The ancient genomes of Chichén Itz show insights into ritual life
The Chichén Itz Chultun collection. The present-day DNA extracted from 68 unrelated adult individuals from Tixcacaltuyub, Yucatán, Mexico
The Council of Archaeology, National Institute of Anthropology and History in Mexico granted a permit to analyse the individuals from the Chichén Itz Chultun collection. 401.1 S.3-2017/482; 10.03.2017). We looked at the material that was recovered from Chichén Itz. The bone samples were collected in a sanitary manner in the osteology lab of the INAH Yucatn. Photographic records of the samples were maintained throughout the whole procedure. The present-day DNA was extracted from blood samples obtained from 68 unrelated adult individuals (TIX) from Tixcacaltuyub, Yucatán, Mexico under a protocol approved by the Committee of Ethics and Research, UADY, granting permit to collect blood samples and carry out analyses on the genetic material obtained from such samples (project: Bienestar Comunitario: Proyecto de capacitación para la autogestión de la salud de personas con DT2 y sus familias, en la comunidad de Tixcacaltuyub y Yaxcabá; official notice no. F-FENC-SAC-14/REV: 04; registry no. 09/17) and performed according to the requisites of the Helsinki Declaration (2008) and the General Health Law of Mexico. In order for YCH to obtain bone powder it was needed from the densest part of each petrous bone in facilities devoted to ancient DNA protocols and a bleach/rinse and ultraviolet decontamination protocol. For TIX, 5 ml of peripheral blood were obtained by venepuncture after each participant was informed about the procedure, the aim of the protocol and the potential risks, and after signing a consent letter. For TIX, the DNA was extracted in a dedicated laboratory at UADY. Their samples were anonymized from this point onwards.
The children Del Castillo-Chávez and her colleagues analysed were found in the 1960s in an underground chamber called a chultún and an adjacent cave, near the Sacred Cenote. The remains showed no signs of violence, but they were found as part of a shrine, now destroyed by construction work.
In the hope of identifying the sex of the remains and to glean other genetic insights, Del Castillo-Chávez teamed up with immunogeneticist Rodrigo Barquera and palaeogeneticist Johannes Krause at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and their colleagues. The team obtained the ancient genome data from 64 skulls of people who were killed in the hultn.
It’s not fully clear why these children were selected for sacrifice. The analysis of their bones suggested that they were typical of ancient Maya. Related individuals tended to have similar isotopic profiles, suggesting that they were raised in a similar way.
It’s likely that it was part of training them for this sacrifice, according to Barquera. Death and sacrifice for them means very different things to us. It was a big honor to be part of this.
Mapping immune-captured sequence data to the human genome assembly GRCh37 (hg19) from the Genome Reference Consortium 142
An HLA allele that has become more than twice as common has been linked to protection against severe Salmonella infections. The previous study by the team had a link to the Salmonella enterica sp. In Mexico and elsewhere, millions of people were killed in a disease outbreak that was brought on in the 16th century.
For both YCH and TIX libraries, we performed analyses of the captured sequence data using nf-core/eager v.2.3.4 (ref. 31). AdapterRemoval v.2 (ref. 141) was used to trim adaptor sequences and to remove adaptor dimers and low-quality sequence reads (minimum length, 30; minimum base quality, 20). Preprocessed sequences were mapped to the human genome assembly GRCh37 (hg19) from the Genome Reference Consortium142 using BWA v.0.7.12 (ref. The seed length was 32. The C to T misincorporation frequencies were obtained using mapDamage 2.0 in the case of ancient samples. To assess the authenticity of the fragments from the libraries. Genetic sex of the analysed individuals was assigned using SNP capture data by calculating the ratio of average X chromosomal and Y chromosomal coverage to the average autosomal coverage normalized by the chromosome length at the targeted SNPs35. Samples with an X rate between 0.35 and 0.55 and a Y rate between 0.4 and 0.7 were confirmed male. Analysis of next generation sequencing data(ANGSD) was used to estimate nuclear contamination, as males are expected to be homozygous at each X chromosome position145. We used a custom script and mpileup to generate a pileup from the merged data of each individual. For each position on our capture panel, a random read was drawn for each individual and the allele of that read was assumed to be the homozygous genotype of the individual at that position. The data from the Central American population was merged to make a comparison with other data from the region. Only people with more than 20,000 SNPs on the assays were kept for downstream analyses.
