Evaluating genotype imputation pipeline for ultra-low coverage ancient genomes

Funder: Sapienza Università di RomaAbstract: Although ancient DNA data have become increasingly more important in studies about past populations, it is often not feasible or practical to obtain high coverage genomes from poorly preserved samples. While methods of accurate genotype imputation from > 1 × coverage data have recently become a routine, a large proportion of ancient samples remain unusable for downstream analyses due to their low coverage. Here, we evaluate a two-step pipeline for the imputation of common variants in ancient genomes at 0.05–1 × coverage. We use the genotype likelihood input mode in Beagle and filter for confident genotypes as the input to impute missing genotypes. This procedure, when tested on ancient genomes, outperforms a single-step imputation from genotype likelihoods, suggesting that current genotype callers do not fully account for errors in ancient sequences and additional quality controls can be beneficial. We compared the effect of various genotype likelihood calling methods, post-calling, pre-imputation and post-imputation filters, different reference panels, as well as different imputation tools. In a Neolithic Hungarian genome, we obtain ~ 90% imputation accuracy for heterozygous common variants at coverage 0.05 × and > 97% accuracy at coverage 0.5 ×. We show that imputation can mitigate, though not eliminate reference bias in ultra-low coverage ancient genomes

Patterns of Inter-Chromosomal Gene Conversion on the Male-Specific Region of the Human Y Chromosome

The male-specific region of the human Y chromosome (MSY) is characterized by the lack of meiotic recombination and it has long been considered an evolutionary independent region of the human genome. In recent years, however, the idea that human MSY did not have an independent evolutionary history begun to emerge with the discovery that inter-chromosomal gene conversion (ICGC) can modulate the genetic diversity of some portions of this genomic region. Despite the study of the dynamics of this molecular mechanism in humans is still in its infancy, some peculiar features and consequences of it can be summarized. The main effect of ICGC is to increase the allelic diversity of MSY by generating a significant excess of clustered single nucleotide polymorphisms (SNPs) (defined as groups of two or more SNPs occurring in close proximity and on the same branch of the Y phylogeny). On the human MSY, 13 inter-chromosomal gene conversion hotspots (GCHs) have been identified so far, involving donor sequences mainly from the X-chromosome and, to a lesser extent, from autosomes. Most of the GCHs are evolutionary conserved and overlap with regions involved in aberrant X–Y crossing-over. This review mainly focuses on the dynamics and the current knowledge concerning the recombinational landscape of the human MSY in the form of ICGC, on how this molecular mechanism may influence the evolution of the MSY, and on how it could affect the information enclosed within a genomic region which, until recently, appeared to be an evolutionary independent unit

Evaluating genotype imputation pipeline for ultra-low coverage ancient genomes

Funder: Sapienza Università di RomaAbstract: Although ancient DNA data have become increasingly more important in studies about past populations, it is often not feasible or practical to obtain high coverage genomes from poorly preserved samples. While methods of accurate genotype imputation from > 1 × coverage data have recently become a routine, a large proportion of ancient samples remain unusable for downstream analyses due to their low coverage. Here, we evaluate a two-step pipeline for the imputation of common variants in ancient genomes at 0.05–1 × coverage. We use the genotype likelihood input mode in Beagle and filter for confident genotypes as the input to impute missing genotypes. This procedure, when tested on ancient genomes, outperforms a single-step imputation from genotype likelihoods, suggesting that current genotype callers do not fully account for errors in ancient sequences and additional quality controls can be beneficial. We compared the effect of various genotype likelihood calling methods, post-calling, pre-imputation and post-imputation filters, different reference panels, as well as different imputation tools. In a Neolithic Hungarian genome, we obtain ~ 90% imputation accuracy for heterozygous common variants at coverage 0.05 × and > 97% accuracy at coverage 0.5 ×. We show that imputation can mitigate, though not eliminate reference bias in ultra-low coverage ancient genomes

Identification and molecular characterisation of an AMEL-X null allele due to an Alu insertion

