A computational analysis was performed to identify full-length lineage specific AluY sequences in the olive baboon (Panu_3.0), rhesus macaque (Mmul_8.0.1), African green monkey (chlSab2), orangutan (P_pygmaeus 2.0.2), chimpanzee (Pan_tro3), and human (GRCH38/hg38) reference genomes, as previously described by Steely et al. (53).

Initial screen for polymorphic Alu insertions. Two methods were used to identify 494 informative Alu insertions, a subset of young elements currently polymorphic within the genus Papio. The first method used BLAT to align AluY sequences obtained from the P. anubis assembly (Panu_2.0) against M. mulatta (RheMac2) and H. sapiens (GRCh38/hg38). Insertions present in P. anubis yet absent from the other two assemblies were subjected to polymerase chain reaction (PCR) analysis to determine whether they were polymorphic across baboon species. A total of 187 loci were retained after this analysis.

Computational analysis of diversity samples. In our second method, whole-genome sequence data generated from six of the diversity panel baboons (16098, 28547, 28755, 34472, 34474, and 97124) were aligned to P. anubis genome (Panu_2.0), as previously described by Jordan et al. (54).

Primer design. Oligonucleotide primers for locus-specific PCR were designed as reported in (53).

Sanger sequencing. Following PCR analysis of candidate Alu insertion polymorphisms on the DNA panel of baboons, a small number of loci required Sanger sequencing for clarification. The first category included eight loci ascertained from either a yellow or kinda baboon genome, computationally absent from the reference genome Panu_2.0, but for which PCR results indicated a filled site, or Alu present amplicon size, for reference DNA sample 27861. These were classified as possible “false-negative” events (supposed to be absent but were not), and PCR fragments were sequenced from 27861 and the ascertained individual to confirm the existence of a shared insertion event. The second category included nine loci in which the Alu present PCR product was either larger or smaller than the predicted filled size amplicon, or displayed both size bands, in one or more Papio species, but not all. All applicable PCR fragments were sequenced to confirm that the ascertained Alu element of interest was present and to determine what the extra sequence contained. Four PCR fragments per locus were gel-purified using a Wizard SV gel purification kit (Promega Corporation, Madison, WI, USA, catalog A9282) according to the manufacturer’s instruction. Cycle sequencing was performed, and resulting products were cleaned by standard ethanol precipitation. Capillary electrophoresis was performed on an ABI 3130xl Genetic Analyzer (Applied Biosystems Inc., Foster City, CA) and evaluated using ABI software Sequence Scanner v1.0. Sequence alignment figures were constructed in BioEdit, and a consensus sequence for each locus was determined from the multiple forward and reverse Sanger sequences obtained for each locus (53).

Phylogenetic analysis. A phylogenetic tree was created using the larger dataset of polymorphic Alu elements as characters. If an Alu insertion was fixed present (homozygous) at a particular locus, it was coded as “1”. If the insertion was fixed absent at a particular locus, it was coded as “0”. Insertions that were found to be heterozygous in an individual were coded as “1,0”. If a locus could not be resolved through PCR, then it was coded as a “?”. Mesquite 3.04 (53, 54) was used to create a Dollo parsimony matrix with all characters set to the character type Dollo.up. A heuristic search was completed using PAUP* 4.0a147 (54) with a total of 10,000 bootstrap replicates. The majority-rule tree does not include bootstrap values below 50% for any branches, but does include values for consistency index, retention index, and homoplasy index. A neighbor-joining phylogenetic tree was also created using the same dataset to illustrate the overall topology. The trees were produced in PAUP* and visualized using FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

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