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Complex sequence organization of heterochromatin in the holocentric plant Cuscuta europaea elucidated by the computational analysis of nanopore reads.

ABSTRACT: Repeat-rich regions of higher plant genomes are usually associated with constitutive heterochromatin, a specific type of chromatin that forms tightly packed nuclear chromocenters and chromosome bands. There is a large body of cytogenetic evidence that these chromosome regions are often composed of tandemly organized satellite DNA. However, comparatively little is known about the sequence arrangement within heterochromatic regions, which are difficult to assemble due to their repeated nature. Here, we explore long-range sequence organization of heterochromatin regions containing the major satellite repeat CUS-TR24 in the holocentric plant Cuscuta europaea. Using a combination of ultra-long read sequencing with assembly-free sequence analysis, we reveal the complex structure of these loci, which are composed of short arrays of CUS-TR24 interrupted frequently by emerging simple sequence repeats and targeted insertions of a specific lineage of LINE retrotransposons. These data suggest that the organization of satellite repeats constituting heterochromatic chromosome bands can be more complex than previously envisioned, and demonstrate that heterochromatin organization can be efficiently investigated without the need for genome assembly.

SUBMITTER: Vondrak T

PROVIDER: S-EPMC8091179 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHuman 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.ResultsWe present a hybrid assembly method, NanoPore Guided REgional Assembly Tool (NPGREAT), which combines Linked-Read data with mapped ultralong nanopore reads spanning subtelomeric segmental duplications to potentially overcome these difficulties. Linked-Read sets of DNA sequences 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). Mapped 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. Our method was tested for a subset of representative subtelomeres with ultralong nanopore read coverage in the haploid human cell line CHM13. A 10X Linked-Read dataset from CHM13 was combined with ultralong nanopore reads from the same genome to provide improved subtelomere assemblies. Comparison of Nanopore-only assemblies using SHASTA with our NPGREAT assemblies in the distal-most subtelomere regions showed that NPGREAT produced higher-quality and more complete assemblies than SHASTA alone when these regions had low ultralong nanopore coverage (such as cases where large segmental duplications were immediately adjacent to (TTAGGG) tracts).ConclusionIn genomic regions with large segmental duplications adjacent to telomeres, NPGREAT offers an alternative economical approach to improving assembly accuracy and coverage using linked-read datasets when more expensive HiFi datasets of 10-20 kb reads are unavailable.

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Complex sequence organization of heterochromatin in the holocentric plant Cuscuta europaea elucidated by the computational analysis of nanopore reads.

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