The amelogenin locus is used in most forensic STR multiplex kits as sex-typing marker. In the present work, we performed the molecular characterisation of an AMEL-X null allele, observed in an Eritrean male profile when using either the PowerPlex® Fusion 6C or the AmpFLSTR™ NGM SElect™ multiplexes. After X-specific PCR amplification of the amelogenin locus using an in-house primer pair and Sanger sequencing, we found that the AMEL-X amplification failure was due to an insertion of a 370 bp Alu element. We recognised all the hallmarks of the Alu elements, i.e. the target specific duplications (TSDs) at the boundaries of the insertion, the 3’ A-tail and the LINE1 endonuclease cleavage site, and we used these elements to precisely identify the insertion point of the retro-transposon. We found that the Alu insertion did not disrupt the primer binding sites of the AMEL-X neither in the PowerPlex® Fusion 6C nor in the AmpFLSTR™ NGM SElect™ system. Instead, the amplification failure of the X-specific amelogenin marker could be explained by the competition of the Y-specific amelogenin locus for the same primer pair, which lead to the sub-optimal amplification of the longer AMEL-X. The analysis of 145 additional eastern African males did not reveal other AMEL-X null alleles. To our knowledge, this is the first reported case of an Alu insertion causing an AMEL-X dropout, which is usually due to point mutations at the primer binding sites

LOW DISCRIMINATION POWER OF THE YFILER™ PLUS PCR AMPLIFICATION KIT IN AFRICAN POPULATIONS. DO WE NEED MORE RM Y-STRs?

Recently, thirteen rapidly mutating Y-STRs (RM Y-STRs), characterized by a mutation rate higher than 10−2/STR/generation, have been proven to be extremely useful in distinguishing among close male relatives. Six of these RM-YSTRs have been included in the Yfiler™ Plus PCR Amplification Kit, which shows the highest discriminatory power among Y-STR commercial multiplex currently available. In this study, we used Yfiler Plus to analyze 1437 males from eastern, northern and sub-Saharan Africa (462, 477 and 498 subjects, respectively). 242 out of 1437 subjects were found to share 102 Y-STR haplotypes, resulting in a low discrimination capacity (DC=0.90) as compared to other continental regions. With few exceptions,Y-STR haplotype sharing was limited to subjects coming from the same country and ethnic group, and belonging to the same binary Y-SNP haplogroup, suggesting possible familial relationships along the male lineage. In order to evaluate the presence of hidden familial relationships, all the 242 subjects sharing a Y-STR haplotype were further genotyped for 16 autosomal STRs using the AmpFℓSTR® NGM SElect™ PCR Amplification Kit. Blind search and simulation test analyses for kinship using the Familias software revealed the presence of a low proportion of close relatives (second degree or closer) in our sample set. These findings show that close relatedness explains only a relatively small proportion of the observed Y-STR haplotype sharing, suggesting that a higher number of RM Y-STRs should be included in commercial kits to improve the discrimination power of male-specific markers in regions characterized by high levels of endogamy

New insights into the evolution of human Y chromosome palindromes through mutation and gene conversion

About one-quarter of the euchromatic portion of the male-specific region of the human Y chromosome consists of large duplicated sequences that are organized in eight palindromes (termed P1–P8), which undergo arm-to arm gene conversion, a proposed mechanism for maintaining their sequence integrity. Although the relevance of gene conversion in the evolution of palindromic sequences has been profoundly recognized, the dynamic of this mechanism is still nuanced. To shed light into the evolution of these genomic elements, we performed a high-depth (50×) targeted next-generation sequencing of the palindrome P6 in 157 subjects belonging to the most divergent evolutionary lineages of the Y chromosome.We found 118 new paralogous sequence variants, which were placed into the context of a robust Y chromosome phylogeny based on 7240 SNPs of the X-degenerate region.We mapped along the phylogeny 80 gene conversion events that shaped the diversity of P6 arms during recent human history. In contrast to previous studies, we demonstrated that arm-to-arm gene conversion, which occurs at a rate of 6.01×10 −6 conversions/base/year, is not biased toward the retention of the ancestral state of sequences.We also found a significantly lower mutation rate of the arms (6.18×10−10 mutations/base/year) compared with the spacer (9.16×10−10 mutations/base/year), a finding that may explain the observed higher inter-species conservation of arms, without invoking any bias of conversion. Finally, by formally testing the mutation/conversion balance in P6, we found that the arms of this palindrome reached a steady-state equilibrium between mutation and gene conversion

