The reference human nuclear mitochondrial sequences compilation validated and implemented on the UCSC genome browser

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 [18]. 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.

Detailed statistics on the NumtS length and similarity distributions are shown in Table 1. Mapping of NumtS (Figure 2) showed that chromosome 2 and chromosome 18 are respectively the most and least densely populated by this class of genomic elements in terms of base pairs overall length (Table 2). A statistical correlation showed that there is a linear relationship between the number of NumtS located on each chromosome and the relative chromosome length (Pearson's r = 0.82). On the other hand, the value obtained by correlating the number of NumtS for each chromosome and the genic density (0.22) suggests that, at chromosome level, there is no evidence of NumtS distribution biased by the presence of genes. NumtS mtDNA coverage (Figure 3) highlights that the fragments add up to several copies of the mitochondrial genome. Over-represented regions encompass many tRNAs, the two ribosomal genes 12S and 16S, large portions of COX1, COX3, CYTB genes and short fragments of ND1, ND5 and ND6 genes. On the other hand, D-loop region, the whole ATP8 gene and most of ND1, ND4 and ND6 genes are under-represented.

	HSP similarities	HSP lengths (% of mt genome)	ΔHSP-span differences	Distances between concatenated HSPs
min	63.52	31 (0.1)	0	-8
1st quartile	73.74	94 (0.5)	0	215.25
median	78.41	215 (1)	1	320
3rd quartile	84.62	619 (3)	5	585.75
max	100	14836 (89)	160	6129

For each HSP, the ΔHSP-span difference is the absolute value of the difference between the HSP length on nuclear genome and the HSP length on the mitochondrial genome. The distance of -8 bp reported indicates overlapping of two concatenated HSPs on nuclear genome.

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 [13] 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.

Design of 355 specific primer pairs was performed, providing amplicons of molecular weight (MW) expected in presence of the NumtS, as confirmed by gel electrophoresis, allowing the validation of 354 HSP_NumtS. Overall 339/354 were successfully sequenced on both strands. Despite yielding an amplicon of expected MW in the case of NumtS presence, the remaining 15 PCR products contained long homopolymeric stretches or heterozygous repetitive satellite-like sequences, which rendered sequencing incomplete. Thus 44% of the total HSP_NumtS (339/766) were sequenced, which is the largest set of NumtS sequenced to date, to the best of our knowledge. Each obtained sequence was multi-aligned to the corresponding mtDNA fragment of the same individual, to the hg18 corresponding nuclear DNA fragment as well as to the rCRS mtDNA fragment. The multi-alignments are available in the additional file 2 Validated_NumtS_multial.txt. HSP_NumtS sequences were submitted to the EMBL databank through the WebIN submission tool (http://www.ebi.ac.uk/webin) available through the INSDC (International Nucleotide Sequence Database Collaboration) organization. To this purpose, a human NumtS EMBL flat-file format template was designed (Figure 4). The entry structure reports in the "Features" table the "misc_feature" data which allow the end-user accessing the INSDC databases to obtain the NumtS location on the nuclear genome and the mtDNA fragments of origin. The accession numbers are reported in additional file 3 NumtS_validation.xls. Overall, 279 assembled NumtS were validated with this approach (Table 3), i.e. those NumtS for which a specific primer pair could be designed.

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 ' NumtS' track shows the mapping of the HSPs returned by BLASTN on the nuclear genome, namely the HSP_NumtS. Items shading reflects the similarity score obtained by BLASTN and the arrows direction is concordant with the strand on which the NumtS is aligned. A link to mtDNA mapping is also provided. The 'NumtS assembled' track shows items obtained by assembling HSPs annotated in the 'NumtS' track fulfilling the conditions described in the Methods section. The 'NumtS on mitochondrion' track shows mapping of the HSP_NumtS on the mitochondrial genome. Items shading reflects the similarity returned by BLASTN, and the direction of the arrows is concordant with the alignment strand. For every item, a link pointing to the nuclear mapping is provided. The 'NumtS on mitochondrion with chromosome placement' track shows the mapping of the HSP_NumtS on the mitochondrial genome, but the items are depicted according to the colours assigned to each human chromosome by the UCSC Genome Browser. For every item, a link pointing to the nuclear mapping is available. The four NumtS tracks annotate the entire RHNumtS.2 compilation. A screenshot example of the browsing of the UCSC NumtS tracks is reported in Figure 5 and 6, while a brief explanation of their surveying is provided in additional file 4 UCSC_NumtS_browsing.pdf.

Blasting of the human nuclear genome versus the mitochondrial genome

BLAST2seq implements the BLASTN program (release 2.2.19 of the BLAST suite) [20] 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 [21]. 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).

NumtS assembling

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).

Primer design

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/) [22]. 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 [18]. 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 [18]. 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").

Multi-alignment

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/) [23]. 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 [18] have been included in the dataset.

Comparison of the RHNumtS.2 sequences versus the FES

The HapMap consortium [24] 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 [25]. 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 [25] 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).