Description and display conventions

NumtS (Nuclear mitochondrial sequences) are mitochondrial fragments inserted in nuclear genomic sequences. The most credited hypothesis concerning their generation suggests that in presence of mutagenic agents, or under stress conditions, fragments of mtDNA escape from mitochondria, reach the nucleus and insert into chromosomes during break repair; although NumtS can also derive from duplication of genomic fragments. NumtS may be a cause of contamination during human mtDNA sequencing and hence frequent false low heteroplasmic evidences have been reported.The Bioinformatics group chaired by M.Attimonelli (University of Bari, Italy) has produced the RHNumtS (Reference Human NumtS) compilation annotating more than 500 Human NumtS. To allow the scientific community to access the compilation and to perform genomics comparative analyses inclusive of the NumtS data, the group has designed the Human NumtS tracks described below.

The NumtS tracks show nuclear and mitochondrial regions, the former derived from the application of the Lift-Over (Galaxy) tool on the High Score Pairs (HSPs) obtained by aligning the mitochondrial reference genome (NC_012920) with the hg18 release of the human genome then converted to hg19.

1. "NumtS (Nuclear mitochondrial Sequences)" Track

   The "NumtS mitochondrial sequences" track shows the mapping of the HSPs returned by BlastN on the nuclear genome. The shading of the items reflect the similarity returned by BlastN, and the direction of the arrows is concordant with the strand of the alignment. For every item, a link pointing to the mitochondrial mapping is provided, thus allowing a fast cross among the NumtS genomic contexts.

2. "NumtS assembled" Track

   The "NumtS assembled" track shows items obtained by assembling HSPs annotated in the "NumtS" track fulfilling the following conditions:

   • The orientation of their alignments must be concordant.
   • The distance between them must be less than 2 kb, on the mitochondrial genome as well as on the nuclear genome.

   Exceptions for the second condition arise when a long repetitive element is present between two HSPs.

3. "NumtS on mitochondrion" Track

   The "NumtS on mitochondrion" track shows the mapping of the HSPs on the mitochondrial genome. The shading of the items reflects the similarity returned by BlastN, and the direction of the arrows is concordant with the strand of the alignment. For every item, a link pointing to the nuclear mapping is provided.

4. "Human NumtS on mitochondrion SNP" Track

   The "Human NumtS SNP" shows the mapping of the HSPs on the mitochondrial genome, with the SNPs which fall within, derived from the comparison with the hg19 build. No shading is provided here. For every item, a link pointing to the nuclear mapping is provided.

Methods

NumtS mappings were obtained by running Blast2seq (program: BlastN) between each chromosome of the Human Genome (hg18 build) and the human mitochondrial reference sequence (rCRS, AC: NC_012920), fixing the e-value threshold to 1e-03. Mapping coordinates were converted from hg18 to the hg19 assemby by using the Lift-Over software part of Galaxy suite. The assembling of the HSPs was performed with spreadsheet interpolation and manual inspection. BED format is used for the first three annotation tracks, while for the last one the BAM format is implemented.

Verification

NumtS predicted in silico were validated by carrying out PCR amplification and sequencing on blood-extracted DNA of a healthy individual of European origin. PCR amplification was successful for 275 NumtS and provided amplicons of the expected length. All PCR fragments were sequenced on both strands, and submitted to the EMBL databank.

Furthermore, 541 NumtS were validated by merging NumtS nuclear coordinates with HapMap annotations. The analysis was carried on eight HapMap individuals (NA18517, NA18507, NA18956, NA19240, NA18555, NA12878, NA19129, NA12156). For each sample, clones with a single best concordant placement (according to the fosmid end-sequence-pair analysis described in Kidd et al., 2008), have been considered. The analysis showed that 541 NumtS (at least 30bp for each one) had been sequenced in such samples.

Credits

These data were provided by Domenico Simone, Francesco Maria Calabrese and Marcella Attimonelli at the Department of Biochemistry and Molecular Biology "Ernesto Quagliariello" (University of Bari, Italy). Primer design was carried out by Francesco Calabrese and Giuseppe Mineccia. PCR validation was carried out by Martin Lang, Domenico Simone and Giuseppe Gasparre. Merging with HapMap annotations was performed by Domenico Simone.

References

Kidd JM, Cooper GM, Donahue WF, Hayden HS, Sampas N, Graves T, Hansen N, Teague B, Alkan C, Antonacci F et al. Mapping and sequencing of structural variation from eight human genomes. Nature. 2008 May 1;453(7191):56-64. PMID: 18451855; PMC: PMC2424287

Lascaro D, Castellana S, Gasparre G, Romeo G, Saccone C, Attimonelli M. The RHNumtS compilation: features and bioinformatics approaches to locate and quantify Human NumtS. BMC Genomics. 2008 Jun 3;9:267. PMID: 18522722; PMC: PMC2447851

Simone D, Calabrese FM, Lang M, Gasparre G, Attimonelli M. The reference human nuclear mitochondrial sequences compilation validated and implemented on the UCSC genome browser. BMC Genomics. 2011 Oct 20;12:517. PMID: 22013967; PMC: PMC3228558