Nanopore sequencing,is an emerging third-generation sequencing technology with great application prospects,but its application in the field of animal pathogens is still in its infancy.To explore its potential in determining the whole genome sequences of avian influenza virus(AIV),RNA was extracted from two AIV-positive chicken swab samples collected from a poultry market.The AIV universal primers were employed for targeted amplification of the entire genome.Following steps such as end repair,addition of A bases at the 3'end,barcode ligation,and sequencing adapter attachment,the sequencing library was prepared.Using the GridION×5 Mk1 sequencer,7.16 gigabytes of data were generated,and the obtained sequencing data underwent quality control,low-quality reads removal,comparison and splicing,and assembly to acquire the complete AIV genome sequences,which took 13 hours to complete the entire experimental process.Simultaneously,first-generation sequencing methods were employed to validate the accuracy of nanopore sequencing results.The quality control results of the sequencing data indicated that all reads had quality scores exceeding 10,demonstrating high sequencing data quality.Of which,AIV1 produced 404474 reads with an average length of 996.7 base pairs and an average quality score of 13.8,while AIV2 generated 76589 reads with an average length of 1095 base pairs and an average quality score of 13.5.The coverage rate and sequencing depth of the eight gene segments of AIV were calculated using samtools software,and the results revealed that the whole genome coverage rate of both AIV1 and AIV2 reached 100%,with an average sequencing depth of 34 473×and 7 470×respectively,indicating a highly reliable assembly results.Through the comparative analysis of Geneious Prime software,the sequencing results obtained from nanopore third-generation sequencing technology are comparable to that from the first-generation sequencing method,with a consistency of 98.78%to 100%.Of which,the consistency observed in seven gene segments of AIV1 and five gene segments of AIV2 reached 100%,.and even the lowest consistency in the MP gene reached 98.78%,indicating the high accuracy of this method.This study successfully explores the application of nanopore third-generation sequencing technology in AIV whole-genome sequencing.By optimizing amplification conditions and data processing,a novel method for determining the complete genome sequence of avian influenza virus has been established,which can play a crucial role in the emergency detection of new avian influenza outbreaks..
维普
万方数据
