Design Genus-Specific Primers And Universal Primers In Specific Environment Based On K-mer

Clarification of microbial community structure and diversity is an important issue in the field of molecular ecology.There are various microorganisms in the environment,and most of them cannot be cultivated in the laboratory,so it is impossible to cultivate microorganisms in the laboratory by simulating the environment to understand the microbial composition in the environment.The existing sequencing tools have achieved the sequencing of microorganisms in the environment,and based on the sequencing data,the species and abundance in the environment can be analyzed.For example,sequencing of human intestinal microorganisms can reveal the main distribution of species and find the relationship between intestinal microorganisms and some human diseases,such as diabetes and obesity.The 16S rRNA gene is an important target in bacterial evolution and ecology research.PCR amplification and sequencing of 16S rRNA sequences in the environment can be used to explore the diversity of bacteria in the environment and determine the phylogenetic relationships between taxa.The accuracy of 16S rRNA sequence analysis largely depends on the selection of primers,which poses great challenges for designing primers for microorganisms in the environment.On the one hand,there are a large number of bacterial genera in the environment and many sequences have high similarity,making it difficult to find highly specific fragments as specific primers in bacterial sequences.On the other hand,universal primers for bacteria are designed for 16S rRNA sequences of all bacteria,but specific environments do not include all bacteria,so it is necessary to design universal primers for specific environments.In response to the above two issues,this article developed algorithms for designing genus-specific primers and universal primers in specific environments based on k-mer.This article develops an algorithm for designing genus specific primers based on k-mer,with the goal of finding specific k-mer as a specific primer in the 16S rRNA sequence of the target bacterial genus.Firstly,relatively specific k-mers were preliminarily screened out.Then,the bacterial genus sets covered by the adjacent point sets of the two k-mers as forward and reverse primers are determined.If the intersection of the two only involves the target genus,then these two k-mers are candidate primers.Finally,the Primer-BLAST tool was used to verify their specificity and determine the final specific primer pairs.This algorithm designed multiple pairs of specific primers for 19 genera in Lachnospiraceae,and their properties were better than those of the specific primers designed with Primer-BLAST tool.This article also developed an algorithm for designing universal primers for specific environments based on k-mer,with the goal of finding conservative k-mer as a universal primer in the 16S rRNA sequences of all bacterial genera in the environment.Similarly,we determine the neighboring point sets of k-mers and the sets of bacterial genera they cover,screen the neighboring point sets that can cover all bacterial genera in the environment,treat the key k-mer in the neighboring point set as candidate primers,and use the Testprimer tool to evaluate their coverage of bacterial genera in the environment to determine the final universal primers.The universal primers in the Silva database have a coverage rate of up to 85%for bacteria in the oral environment,and the above algorithm has designed six pairs of universal primers with a coverage rate of over 90%.The innovation of the above two algorithms lies in:(1)cleverly defining the neighboring points of k-mer,which may be combined with k-mer in other bacterial sequences.(2)Provided a new approach to design primer,and preliminarily determined k-mers that can serve as forward and reverse primers.If the intersection of the bacterial genus set covered by neighboring point sets of these two k-mer only includes the target bacterial genus,then these two k-mers are candidate specific primer pairs.If both sets of bacterial genera contain all the bacterial genera in the environment,then these two k-mers are candidate universal primer pairs.