We mapped sequence data from the immune-captured libraries against a custom panel containing over 1,250 alleles in CIWD 3.0, in order to create a development version of OptiType151. There was a set number of possible best matches. There are two classes of alleles for the genes: class I and class II. Of the 882 raw allele calls, 72 (8.1%) with anomalous coverage patterns induced by reads cross-mapping to several loci were overruled in favour of secondary allele predictions. The frequencies and properties of linkage were used to assign haplotypes. kinship when possible To compare the HLA frequencies we performed Fisher tests and adjusted the P values according to the Benjamini–Hochberg correction. The FDR adjustment controls for a low number of false positives compared to the Bonferroni correction, which ensures the existence of absolutely no false positives, resulting in increased statistical power. It has been argued74 that certain types of non-overlapping association between HLA loci may be a signature of pathogen selection. We used Y CH and TIX haplotypes to calculate the (f_rmadj ) metric, which was used to estimate the number of HLA genes in the dataset. To provide a point of comparison for the ({f}_{{\rm{adj}}}^{ }) score of each locus pair for both YCH and TIX, we generated 5,000 random permutations of the order of the total alleles at one of the loci in each pair and recalculated ({f}_{{\rm{adj}}}^{ }) for each set of randomized data. We generated distributions of possible ({f}_{{\rm{adj}}}^{ }) scores for each pair of loci in the dataset that would be obtained if the alleles at those loci were associated entirely at random. The difference between the f_rmadj and the mean of the (f_rmadj ) scores was calculated. The scores gave us the ability to rank the pairs according to how much non-overlap they showed relative to random associations between the different alleles. To further assess the role of HLA class II alleles in resistance to S. enterica infection, we ran in silico binding predictions for the HLA-DRB1/3/4/5 alleles and -DQA1/DQB1 allele pairs found in YCH individuals and peptides derived from 18 Salmonella spp. proteins that have been previously reported to be highly immunogenic in humans (Supplementary Methods: ‘In silico binding prediction assays’) using NetMHCIIpan-4.0 (ref. 82) as implemented in the IEDB Analysis Resource virtual machine image157,158. The adjusted rank values are what the authors recommended in order for binders to be classified as strong or weak.
The mtDNA haplogroups were determined mapping reads to the revised Cambridge reference sequence147. HaploGrep2 and HAPLOFIND149 were used to confirm the corresponding haplogroups. For the Y-haplogroup assignments, we created pileups of reads for each individual which mapped to Y chromosome (Y-Chr) SNPs as listed on the ISOGG Y-DNA Haplogroup Tree (v.15.73; https://isogg.org/tree/). We then manually assigned Y-Chr haplogroups for each individual based on the most downstream SNP retrieved after evaluating the presence of upstream mutations along the Y-Chr haplogroup phylogeny (Supplementary Fig. 6). Full mtDNA haplogroups and Y-Chr genotypes can be found in Supplementary Tables 9 (YCH) and 10 (TIX).
Using an in-solution capture approach based on modified immortalized probe sequences136, target immunity genes sequences, mtDNA, Y chromosome or a panel of 1,237,207 SNPs were enriched from the total DNA in the sequencing libraries32,33,137,138,139,140. The library pools were matched end to end on the Illumina platform with 75 cycles for about 20 million reads.
We used the characteristic damage plots associated with aDNA129 to build libraries using 15 L of each YCH extract. We used 20 l of YCH extracts to build UDG half libraries using illuminn-specific adaptors with a modified double-stranded library preparation protocol. We built libraries that were non-UDG-treated. Both YCH and TIX libraries were quantified using quantitative PCR (qPCR) with the IS7 and IS8 primers in a quantification assay using a DyNAmo SYBR Green qPCR Kit (Thermo Fisher Scientific) on the LightCycler 96 platform (Roche Diagnostics).
