NPGREAT: Assembly of the human subtelomere regions with the use of ultralong Nanopore reads and Linked-Reads

Background: Human subtelomeric DNA regulates the length and stability of adjacent telomeres that are critical for cellular function, and contains many gene/pseudogene families. Large evolutionarily recent segmental duplications and associated structural variation in human subtelomeres has made complete sequencing and assembly of these regions difficult to impossible for many loci, complicating or precluding a wide range of genetic analyses to investigate their function. Results We present a hybrid assembly method, NanoPore Guided REgional Assembly Tool (NPGREAT), which combines Linked-Read data with ultralong nanopore reads spanning subtelomeric segmental duplications to potentially overcome these difficulties. Linked-Read sets identified by matches with 1-copy subtelomere sequence adjacent to segmental duplications are assembled and extended into the segmental duplication regions using Regional Extension of Assemblies using Linked-Reads (REXTAL). Telomere-containing ultralong nanopore reads are then used to provide contiguity and correct orientation for matching REXTAL sequence contigs as well as identification/correction of any misassemblies (associated primarily with tandem repeats). While we focus on subtelomeres, the method is generally applicable to assembly of segmental duplications and other complex genome regions. Our method was tested for a subset of representative subtelomeres with ultralong nanopore read coverage in GM12878. 10X Linked-Read datasets with high depth of coverage and a TELL-seq Linked-Read dataset with lower depth of coverage were each combined with the ultralong nanopore reads from the same genome to provide improved assemblies. Tandem repeat regions of the short-read assemblies, which are especially prone to misassembly due to collapse of matching tandemly repeated reads, were readily identified and properly sized by comparison with the nanopore reads. Conclusion The NPGREAT method resulted in extension of high-quality assemblies into otherwise inaccessible segmental duplication regions near telomeres, enhancing our ability to accurately assemble human subtelomere DNA. This information will enable improved analyses of the structure, function, and evolution of these key regions.