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However, as for the current ancient samples, we must also admit some technical limits of haplogroup determination based on coding region SNP analysis.
TMRCA estimates were calculated using the rho statistic and its standard deviation (SD) [ 55] with three previously described mutation rates based on coding region mutations.
Low diversity in haplogroup L1b based on HVS1 and 2 sequences was also observed by [37] as six out of nine known L1b subclades can only be characterized based on coding region mutations.
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Tree topologies based on genomic sequences were identical to those based on coding regions only, but posterior probabilities were higher using genomic sequences (data not shown).
This confirms previous findings that markers based on cDNA sequences are more transferable among species than random markers, such as genomic SSRs, since they are based on coding regions, which are generally more conserved that non coding regions [ 49- 54].
The age for L3f3 clade, based on mtDNA coding region diversity (8.0 ± 2.5 kya), is remarkably concordant with archaeological findings for the proto-Chadic migration.
Out of the total 2,783 samples, M haplogroup samples (1751) were differentiated based on their specific coding region mutations.
Hg assignment was then confirmed, based on control and coding region Hg defining polymorphisms (Table S2) determined by means of restriction fragment length polymorphisms or direct sequencing.
By virtue of the updated phylogeny of haplogroup M9a'b, we further classified the remaining M9a'b candidates based on the specific coding region motifs (for our own samples; see Additional file 1) and/or by matching and near-matching [ 32, 47] with the well-defined M9a'b lineages (for the reported mtDNAs from the literature).
Based on these results, one coding region (rnl) and two intergenic regions (nad1-nad4 Rps3- putative N-acetyl-transferase) were selected.
Based on the coding region analysis, we extended the initial number of 49 putative lineages (Table S4) to 53 potentially founding lineages, because the coding region analysis suggested that four putative founding lineages (J1b'e/J1e, H4a1a, H30 and R0a1c) were each comprised of 2 daughter sub-lineages.
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