Complete Control Region Sequences Research Articles

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

As a first step towards integrating next generation sequencing (NGS) technology into the FBI Laboratory’s operational casework, the PowerSeq™ CRM Nested System, an NGS-based mitochondrial DNA (mtDNA) control region assay, was developmentally and internally validated. The validation studies were conducted in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for Forensic DNA Analysis Methods, and the FBI’s Quality Assurance Standards (QAS) for Forensic DNA Testing Laboratories. The assay was shown to be highly reproducible, with variant frequencies across intra and inter-run replicates of the same sample differing, on average, by just 0.3% for substitutions and point heteroplasmies and 1.5% for insertions and deletions. The assay was also shown to be extremely sensitive, yielding complete control region sequence data from as few as 2000 copies of mtDNA. This is a more than 20-fold increase in sensitivity when compared to the FBI Laboratory’s current Sanger sequencing-based protocols and, based on mtDNA quantitation values of samples routinely encountered in mtDNA casework, suggests that the percentage of questioned samples from which full control region data can be recovered will increase from our current 20% to approximately 90% success with NGS technology. In addition, the assay requires on average only 30% of the extract volume typically required to develop control region profiles from degraded samples via Sanger sequencing. Overall, these studies establish the reliability of the PowerSeq™ CRM Nested System for accurate mtDNA control region typing and can serve as a model for laboratories seeking to validate NGS protocols for forensic mtDNA analysis.

Variation of the Bovidae (Mammalia: Artiodactyla) mitochondrial DNA control region and their phylogenetic relationship

The control region is the major noncoding segment of animal mitochondrial DNA. The mammal family Bovidae comprises all artiodactyl ungulates. To infer the organization and variation of Bovidae mitochondrial DNA control region, the complete control region sequences of 91 species were analysed. The control region ranged from 677 bp (Saiga tatarica) to 1329 bp (Oryx dammah) in length and can be separated into three domains among these species. The control region has the same flanking gene order from tRNApro to tRNAPhe. Genetic distances between species ranged from 0.44% (between Bos javanicus and Bos taurus) to 24.05% (between Syncerus caffer and Gazella subgutturosa). The average genetic distances among the species within the genera varied from 2.78% (Procapra) to 22.07% (Hemitragus). The average genetic distances showed significantly negative correlation with ts/tv. The maximum-likelihood method was used to construct a phylogenetic tree. Members of Bovinae appear in basal position among the Bovidae lineage. Analysis of control region genes supported the hypothesis of polyphyly for Antilopinae.

Effective Characterisation of the Complete Orang-Utan Mitochondrial DNA Control Region, in the Face of Persistent Focus in Many Taxa on Shorter Hypervariable Regions.

The hypervariable region I (HVRI) is persistently used to discern haplotypes, to distinguish geographic subpopulations, and to infer taxonomy in a range of organisms. Numerous studies have highlighted greater heterogeneity elsewhere in the mitochondrial DNA control region, however–particularly, in some species, in other understudied hypervariable regions. To assess the abundance and utility of such potential variations in orang-utans, we characterised 36 complete control-region haplotypes, of which 13 were of Sumatran and 23 of Bornean maternal ancestry, and compared polymorphisms within these and within shorter HVRI segments predominantly analysed in prior phylogenetic studies of Sumatran (~385 bp) and Bornean (~323 bp) orang-utans. We amplified the complete control region in a single PCR that proved successful even with highly degraded, non-invasive samples. By using species-specific primers to produce a single large amplicon (~1600 bp) comprising flanking coding regions, our method also serves to better avoid amplification of nuclear mitochondrial insertions (numts). We found the number, length and position of hypervariable regions is inconsistent between orang-utan species, and that prior definitions of the HVRI were haphazard. Polymorphisms occurring outside the predominantly analysed segments were phylogeographically informative in isolation, and could be used to assign haplotypes to comparable clades concordant with geographic subpopulations. The predominantly analysed segments could discern only up to 76% of all haplotypes, highlighting the forensic utility of complete control-region sequences. In the face of declining sequencing costs and our proven application to poor-quality DNA extracts, we see no reason to ever amplify only specific ‘hypervariable regions’ in any taxa, particularly as their lengths and positions are inconsistent and cannot be reliably defined–yet this strategy predominates widely. Given their greater utility and consistency, we instead advocate analysis of complete control-region sequences in future studies, where any shorter segment might otherwise have proven the region of choice.

