The reference human nuclear mitochondrial sequences compilation validated and implemented on the UCSC genome browser
© Simone et al; licensee BioMed Central Ltd. 2011
Received: 13 April 2011
Accepted: 20 October 2011
Published: 20 October 2011
Eukaryotic nuclear genomes contain fragments of mitochondrial DNA called NumtS (Nuclear mitochondrial Sequences), whose mode and time of insertion, as well as their functional/structural role within the genome are debated issues. Insertion sites match with chromosomal breaks, revealing that micro-deletions usually occurring at non-homologous end joining loci become reduced in presence of NumtS. Some NumtS are involved in recombination events leading to fragment duplication. Moreover, NumtS are polymorphic, a feature that renders them candidates as population markers. Finally, they are a cause of contamination during human mtDNA sequencing, leading to the generation of false heteroplasmies.
Here we present RHNumtS.2, the most exhaustive human NumtSome catalogue annotating 585 NumtS, 97% of which were here validated in a European individual and in HapMap samples. The NumtS complete dataset and related features have been made available at the UCSC Genome Browser. The produced sequences have been submitted to INSDC databases. The implementation of the RHNumtS.2 tracks within the UCSC Genome Browser has been carried out with the aim to facilitate browsing of the NumtS tracks to be exploited in a wide range of research applications.
We aimed at providing the scientific community with the most exhaustive overview on the human NumtSome, a resource whose aim is to support several research applications, such as studies concerning human structural variation, diversity, and disease, as well as the detection of false heteroplasmic mtDNA variants. Upon implementation of the NumtS tracks, the application of the BLAT program on the UCSC Genome Browser has now become an additional tool to check for heteroplasmic artefacts, supported by data available through the NumtS tracks.
Human mitochondrial DNA (mtDNA) is widely used for phylogenetic, forensic and clinical studies and many features like maternal inheritance, absence of recombination and lack of efficient repair systems are well known and extensively studied. Recent advances in genetics provide researchers with mitochondrial DNA sequences located within the nuclear genome, thus allowing the investigation of intriguing aspects of genome organization. Fragments of mtDNA that give rise to nuclear mitochondrial sequences (NumtS) are found in many eukaryotic nuclear genomes and believed to derive from damaged mitochondria . The discovery of these genomic elements dates back to 1967 through hybridization experiments on mouse liver between mtDNA and nuclear genome . NumtS generation may have started soon after the endosymbiontic event  although the underlying mechanisms are still unclear and time and mode of arrival from mitochondria to nucleus have not been defined. As far as the mode, the most credited hypothesis suggests that in presence of mutagenic agents or under stress conditions, fragments of mtDNA may escape the organelles, reach the nucleus and likely insert into nuclear DNA during double-strand breaks (DSB) repair by the non-homologous end joining (NHEJ) machinery, although other mechanisms have been proposed [4, 5]. It is commonly accepted that most human NumtS have originated before modern man, although evidences of NumtS recent insertions as well as their duplication in human genomes have been reported [6–15]. As a consequence, some NumtS display a highly polymorphic behaviour, as they can occur in homo- or heterozygosis, or be absent in different individuals at specific loci. These features render them candidates as population markers, as already suggested .
The RHNumtS.2 compilation
The RHNumtS.2 compilation was obtained through an in silico hybridization between each human chromosome (build hg18) and the reference human mitochondrial genome rCRS [GenBank:NC_012920]. The process returned 766 High Scoring Pairs (HSPs), i.e. mitochondrial fragments similar to nuclear sequences, hereafter named HSP_NumtS, whose alignment lengths ranged from 31 to 14904 bp. The similarity percentage of each fragment versus the rCRS sequence ranged from 63% to 100%. HSP_NumtS showing evident neighbourhood on both nuclear and mitochondrial genomes were merged in a single NumtS (assembled NumtS) according to the criteria described in the Methods section. NumtS covering the D-loop region were returned by BLASTN as different HSPs, as in the rCRS EMBL/GenBank/DDBJ databank entry the D-loop is split into the end (positions 16024-16569) of the sequence followed by the start (positions 1-576). Therefore, HSP_NumtS close in the nuclear genome and mapping on the D-loop region, were also merged in a single assembled NumtS: this device fitted our joining protocol to the circularity of the mitochondrial genome. Overall, RHNumtS.2 annotates 766 human HSP_NumtS corresponding to 585 assembled Human NumtS inclusive of the 190 annotated in RHNumtS.1 . Covered genome amounts to 627410 bases. The complete RHNumtS.2 compilation is reported here in the additional file 1 RHNumtS.2.xls. A NumtS ID was assigned to each assembled NumtS with a format HSA_NumtS_xxx, where HSA stands for H. sapiens and xxx is a three-digit code.