Information on the mutations here reported and analyzed for the first time.

a<p>Position according to the February 2009 human Y-chromosome reference sequence (GRCh37).</p>b<p>PCR primers amplify three paralogous MSY regions.</p

The peopling of the last Green Sahara revealed by high-coverage resequencing of trans-Saharan patrilineages

Abstract Background Little is known about the peopling of the Sahara during the Holocene climatic optimum, when the desert was replaced by a fertile environment. Results In order to investigate the role of the last Green Sahara in the peopling of Africa, we deep-sequence the whole non-repetitive portion of the Y chromosome in 104 males selected as representative of haplogroups which are currently found to the north and to the south of the Sahara. We identify 5,966 mutations, from which we extract 142 informative markers then genotyped in about 8,000 subjects from 145 African, Eurasian and African American populations. We find that the coalescence age of the trans-Saharan haplogroups dates back to the last Green Sahara, while most northern African or sub-Saharan clades expanded locally in the subsequent arid phase. Conclusions Our findings suggest that the Green Sahara promoted human movements and demographic expansions, possibly linked to the adoption of pastoralism. Comparing our results with previously reported genome-wide data, we also find evidence for a sex-biased sub-Saharan contribution to northern Africans, suggesting that historical events such as the trans-Saharan slave trade mainly contributed to the mtDNA and autosomal gene pool, whereas the northern African paternal gene pool was mainly shaped by more ancient events

Phylogeographic Refinement and Large Scale Genotyping of Human Y Chromosome Haplogroup E Provide New Insights into the Dispersal of Early Pastoralists in the African Continent

Haplogroup E, defined by mutation M40, is the most common human Y chromosome clade within Africa. To increase the level of resolution of haplogroup E, we disclosed the phylogenetic relationships among 729 mutations found in 33 haplogroup DE Y-chromosomes sequenced at high coverage in previous studies. Additionally, we dissected the E-M35 subclade by genotyping 62 informative markers in 5,222 samples from 118 worldwide populations. The phylogeny of haplogroup E showed novel features compared with the previous topology, including a new basal dichotomy. Within haplogroup E-M35, we resolved all the previously known polytomies and assigned all the E-M35* chromosomes to five new different clades, all belonging to a newly identified subhaplogroup (E-V1515), which accounts for almost half of the E-M35 chromosomes from the Horn of Africa. Moreover, using a Bayesian phylogeographic analysis and a single nucleotide polymorphism-based approach we localized and dated the origin of this new lineage in the northern part of the Horn, about 12ka. Time frames, phylogenetic structuring, and sociogeographic distribution of E-V1515 and its subclades are consistent with a multistep demic spread of pastoralism within north-eastern Africa and its subsequent diffusion to subequatorial areas. In addition, our results increase the discriminative power of the E-M35 haplogroup for use in forensic genetics through the identification of new ancestry-informative markers.The authors are grateful to all the donors for providing DNA samples and to the people that contributed to the sample collection. In particular, they thank João Lavinha (for the Portuguese samples); Farha El Chennawi, Anne Cambon-Thomsen, M.S. Issad, Eric Crubézy, Abdellatif Baali, Mohammed Cherkaoui, and Mohammed Melhaoui for their help in the collection of the Moroccan, Algerian, and Egyptian Berbers samples; and the National Laboratory for the Genetics of Israeli Populations. This work was supported by the Italian Ministry of Education, Progetti PRIN (grant numbers 2012JA4BTY_004 to F.C. and 2012JA4BTY_003 to A.N.) and by Sapienza University of Rome (grant number C26A13S9AR to F.C.). The sampling of the Berbers was made within the framework of the Inserm "Réseau Nord/Sud" No. 490NS1 (Mozabite Berbers), "The Origin of Man, Language and Languages," EUROCORES Programme and benefited from funding by the Région Midi-Pyrénées (Toulouse, France), the CNRS, and the E.C. Sixth Framework Programme under Contract ERASCT-2003-980409