Human-specific tandem repeat expansion and differential gene expression during primate evolution Arvis Sulovaria, Ruiyang Lia, Peter A. Audanoa, David Porubskya, Mitchell R. Vollgera, Glennis A. Logsdona, Human Genome Structural Variation Consortium1, Wesley C. Warrenb, Alex A. Pollenc, Mark J. P. Chaissona,d, and Evan E. Eichlera,e,2 aDepartment of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195; bBond Life Sciences Center, University of Missouri, Columbia, MO 65201; cDepartment of Neurology, University of California, San Francisco, CA 94143; dQuantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089; and eHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195 Edited by Stephen T. Warren, Emory University School of Medicine, Atlanta, GA, and approved October 1, 2019 (received for review July 17, 2019) Short tandem repeats (STRs) and variable number tandem repeats Despite their established importance in population genetics (VNTRs) are important sources of natural and disease-causing and disease association, tandem repeats, particularly VNTRs, variation, yet they have been problematic to resolve in reference are among the least characterized forms of genetic variation in genomes and genotype with short-read technology. We created a the human genome (13, 14). Their repetitive nature and some- framework to model the evolution and instability of STRs and VNTRs times extreme GC content make them particularly challenging to in apes. We phased and assembled 3 ape genomes (chimpanzee, sequence and assemble with standard whole-genome shotgun gorilla, and orangutan) using long-read and 10x Genomics linked- sequencing assembly strategies, including next generation se- read sequence data for 21,442 human tandem repeats discovered in quencing approaches that depend on bridge amplification (15). 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded Their large size, often many kilobases in length, and their in- Escherichia specifically in humans, including large tandem repeats affecting herent instability during clonal propagation through coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). coli vectors have limited their accurate representation in the We show that short interspersed nuclear element–VNTR–Alu (SVA) human reference genome, which was largely dependent on hi- GENETICS retrotransposition is the main mechanism for distributing GC-rich erarchical bacterial artificial chromosome (BAC) clone sequence human-specific tandem repeat expansions throughout the ge- and assembly (16, 17). As a result, recent surveys of human ge- nome but with a bias against genes. In contrast, we observe that nomes using orthogonal single-molecule long-read sequencing VNTRs not originating from retrotransposons have a propensity to technologies (18, 19) have shown that the length and number of cluster near genes, especially in the subtelomere. Using tissue- these repeats have been systematically underestimated. Be- specific expression from human and chimpanzee brains, we iden- cause their length and purity are critical to determining their tify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using Significance single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles anal- ogous to intermediate progenitor cells. Finally, we compare the Short tandem repeats (STRs) and variable number tandem re- sequence composition of some of the largest human-specific re- peats (VNTRs) are among the most mutable regions of our ge- peat expansions and identify 52 STRs/VNTRs with at least 40 un- nome but are frequently underascertained in studies of disease interrupted pure tracts as candidates for genetically unstable and evolution. Using long-read sequence data from apes and regions associated with disease. humans, we present a sequence-based evolutionary framework for ∼20,000 phased STRs and VNTRs. We identify 1,584 tandem tandem repeat | STR | VNTR | tandem repeat expansion | repeats that are specifically expanded in human lineage. We genome instability show that VNTRs originate by short interspersed nuclear ele- ment–VNTR–Alu retrotransposition or accumulate near genes in subtelomeric regions. We identify associations with expanded hort tandem repeats (STRs) and variable number tandem re- tandem repeats and genes differentially spliced or expressed Speats (VNTRs), also referred to as micro- and minisatellites (1, between human and chimpanzee brains. We identify 52 loci with 2), are operationally defined as tandemly repeating units of DNA long, uninterrupted repeats (≥40 pure tandem repeats) as can- ≥ of 1 to 6 and 7 bp in length, respectively (3). The mutation rates didates