuracy, can significantly reduce the false positive rate of identification, improve the accuracy of nanopore sequencing as a whole, and reduce the cost of sequencing.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a position-anchored barcode system for nanopore sequencing library construction. Background technique [0002] At present, there are many clinically infected patients in the world and there are various sources of infection. In China, infectious diseases even account for 49% of the total incidence of all diseases. At present, the conventional clinical diagnosis method is the doctor's empirical judgment plus microscopic examination, biochemical analysis, etc. to determine the source of infection of the symptoms, but human factors, detection cycle and detection range limitations can easily lead to false detection and missed judgment, especially not conducive to acute Diagnosis and treatment of infection. With the vigorous development of high-throughput sequencing and genomics, metagenomic sequencing technology is flourishing in the field of infection diagnosis because it can quickly,...

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Problems solved by technology

[0003] The development of Illumina next-generation sequencing is in full swing in China, but there are the following problems in the application of microbial detection: First, the read length of next-generation sequencing is less than a few hundred bp, and there will be high homologous sequences among different species of microorganisms, resulting in The accuracy of metagenomic species analysis is poor, and irrelevant microbial information is fed back in the data report, which instead causes greater diagnostic interference to doctors; secondly, the identification of deeper disease-causing genes and drug-resistant genes requires assembly and splicing of sequencing sequences , so complex analysis requires higher time and capital costs to make up for the read length defect of next-generation sequencing data; in addition, the instruments related to next-generation sequencing are expensive, cumbersome to operate, and the initial investment is high, and the entire sequencing time is long, making it difficult to Applicable to the needs of acute infection

The third-generation sequencing technology PacBio has greatly improved the sequencing read length, and can detect 8-12kb, or even 40-70kb long fragment data, but its disadvantage is that the library construction process is relatively complicated

However, when distinguishing samples according to the barcode sequence, it was found that the 24bp barcode included in the library construction kit was seriously confused. The reason was that the Oxford Nanopore sequencer would cause reads The error rate of single base in the medium is as high as 10-15%. Therefore, when the reads are classified according to the barcode sequence in the downstream data analysis, the cross-contamination of data between samples will be caused by the error of barcode identification, which will lead to false positive identification of microorganisms, which will bring negative consequences for clinical decision-making. come to great trouble

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