HSPs statistics concerning the Blast2seq application of the rCRS sequence (J01415)
HSP lengths (% of mt genome)
Distances between concatenated HSPs
NumtS number and percentage for each chromosome
NumtS per Chr
Chr length (bp)
Total NumtS span (bp)
NumtS bp % per Chr
Bench and in silico validation of RHNumtS.2
The in silico hybridization was based on a consensus human genome (build hg18) derived from the DNA sequencing from 6 different samples. Due to the polymorphic nature of human NumtS  and to the technical difficulties posed by repetitive sequences during assembly of the consensus genome, we proceeded to validate RHNumtS.2 on an individual of European origin, through amplification and sequencing (bench validation). HSP_NumtS not present in the European sample (HSA_NumtS_009, HSA_NumtS_426, HSA_NumtS_522) were validated in an Ethiopian sample. Additionally, in silico validation was carried out on genomic annotations from eight HapMap samples.
Validated Assembled NumtS (%)
PCR and HapMap
PCR and Seq
PCR, Seq and HapMap
In silico validation on HapMap FES data
FES with NumtS
The UCSC human NumtS tracks
In order to facilitate browsing of NumtS sequences, we implemented human NumtS tracks using the UCSC Genome Browser tools upon mapping on the hg18 build. Four different NumtS tracks were designed and implemented under the section 'Variation and Repeats'.
The number of NumtS reported in this release, RHNumtS.2, has increased about by three fold over the first one , while the amount of bases covered has increased about by 1.6 fold. More details about the extension of the previous release are provided in the additional file 1 RHNumtS.2.xls (sheet "RHNumtS.1_extension"). RHNumtS.2 includes all the NumtS annotated in RHNumtS.1; 79 of them weren't extended at all, while the median value of the extension ratio is 1.05. For ten NumtS, the extension was quite considerable (extension ratio > 6). Indeed the protocol designed for the production of the first release was aimed to produce a reference compilation, i.e. a collection of sequences located on the reference human genome build and showing strong evidences allowing to define them as "NumtS". The less stringent protocol here applied has allowed us to recognize 585 NumtS, over 95% of which have been validated here for the first time, either by bench approaches (PCR and sequencing) or in silico on eight samples collected within the international HapMap project.
A result that it is worth commenting concerns the coverage of the mitochondrial genome provided by NumtS. mtDNA fragments related to tRNAs and extended portions of the two ribosomal genes as well as of COX1, COX3 and CYTB genes are over-represented, whereas the D-loop region and other genes such as ATP8, ND1, ND4 and ND6 were the least represented within the nuclear genome. These data are in agreement with the conservation degree of the mitochondrial genes in mammals reported in . The diversified observed frequencies of mtDNA fragments could be justified by taking into account that the RHNumtS.2 compilation results from an in silico hybridization between the modern human mtDNA and the human nuclear genome, therefore more evolutionary conserved mitochondrial regions may have higher chances to be detected with such strategy. Further bioinformatics analyses based on different approaches could also contribute to recognize more ancestral events that had led mitochondrial fragments to insert into nuclear genome. Finally, with respect to the apparently unbalanced distribution of the NumtS on the different chromosomes, although preliminary evidence showed a preferential NumtS localization in non-coding regions, a more detailed analysis of each chromosome and NumtS is required to explain the insertion bias towards specific chromosomal regions.
In this paper we present the most exhaustive overview on the human NumtSome implemented in the RHNumtS.2 compilation, a resource whose aim is to support different research applications ranging from studies concerning human structural variation, diversity, and disease, as well as the detection of false heteroplasmic mtDNA variants.
Blasting of the human nuclear genome versus the mitochondrial genome
BLAST2seq implements the BLASTN program (release 2.2.19 of the BLAST suite)  applied to the comparison between two sequences. The run was performed on a local server. Twenty-four runs were launched, one for each human chromosome sequence available through the hg18 build. The human mtDNA reference sequence rCRS [GenBank:NC_012920] was used as query. Scoring parameters were fixed as follows: 2 for match reward, -3 for mismatch penalty; -5 for gap opening, -2 for gap extension. The e-value was fixed to 1e-03. The hg18 chromosome sequences were fetched using the get-genome program of the GMAP package . Each fragment of each chromosome aligned with the J01415.2 mtDNA whose e-value was lower than the fixed threshold produced an HSP (High Scoring Pair).
