Amplification and Sequencing of VNTRs

Elements were amplified from genomic DNA using primers in the flanking sequence and Phusion HSII (Thermo Scientific). Orangutan DNA was obtained from the Gene Bank of Primates at the German Primate Center. Primer sequences are provided in Additional file 2: Table S6. To facilitate melting of the VNTR, the denaturation time was extended to 30s and 3% DMSO was added to the reaction mix. Amplicons were subcloned into pJET 1.2 (Thermo Scientific) and sequenced. To obtain complete VNTR sequences, subclones containing the VNTR 5′ and 3′ ends, respectively, were generated using SmaI (H8_43) or MscI (OU3, OU4). 5′ primers localized directly upstream of the CT hexameric repeats and 3′ primers designed to exclude the elements’ polyadenylation signals were used for re-amplification. KpnI and NheI recognition sites, respectively, were introduced into the upstream and downstream re-amplification primers. Amplicons were again subcloned into pJET 1.2, sequenced and transferred into pCEP Neo [12 (link)] and pCEP_mneoM via KpnI/NheI. The human SVA H8_43 displays an 11 bp deletion in the SINE-R region when compared to SVA_E and to the subgroup consensus sequences. To obtain a plasmid with a consensus SVA_E SINE-R for cross-species comparison, the missing 11 bp were introduced by site-directed mutagenesis (NEB Q5 kit).