Breed-specific Reference Sequence Optimizes Mapping Accuracy of NGS Analyses for Pigs

Abstract

Background: The reference sequence plays a key role in next-generation sequencing (NGS), which impacts the mapping quality during genome analyses. Especially for mitochondrion which contains plentiful amounts of innate DNA, the optimal reference sequence makes mitochondrial genome (mitogenome) alignment accurate and efficient. In this study, different mapping reference sequences, the commonly used reference sequence (CU-ref), the breed-specific reference sequence (BS-ref) and the sample-specific reference sequence (SS-ref) were compared to test the accuracy of mapping quality in the NGS analyses of pigs.Results: Four pigs from three breeds were high-throughput sequenced and subsequently mapped using three different reference mentioned above, which indicated that the BS-ref produced the largest number of mappable reads and coverages at acceptable run-times. After that, the SNP calling accuracy was evaluated by 18 detection strategies with three tools SAMtools, VarScan and GATK with different parameters under the BS-ref mapping strategy. Results showed that nine detection strategies achieved the same best specificity and sensitivity, which suggested a high accuracy of mitogenome alignment by the BS-ref alignment strategy, with a low requirement for SNP calling tools and parameter choices. Conclusions: Overall, using the breed-specific reference sequences in NGS analyses optimized the mapping quality and the SNP calling accuracy. This study indicates that the different reference sequences which represent different genetic distances between reference sequences and samples in mitogenome alignments influence alignment quality.