De novo genome assemblies from two Indigenous Americans from Arizona identify new polymorphisms in non-reference sequences

There is a collective push to diversify human genetic studies by including underrepresented populations. However, analyzing DNA sequence reads involves the initial step of aligning the reads to the GRCh38/hg38 reference genome which is inadequate for non-European ancestries. To help address this issue, we created a modified hg38 reference map using de novo sequence assemblies from Indigenous Americans living in Arizona (IAZ). Using HiFi SMRT long-read sequencing technology, we generated de novo genome assemblies for one female and one male IAZ individual. Each assembly included [~]17 Mb of DNA sequence not present (non-reference sequence; NRS) in hg38, which consists mostly of repeat elements. Forty NRSs totaling 240 kb were uniquely anchored to the hg38 primary assembly generating a modified hg38-NRS reference genome. DNA sequence alignment and variant calling were then conducted with WGS sequencing data from 387 IAZ cohorts using both the hg38 and modified hg38-NRS reference maps. Variant calling with the hg38-NRS map identified [~]50,000 single nucleotide variants present in at least 5% of the WGS samples which were not detected with the hg38 reference map. We also directly assessed the NRSs positioned within genes. Seventeen NRSs anchored to regions including an identical 187 bp NRS found in both de novo assemblies. The NRS is located in HCN2 79 bp downstream of exon 3 and contains several putative transcriptional regulatory elements. Genotyping of the HCN2-NRS revealed that the insertion is enriched in IAZ (MAF = 0.45) compared to Caucasians (MAF = 0.15) and African Americans (MAF = 0.03). This study shows that inclusion of population-specific NRSs can dramatically change the variant profile in an under-represented ethnic groups and thereby lead to the discovery of previously missed common variations. AUTHOR SUMMARYGRCh38/hg38 reference genome has been the standard reference for large-scale human genetics studies. However, it does not adequately represent sequences of non-European ancestry. In this study, using long-read sequencing technology, we constructed de novo sequence assemblies from two Indigenous Americans from Arizona. We then compared the de novo assemblies to the hg38 reference genome to identify non-reference sequences (NRSs). We integrated these NRSs into our whole-genome sequencing (WGS) variant calling pipeline to improve read alignment and variant detection. We also directly assessed the NRSs positioned within genes. Inclusion of population-specific NRSs dramatically changed the variant profile of our study group with under-represented ethnicity, revealing common variation not detected by our previous population-level WGS and genotyping studies.