New insights into the novel sequences of the chicken pangenome by liquid chip.

Increasing evidence indicates that the missing sequences and genes in the chicken reference genome are involved in many crucial biological pathways, including metabolism and immunity. The low detection rate of novel sequences by resequencing hindered the acquisition of these sequences and the exploration of the relationship between new genes and economic traits. To improve the capture ratio of novel sequences, a 48K liquid chip including 25K from the reference sequence and 23K from the novel sequence was designed. The assay was tested on a panel of 218 animals from 5 chicken breeds. The average capture ratio of the reference sequence was 99.55%, and the average sequencing depth of the target sites was approximately 187 X, indicating a good performance and successful application of liquid chips in farm animals. For the target region in the novel sequence, the average capture ratio was 33.15% and the average sequencing depth of target sites was approximately 60X, both of which were higher than that of resequencing. However, the different capture ratios and capture regions among varieties and individuals proved the difficulty of capturing these regions with complex structures. After genotyping, GWAS showed variations in novel sequences potentially relevant to immune-related traits. For example, a SNP close to the differentiation of lymphocyte-related gene IGHV3-23-like was associated with the H/L ratio. These results suggest that targeted capture sequencing is a preferred method to capture these sequences with complex structures and genes potentially associated with immune-related traits.