for genetically unstable regions associated with disease. among these tandem repeats can be several orders of magnitude −6 higher than the unique portions of the genome, ranging from 10 Author contributions: A.S. and E.E.E. designed research; A.S. performed research; A.S., − to 10 2 nucleotides per generation in STRs (4, 5). The mutation R.L., G.A.L., W.C.W., A.A.P., and M.J.P.C. contributed new reagents/analytic tools; A.S., R.L., P.A.A., D.P., and M.R.V. analyzed data; D.P. helped with enrichment analysis; HGSVC rate for a given locus can vary widely, while the longest and purest provided early data access; A.A.P. helped with brain organoid expression analysis; and tandem repeat tract often defines the most unstable STRs and A.S. and E.E.E. wrote the paper. VNTRs (6–8). As a result, STRs/VNTRs have long been recog- Competing interest statement: E.E.E. is on the scientific advisory board of DNAnexus, Inc. nized among the most polymorphic markers of genomes. They are This article is a PNAS Direct Submission. also an important source of genomic instability associated with Published under the PNAS license. several human disorders, including repeat expansion disorders, Data deposition: All long-read human and nonhuman primate assemblies of the short due to their tendency to expand through replication slippage, tandem repeats/variable number tandem repeats presented in this study were aligned against the GRCh38 and deposited in Zenodo (https://zenodo.org/record/3401477). DNA repair, or nonallelic homologous recombination (9). 1A complete list of the Human Genome Structural Variation Consortium can be found in Tandem repeats can also harbor cryptic disease-causing vari- the SI Appendix. ation in the form of single-nucleotide variants (SNVs) (10) or 2To whom correspondence may be addressed. Email: [email protected]. short insertions and deletions (indels) (11, 12), emphasizing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the importance of accurately predicting both their size and 1073/pnas.1912175116/-/DCSupplemental. sequence composition. First published October 28, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1912175116 PNAS | November 12, 2019 | vol. 116 | no. 46 | 23243–23253 Downloaded by guest on October 2, 2021 mutability and inherent instability, accurate haplotype resolution the 7 human and 6 NHP haplotypes, with the goal of identifying human- of larger alleles is important (20). specific expansions (HSEs) of tandem repeats. SI Appendix has more details. The goal of this study is 2-fold: 1) produce a high-quality set of We also identified ab initio human repeats; these consisted of loci with haplotype-resolved tandem repeat loci in the human genome tandem repeats content in all human samples and no tandem repeat content in any of the NHPs’ homologous loci. Due to the filters used above for both with a specific emphasis on those that are misrepresented in the ab initio and HSE, we expect to observe a set of tandem repeats that are current human reference genome and 2) generate an evolu- differently expanded in humans while also being ab initio, which leads to tionary framework for their origin by establishing the likely ape some STRs/VNTRs being classified in both categories. Thus, we took into ancestral state of each allele. As a starting point, we leveraged account this redundancy between the 2 categories to avoid double counting the haplotype-resolved structural variants (SVs) from 3 diverse in the downstream analyses. individuals generated as part of the Human Genome Structural A subset of the tandem repeats is expected to differ in both length and Variation Consortium (HGSVC) (20). Next, we generated sequence composition from the human genome reference. To estimate the number of reference collapses or misassemblies in GRCh38, we counted STRs/ haplotype-resolved sequences for the homologous loci in non- ≥ human primates (NHPs) using a combination of PacBio long VNTRs with 2-fold as many tandem copies in the shortest human haplotype than in GRCh38 (i.e., collapsed regions) and repeat unit sequence with ≤90% reads and 10x Genomics linked reads from individuals of 3 sequence identity between the human haplotypes and the reference (i.e., species: chimpanzee, gorilla, and orangutan (21). This compar- misassembled). Lastly, a tandem repeat locus was classified as polymorphic if ative analysis allowed us to delineate human-specific tandem its standard deviation of copy numbers across the human haplotypes repeat expansions and further investigate their potential effects was ≥10th percentile. on gene expression and splicing using single-cell and tissue-