High-throughput sequencing method of macrobenthos COI genes and application of high-throughput sequencing method of macrobenthos COI genes

Abstract

The invention belongs to the technical field of macrobenthos identification, and particularly relates to a high-throughput sequencing method of macrobenthos COI genes and application of the high-throughput sequencing method of the macrobenthos COI genes. The high-throughput sequencing method of the macrobenthos COI genes comprises the following steps: (1) with DNA samples of mixed macrobenthos samples as templates, conducting PCR amplification on the COI genes; (2) with PCR amplification products in the step (1) as templates, conducting PCR amplification to obtain PCR products as DNA libraries, wherein an amplification primer pair comprises pyrophosphoric acid sequencing high-throughput sequencing connectors, and each sample corresponds to one pyrophosphoric acid sequencing high-throughputsequencing connector; and (3) conducting quality detection on the obtained DNA libraries, and then choosing qualified DNA libraries for emPCR amplification and high-throughput sequencing. According to the method, through one-time sequencing, high-throughput ~658 bp sequences with reads covering the macrobenthos community full-length COI genes can be obtained, high taxonomy resolution is provided,and the false positive problem brought by excess throughput is avoided.

Description

technical field [0001] The invention belongs to the technical field of benthic animal identification, and in particular relates to a high-throughput sequencing method for the COI gene of benthic animals and an application thereof. Background technique [0002] Benthic invertebrates generally refer to invertebrate groups that live at the bottom of water bodies for all or most of their life history, mainly including molluscs, aquatic insects, annelids, and crustaceans. In aquatic ecological monitoring, benthic invertebrates are crucial for evaluating the ecological status of freshwater ecosystems. Although morphological characteristics play a very important role in species identification, they are easily restricted by biological sex and specific developmental stages, as well as affected by phenotypic plasticity and genetic variability, and due to the ubiquity of cryptic species and the impact of morphological identification on High professional requirements and other reasons ...

AI Technical Summary

Problems solved by technology

However, the Illumina platform for next-generation sequencing is limited by the read length, and most of them are sequenced based on the ~313bp COI fragment. Even with the longest read length of 300PE, the 658bp COI barcode still has a 100bp gap, and the barcode cannot be obtained by one-time sequencing. The full length of the region, the existence of Gap greatly affects the integrity of the data, and cannot meet the requirements of species identification

Moreover, due to the high throughput of Illumina sequencing, it is very easy to detect non-target DNA amplicons with very low copy number

Third-generation sequencing, such as Oxford nanopore and PacBio SMAT, has a longer read length, which can reduce the cost of splicing. However, the error rate of a single read length is high, and repeated sequencing is required to correct errors, which increases the cost of sequencing. Bioinformatics software is not rich enough, and data accumulation is small

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