MtlA gene sequencing based on 3rd -generation sequencing for Vibrio typing.

Abstract

Background: Traditional serotyping and multilocus sequence typing (MLST) for Vibrio parahaemolyticus have limitations in resolution and accuracy, particularly for strains with close genetic relationships. The long read length and high sequencing quality of a new type of nanopore sequencer G-seq500 make it possible to type V. parahaemolyticus strains using mtlA, a new identified typing gene for Vibrio. This gene not only offers advantages in cost-effectiveness and speed relative to MLST but also demonstrates comparable resolution.

Results: We sequenced the full-length mtlA gene (1,953 bp) of 33 V. parahaemolyticus strains using Geneus G-seq500. A total of 199,658 long reads with an average length of 17,306 bp were produced by one sequencing chip. After recognizing adaptor and barcode, each long read can be split into 12 subreads with mean accuracy of 93.25%, which were then aligned into 64,086 consensus sequences with a mean quality Q35 (sequence accuracy 99.88-99.93%). The final mtlA gene was generated by clustering all the consensus sequencing from same strain, and its accuracy reached 100% with over four consensus sequences clustered together. Then we developed a naming rule based on mtlA clustering of 7,573 mtlA sequences from NCBI together with the 33 mtlA gene sequenced by G-seq500, with the largest cluster composed of 2,321 strains designated as mtlA0001.

Conclusions: Our study confirms the high sequencing accuracy of Geneus G-seq500, and the effectiveness of using the full-length mtlA gene for V. parahaemolyticus strain typing and epidemiological surveillance.