Reply to Sangster, G.; Luksenburg, J.A. Contamination of Consensus Sequences in Next-Generation Mitogenomics: The Published Mitochondrial Genome of Haliastur indus Is a Chimera with DNA from Butastur indicus. Comment on “Sonongbua et al. Insights into Mitochondrial Rearrangements and Selection in Accipitrid Mitogenomes, with New Data on Haliastur indus and Accipiter badius poliopsis. Genes 2024, 15, 1439”

We thank Dr. George Sangster and Dr. Jolanda A. Luksenburg for their careful assessment concerning the mitochondrial genome sequence of Haliastur indus (GenBank accession number: OP133375.1) [1], published in our article entitled “Insights into Mitochondrial Rearrangements and Selection in Accipitrid Mitogenomes, with New Data on Haliastur indus and Accipiter badius poliopsis” [2]. We appreciate the opportunity to respond and provide clarification.

After thorough re-examination, we identified an error during the reference-based assembly process. In the original de novo assembly, two scaffolds were obtained, but only one derived from mitochondrial DNA. When these scaffolds were merged, the mitogenome of the grey-faced buzzard (Butastur indicus, GenBank: AB830616.1, identical to the NCBI Reference Sequence NC_032362.1) was used as a reference. Consequently, the reference-based assembly erroneously incorporated a sequence from the B. indicus reference mitogenome, resulting in a chimeric mitogenome. Specifically, 10,078 bp of OP133375.1 originated from H. indus (corresponding to positions 1674–11,751), while the remaining 8977 bp were derived from the B. indicus (positions 1–1673 and 11,752–19,055 of OP133375.1), leading to the erroneous inclusion of mitochondrial genes (e.g., ND5, Cytb, ND6, both rRNAs, and eight tRNAs, including tRNA-Phe, tRNA-Val, tRNA-Ser, tRNA-Leu, tRNA-Thr, tRNA-Pro, and tRNA-Glu) from the latter species.

To correct this issue, we resequenced the same sample using 100 ng/μL DNA on an Illumina NovaSeq™ 6000 platform (2 × 150 bp paired-end, 15× coverage) and combined the new reads with previous data to increase depth. De novo assembly with GetOrganelle (v1.7.4.1) [3] produced a 17,968 bp backbone containing 13 protein-coding genes, rRNAs, tRNAs, and partial control regions. To resolve the repeat-rich control regions, additional PCR amplification and sequencing were performed using Sanger sequencing on an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) and long-read sequencing on a PromethION P2i (Oxford Nanopore Technologies, Oxford, UK). All fragments from Illumina, Sanger, and Nanopore data were merged in Geneious Assembler (v2025.0.3), yielding a 19,986 bp circular mitogenome with no ambiguous bases or structural gaps.

Gene annotation identified the standard avian mitochondrial gene complement, comprising 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and duplicated control regions, as previously reported in Accipitriformes [4]. Read-mapping analyses confirmed uniform coverage across the corrected assembly (mean coverage 96x; median 93x), with reduced depth within the pseudo-control region. This low-coverage segment, caused by tandem repeats that impede short-read mapping, was resolved by incorporating long-read Nanopore data, confirming the completeness and reliability of the final assembly. Alignment of the corrected H. indus mitogenome (OP133375.2) against the previous chimeric version (OP133375.1) and the B. indicus reference (NC_032362.1) confirmed complete removal of the B. indicus-derived fragment (Figure 1). Pairwise identity analysis further supported this correction: OP133375.1 showed 93.9% identity with B. indicus, whereas OP133375.2 exhibited lower identity (80.5%) with B. indicus, consistent with successful removal of the chimeric fragment. Comparative phylogenetic analyses were conducted using the maximum likelihood (ML) method with 1000 ultrafast bootstrap replicates, implemented in the IQ-TREE web server [5], based on 13 protein-coding genes and two rRNAs from 24 Accipitridae species, with Pandion haliaetus (Pandionidae) designated as the outgroup. All genes placed H. indus mitogenome (OP133375.2) in the same clade as Milvus migrans, consistent with the true phylogenetic position of H. indus (Figure 2) [6].

Figure 1.

Sequence alignment showing that the Butastur indicus-derived fragment present in the previous chimeric version (OP133375.1) was removed in the corrected Haliastur indus mitogenome (OP133375.2). Black bars indicate sequence differences, while grey shading denotes positions where the sequences are identical. Colored arrows indicate annotated genomic features: yellow, protein-coding genes; red, rRNAs; pink, tRNAs; and grey arrows indicate control and pseudo-control regions.

Figure 2.

Maximum likelihood phylogenies of mitochondrial genes in Accipitridae, with Pandion haliaetus as outgroup (a) ATP6, ATP8, COI, and COII; (b) COIII, ND1, ND2, and ND3; (c) ND4, ND4L, ND5, and ND6; (d) Cytb, 12S rRNA and 16S rRNA. Green branches indicate the Butastur indicus reference mitogenome (NC_032362.1), red branches indicate the chimeric Haliastur indus mitogenome (OP133375.1), and blue branches indicate the corrected H. indus mitogenome (OP133375.2).

In summary, our re-examination and reassembly confirm that the corrected H. indus mitogenome (OP133375.2) represents the authentic mitochondrial genome of H. indus. The updated sequence has been submitted to GenBank and will be accompanied by a correction publication. We sincerely appreciate the constructive feedback, which has helped improve the accuracy and clarity of our study.

Conflicts of Interest

The authors declare no conflicts of interest.