AgriSeqTM GBS technology to detect multiple species

SHRESTHA S.; CARRASCO C.; SEPÚLVEDA G.; FORLANI L.; POSIK D.M.; BRUNO M.C.; ZAPPA M.E.; CASTILLO N.S.; BARBISAN G; VILLEGAS CASTAGNASSO E E; CRESPI J.A.; PERAL GARCIA P.; GIOVAMBATTISTA G.; SUREN H.

San Diego

Congreso; Plant and Animal Genome Conference (PAG30); 2023

Plant and Animal Genome Conference

ABSTRACTAgriSeqTM GBS technology was used in sequencing specific genes of plant and animals for detection of the species. Species specific unique primers based on genes were designed which were multiplexed together for sequencing. Based on preliminary analysis of positive controls, the reads from mixture of species mapped to their respective genes confirming the presence of the species mixed.INTRODUCTIONAdulteration of food is a matter of concern as it can decrease the quality of the food as well as can have health impacts on humans and economic loss. Hence, the assessment of food is required for detection of adulterants and maintain the quality and safety of foods. There are various methods to detect adulteration in food such as physical, chemical and molecular techniques (1). Here, we utilized next-generation sequencing (NGS) to amplify selected genic regions and detect different constituents of species. Multiplexed markers were applied to identify multiple species within the sample.MATERIALS AND METHODSSamplesA total of 192 samples were studied to identify species within each sample. In order to evaluate the identification using molecular markers, positive controls were developed by mixing DNA of known species. In total, 22 known species with DNA concentration ranging from 0.001 to 10 ng were used in creating mixture which included domestic and wild mammals, birds, sea animals, freshwater fishes, invertebrates and commercial crops. The DNA of 7 or 10 or 11 species were mixed to develop 18 different known mixture. These samples were replicated 4 times with total samples of 72. Commercially available processed foods such as broth, salad dressing, sauce, milk, milk powder, hamburger, canned tuna, etc. were also included in this study. There were 22 different processed product sampled and replicated thrice. In addition, 10 different samples of commercially available pepper were also included with 2-4 replications along with 2 negative controls. AgriSeqTM GBS Panel DesignWe designed 319 multiplexed AgriSeqTM GBS panel representing 177 unique genera. The unique sequence representing the respective species were used in developing markers. For plants, two genes present in the chloroplast were targeted: Maturage K (MATK) and Ribulose-1,5-Biphosphate Carboxylase (RBCL) genes. While for animals two mitochondrial genes were targeted: cytochrome b (CYTB) and cytochrome oxidase subunit 1 (COI) genes. In silico analysis was performed to detect cross amplification from each marker in nt database from NCBI and publicly available complete reference genome from 64 species. There were 135 markers without any cross amplification in both databases, 128 markers with cross amplification in nt database but did not cross amplified within 177 genera of interest, 31 markers had cross amplification in nt database and had cross amplification within 177 genera of interest and 25 markers had cross amplification against RefSeq. All markers categorized into four groups were multiplexed and used for targeted sequencing.