Use of targeted next generation re-sequencing in the identification of polymorphisms in the bovine collagenous lectin gene family

Collagenous lectins bind carbohydrate motifs on pathogens, leading either to the activation of the lectin complement pathway or to the opsonization or agglutination of pathogens. They play an important role in innate immunity against a variety of bacteria, viruses, and fungi. Functionally altered proteins resulting from genetic mutations in collagenous lectins have been shown in other species to predispose animals to infectious disease. This study aimed to 1) identify genetic variation in the bovine collagenous lectin genes; and 2) determine whether polymorphisms were associated with an increased susceptibility to infectious disease. We used pooled, targeted next generation re-sequencing to identify variants in the bovine collagenous lectin genes. Cattle submitted for post-mortem examination at the University of Guelph were classified as normal (n = 40) or diseased (n = 80) based on the presence or absence of infectious disease. Cattle were placed into groups of 5 based on the similarity of diagnosis, and an equal amount of DNA from each animal was pooled. The collagenous lectin genes (including three unique to bovids) along with 3 kb of downstream DNA and up to 50 kb of upstream DNA were targeted for re-sequencing. The sequencing library was prepared with a Roche Nimblegen EZ Developer kit and sequenced on an Illumina MiSeq. In total, 4.6 Gb of usable sequence data was obtained with an average read depth of 42x/cow over the target region. Following application of quality control filters, 6525 single nucleotide variants (SNVs) were found, including 510 not reported in dbSNP. This included 3948 upstream region SNVs, 2672 downstream region SNVs, and 411 intronic SNVs. Within exons, 107 SNVs, including 54 missense mutations, were identified. In silico analysis of the missense mutations identified 16 SNVs with significant potentially disruptive effects on protein structure. A mutation in MBL2, resulting in a P42Q change in the collagen-like domain, holds particular interest, as similar mutations in orthologous genes have been shown to have impact on susceptibility to disease. Allele frequencies between the normal and diseased populations were compared and the potential impact of promoter SNVs investigated. This study demonstrates that pooled, targeted re-sequencing is a cost effective method of polymorphism identification and discovery in cattle. We identified 510 previously unreported SNVs, as well as 16 mutations potentially affecting collagenous lectin stru