Abstract

BackgroundReference sequences play a vital role in next-generation sequencing (NGS), impacting mapping quality during genome analyses. However, reference genomes usually do not represent the full range of genetic diversity of a species as a result of geographical divergence and independent demographic events of different populations. For the mitochondrial genome (mitogenome), which occurs in high copy numbers in cells and is strictly maternally inherited, an optimal reference sequence has the potential to make mitogenome alignment both more accurate and more efficient. In this study, we used three different types of reference sequences for mitogenome mapping, i.e., the commonly used reference sequence (CU-ref), the breed-specific reference sequence (BS-ref) and the sample-specific reference sequence (SS-ref), respectively, and compared the accuracy of mitogenome alignment and SNP calling among them, for the purpose of proposing the optimal reference sequence for mitochondrial DNA (mtDNA) analyses of specific populationsResultsFour pigs, representing three different breeds, were high-throughput sequenced, subsequently mapping reads to the reference sequences mentioned above, resulting in a largest mapping ratio and a deepest coverage without increased running time when aligning reads to a BS-ref. Next, single nucleotide polymorphism (SNP) calling was carried out by 18 detection strategies with the three tools SAMtools, VarScan and GATK with different parameters, using the bam results mapping to BS-ref. The results showed that all eighteen strategies achieved the same high specificity and sensitivity, which suggested a high accuracy of mitogenome alignment by the BS-ref because of a low requirement for SNP calling tools and parameter choices.ConclusionsThis study showed that different reference sequences representing different genetic relationships to sample reads influenced mitogenome alignment, with the breed-specific reference sequences being optimal for mitogenome analyses, which provides a refined processing perspective for NGS data.

Highlights

  • Reference sequences play a vital role in next-generation sequencing (NGS), impacting mapping quality during genome analyses

  • We explored whether the genetic relationships between reference and sample sequence would impact mapping accuracy of pig mitogenomes

  • The former strategy yielded no polymorphic site, while the latter created a large number of polymorphisms (193 sites), potentially caused by nuclear mitochondrial sequences (NUMTs)

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Summary

Introduction

Reference sequences play a vital role in next-generation sequencing (NGS), impacting mapping quality during genome analyses. Reference genomes usually do not represent the full range of genetic diversity of a species as a result of geographical divergence and independent demographic events of different populations. Reference genomes often cannot represent the full range of genetic diversity of a species as a result of geographical divergence and independent demographic events in different populations. To comprehensively characterize genetic variation, different references for different populations may be necessary This perspective has fueled the research on pan-genomes, currently focusing on nuclear genomes, and not involving mitogenomes [16, 17]. This lack of studies on the influence of reference sequences on mitogenome alignment requires addressing

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