Research on the Classification of Cutaneous Bacteria on Roughskin Sculpin with NGS

A common chronic inflammatory infection occurred on the skin of roughskin sculpin-skin ulcer caused by microbe, is one of the main causes leading to the declined fish production in the aquaculture. The modern research method of microbial community is culture-free high-throughput sequencing. To improve the sequencing efficiency, barcode has been introduced to the high-throughput sequencing to distinguish the samples. In this study, we explored the feasibility of sequencing the 16S rRNA gene V1~V3 regions of cutaneous bacteria of roughskin sculpin dually with Illumina MiSeq platform. We studied the impacts of barcode length and sequencing on the classification of the bacteria. Results showed that the splicing of dual-sequencing is up to 93% and the V1~V3 regions can be recognized successfully. Length of barcode had a great influence on the sequences de-multiplexing, and we suggested that barcodes with no less than 12 bp are better for the sequencing. The number of valid sequences had a great influence on the OTU division, and the number of OTUs increased linearly along with the increase of the sequences. While the sequencing of sample reached 6 000~9 000, the classification curves in different levels entered the plateau gradually, illustrating that the data was large enough to cover most bacteria. Flavobacterium, Shewanella and Pseudomonas are probably related to the roughskin sculpin's skin ulcer by analyzing the results of sequencing and the reported references.