For YCH, the bone powder was decalcified and proteins were digested by an overnight incubation (more than 16 h) at 37 °C in a buffer containing EDTA and Proteinase K30. The DNA was purified from the supernatant by a silica column-based method using a silica column for high volumes assay (High Pure Viral Nucleic Acid Large Volume Kit, Roche Molecular Systems). DNA was eluted in 100 µl of TET (10 mM Tris, 1 mM EDTA and 0.05% Tween) and frozen at −20 °C until library preparation128. 5 liters of peripheral blood was collected in the blood collection tubes, with K2edTA and Quick- DNA Miniprep Plus kits used, following the instructions from the developers. Because our protocols are optimized for short-length aDNA and to avoid potential bias through laboratory methods, we sheared the DNA extracted from modern individuals using ultrasonic DNA shearing to an average length comparable to that of aDNA. The average fragment length of 150 base pairs was sheared in a Covaris M 220 Focused Ultrasonicator, which was used to dilution the modern DNA samples.
Direct insight into dietary trends among past populations enables the investigation of connections between diet and social status, cultural customs linked to food, environmental impacts on subsistence and perhaps even individual mobility122,123. The bone collagen 13C and 15N reflect the different parts of the diet of an individual during the time of tissue formation and the various types of fruits and vegetables. To reconstruct the diet of the subadult individuals from Chichén Itzá studied, we analysed bone collagen from temporal and petrous bones (Supplementary Fig. 4). The procedure used to extract the Collagen was 125 (Supplementary Methods). The C:N ratio is used to determine the quality of collagen preservation. If the Collagen yield is more than 1% in weight, it should be acceptable for the values of carbon and nitrogen.
The petrous bone of 26 YCH samples were pretreated and analysed using a standardized procedure. Collagen was extracted from the bone samples (approximately 1 g, using a modified version of the Longin method118), purified by ultrafiltration (fraction greater than 30 kD) and freeze-dried. The collagen was then combusted to CO2 in an elemental analyser. The CO2 was then converted catalytically to graphite and analysed using a MICADAS-type AMS system (Ionplus AG). The blanks and control standards are measured simultaneously in the AMS system. 14C-ages are normalized to δ13C = −25‰ (ref. 119) with a typical uncertainty of 2‰ and calibrated using the dataset IntCal20 and the software Oxcal (v.4.3.2)120,121.
Source: Ancient genomes reveal insights into ritual life at Chichén Itzá
Communities engagement activities in Tixcacaltuyub: A collaborative approach from the community to community evaluation of genetic research results and interventions
The community of Tixcacaltuyub self-identifies as a Mayan community and has been in a years-long cooperative relationship with the Chemistry and Nursing Faculties of UADY, Mérida, Yucatán, following projects investigating the relationship between health and lifestyle in the community. J.C.L.R., M.E.M.M. and J.C.T.R., together with C. Tzec-Puch, have been working in collaboration with the community to communicate results of clinical tests and to develop health interventions in Tixcacaltuyub. As a result of these interventions, healthcare barriers and opportunities have been identified and work is now underway with the active participation of the community to implement a co-responsible model of healthcare. In April 1993, J.C.L.R and R.B. traveled to Tixcacaltuyub to collect community feedback after their genetic research and to return the results to participants and students. Feedback from these engagements has been incorporated into the final manuscript (Supplementary Methods: ‘Community engagement activities’). As part of our strategies for outreach and making our results available to a broader audience, we included a non-peer reviewed, Spanish translation of the main manuscript (Supplementary Note 1).
The present-day DNA of 68 unrelated individuals was extracted from blood samples obtained from Tixcacaltuyub, Yucatn, Mexico under a protocol approved by the Committee of Ethics and Research, UADY, granting permit to collect blood samples and carry out analyses on the genetic material obtained from such samples. The present-day DNA was extracted from 68 unrelated adult individuals from Tixcacaltuyub.