Portuguese mitochondrial DNA genetic diversity—An update and a phylogenetic revision

In recent years a large amount of mitochondrial population data for forensic purposes has been produced. Current efforts are focused at increasing the number of studied populations while generating updated genetic information of forensic quality. However, complete mitochondrial control region sequences are still scarce for most populations and even more so for complete mitochondrial genomes. In the case of Portugal, previous population genetics studies have already revealed the general portrait of HVS-I and HVS-II mitochondrial diversity, becoming now important to update and expand the mitochondrial region analysed. Accordingly, a total of 292 complete control region sequences from continental Portugal were obtained, under a stringent experimental design to ensure the quality of data through double sequencing of each target region. Furthermore, H-specific coding region SNPs were examined to detail haplogroup classification and complete mitogenomes were obtained for all sequences belonging to haplogroups U4 and U5. In general, a typical Western European haplogroup composition was found in mainland Portugal, associated to high level of mitochondrial genetic diversity. Within the country, no signs of substructure were detected. The typing of extra coding region SNPs has provided the refinement or confirmation of the previous classification obtained with EMMA tool in 96% of the cases. Finally, it was also possible to enlarge haplogroup U phylogeny with 28 new U4 and U5 mitogenomes.

The complete mitochondrial genome of the Japanese ornamental koi carp (Cyprinus carpio) and its implication for the history of koi

Complete mitochondrial genome (mitogenome) sequences were determined for two individuals of Japanese ornamental koi carp. Interestingly, the obtained mitogenomes (16,581 bp) were both completely identical to the recently reported mitogenome of Oujiang color carp from China. Control region (CR) sequences in DNA database demonstrated that more than half (65%) of the koi carp individuals so far reported had partial or complete CR sequences identical to those from Oujiang color carp. These results might suggest that the Japanese koi carp has been originated from Chinese Oujiang color carp, contrary to the belief in Japan that the koi carps have been developed directly from carp stocks in Japan. In any case, the present results emphasize the importance of analyzing Oujiang color carp when studying the origin of koi carp.

Genetic Identification of Subspecies and Stocks of Minke Whale Meats Purchased from Korean Markets during 2005-2007

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as the products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appear to be originated from the J stock within this subspecies. Thus, it seems to be urgent to fulfill strict regulation measures to reduce their accidental fisheries bycatch because the J stock has been protected since 1986. Additionally, B. a. scammoni is distinct from B. a. acutorostrata, with average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% distance in the complete cytochrome b gene sequence analysis, and these present results support the present subspecies classification, although further sequencing analyses with nuclear genes are necessary.

Mitochondrial DNA control region variation in an autochthonous Basque population sample from the Basque Country

Mitochondrial control region (16024–576) sequences were generated from 106 samples from autochthonous Basques from the Autonomous Community of the Basque Country. It is especially important to generate mtDNA databases from isolated populations in order to maximize the power of discrimination of this molecular marker. It also represents a useful approach to carry out a more accurate haplogroup classification. This is the first database report of complete control region sequences in an autochthonous Basque population sample. Strict selection criteria of autochthonous individuals, automation of laboratory processing and independent reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data of the autochthonous Basque population.

Variation in mitochondrial DNA and maternal genetic ancestry of Ethiopian cattle populations

This study describes complete control region sequences of mitochondrial DNA (mtDNA) from 117 Ethiopian cattle from 10 representative populations, in conjunction with the available cattle sequences in GenBank. In total, 79 polymorphic sites were detected, and these defined 81 different haplotypes. The haplotype and nucleotide diversity of Ethiopian cattle did not vary among the populations studied. All mtDNA sequences from Ethiopian cattle converged into one main maternal lineage (T1) that corresponds to African Bos taurus cattle. According to the results of this study, no zebu mtDNA haplotypes have been found in Ethiopia, where the most extensive hybridization took place on the African continent.

Structure and evolution of the mitochondrial DNA complete control region in the lizard Lacerta dugesii (Lacertidae, Sauria).

We sequenced the complete control region (CR) and adjacent tRNAs, partial 12S rRNA, and cytochrome b (over 3100 bp) from eight individuals of Madeiran wall lizards, Lacerta dugesii, from four distinct island populations. The tRNAs exhibit a high degree of intraspecific polymorphisms compared to other vertebrates. All CR sequences include a minisatellite that varies in length between populations but is apparently fixed within them. Variation in minisatellite length appears between populations separated by apparently very short evolutionary time spans. Many motifs identified in the CR of other vertebrates are not highly conserved, although conserved blocks are identifiable between the few published reptile CR sequences. Overall there are extensive differences in the internal organization of the reptile CR compared to the more widely studied mammals and birds. Variability in the CR is lower than in cytochrome b, but higher than in 12S rRNA. Phylogenetic analysis of these sequences produces a well-resolved estimate of relationships between populations.