The assembling of the HSPs was performed with spreadsheet interpolation and manual inspection, strongly supported by graphical display of the HSP_NumtS with a custom annotation tool available on the UCSC Genome Browser. HSP_NumtS located less than 2000 bp apart on a specific chromosome and corresponding to two mtDNA fragments, not more than 2000 bp apart and oriented in the same direction, were merged in a single NumtS and here named as 'assembled NumtS'. The fragment joining protocol was slightly modified for HSPs interposed by long repetitive elements (see HSA_NumtS_014 in Figure 5 for an example).
Primers were designed with primer BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) using as a template each NumtS extended by 1000 nucleotides from the 5' and 3' ends, and specifically locating primers in NumtS flanking regions. The NumtS sequences with their flanking regions were extracted from the UCSC Genome Browser (http://genome.ucsc.edu/) . Primers were designed to ensure amplification, even in case of NumtS absence. For NumtS longer than 1200 bp, external/internal, internal/external, and/or internal/internal primer pairs were designed. To exclude co-amplification of mtDNA, primers were validated through BLAST analysis, by comparison with the mitochondrial sequence of the European individual . Moreover, in order to avoid primers self-hybridization, the Oligo Analysis tool available at Operon web site (http://www.operon.com/technical/toolkit.aspxis), was used. Sequences of primer pairs are provided in additional file 3 NumtS_validation.xls (sheet "Validated_NumtS").
PCR amplification and sequencing
The validation of RHNumtS.2 was carried out on DNA extracted from blood of an individual of European origin belonging to a typical European mitochondrial haplogroup H2b. NumtS were amplified and sequenced as previously described . HSP_NumtS not present in the European sample were validated in an Ethiopian sample whose mtDNA belonged to the L0 haplogroup. NumtS sequences were submitted to the EMBL databank and hence to the three Nucleic Acids databases joint in the international collaboration INSDC. The WebIn tool available on-line was utilized (http://www.ebi.ac.uk/embl/Submission/). Accession numbers are provided in additional file 3 NumtS_validation.xls (sheet "Validated_NumtS").
NumtS sequences were multialigned to the corresponding hg18 sequence inclusive of the NumtS flanking region and to the mtDNA fragments from rCRS and from the validation sample. The multi-alignment has been produced using the ClustalW program (http://www.ebi.ac.uk/Tools/ClustalW/) . The whole data set of the multialigned NumtS is available in the additional file 2 Validated_NumtS_multial.txt. Multi-alignment of the sequences already published in  have been included in the dataset.
Comparison of the RHNumtS.2 sequences versus the FES
The HapMap consortium  has made available 270 samples from Nigeria, China, Japan and North/West Europe. With the aim to study human structural variation, eight HapMap samples (Table 4) were selected and their genomic DNA was cloned using a fosmid subcloning strategy . For each individual library the paired ends were sequenced and the obtained FES (Fosmid End Sequences) data were made publicly available (http://hgsv.washington.edu/).
The in silico validation of NumtS was based on a merging protocol carried out using the Galaxy package available at http://main.g2.bx.psu.edu/. For each sample, only clones with a single best concordant placement according to the FES-pair analysis previously described  were considered.
NumtS tracks implementation
The NumtS tracks and the external links were produced starting from the RHNumtS.2 compilation spreadsheet, with manual manipulation and by using in-house shell and Python scripts. The human mitochondrial reference genome at the UCSC Genome Browser derives from an African individual [GenBank:NC_001807] and shows three insertions with respect to the rCRS. Therefore, the mitochondrial coordinates annotated in the additional file 1 RHNumtS.2.xls were re-mapped to NC_001807. The tracks have been released in bed format, one of the formats allowing the display of the tracks at the UCSC Genome Browser. Templates of the bed file format and the chromosome colour key are available on the UCSC Genome Browser help pages (http://genome.ucsc.edu).
We thank E. Picardi and G. Pesole who have offered access to the PesoleLab server where most analyses have been performed; P. ten Hoopen of the EMBL data library annotators group who has supported us in the sequence submission; A. Zweig and C. Li of the UCSC tracks annotation staff who have allowed us to publish the NumtS tracks at the UCSC Genome Browser; G. Mineccia for contribution to primer design and NumtS sequences multi-alignment.
This work was supported by "Fondo di Ateneo" (University of Bari); by the contribution obtained by prof. Herawati Sudoyo of the Eijkman Institute of Molecular Biology, Jakarta (Indonesia) to partially fund G.G. salary; and partially by the Italian Ministry of University and Research FIRB "Futuro in Ricerca" grant number J31J10000040001 to G.G.
The authors declare no conflict of interest.
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