Long-read Data Revealed Structural Diversity in Human Centromere Sequences

Abstract

Centromeres invariably serve as the loci of kinetochore assembly in all eukaryotic cells, but their underlying DNA sequences evolve rapidly. Human centromeres are characterized by their extremely repetitive structures, i.e., higher-order repeats, rendering the region one of the most difficult parts of the genome to assess. Consequently, our understanding of centromere sequence variations across human populations is limited. Here, we analyzed chromosomes 11, 17, and X using long sequencing reads of two European and two Asian genomes, and our results show that human centromere sequences exhibit substantial structural diversity, harboring many novel variant higher-order repeats specific to individuals, while frequent single-nucleotide variants are largely conserved. Our findings add another dimension to our knowledge of centromeres, challenging the notion of stable human centromeres. The discovery of such diversity prompts further deep sequencing of human populations to understand the true nature of sequence evolution in human centromeres. MainCentromeres have been one of the most mysterious parts of the human genome since they were characterized, in the 1970s, as large tracts of 171 bp strings called alpha-satellite monomers 1, 2 . With a growing body of evidence suggesting their relevance to human diseases as sources of genomic instability or as repositories of haplotypes containing causative mutations 3-8 , it has become more important to investigate the underlying sequence variations in centromeres 9, 10 .Human centromeres have nested repeat structures. Namely, a series of distinctively divergent alpha-satellite monomers compose a larger unit called higher-order repeat (HOR) unit, and copies of an HOR unit are tandemly arranged thousands of times to form large, homogeneous HOR arrays. While HOR units are chromosome-specific and consist of two to 34 alphasatellite monomers, copies of an HOR unit are almost identical (95 ∼ 100%) within a chromosome (Figure 1a) [11][12][13][14][15][16][17] . The total HOR array length in each chromosome is known to differ dramatically among individuals 7, 18 or across human populations 19,20 . In addition to array length, other types of variation are known to exist within an HOR array, such as structurally variant HORs that consist of different numbers and/or types of alpha-satellite monomers 20-23 and also single-nucleotide variations (SNVs) between the HOR units 20, 24, 25 . One of the major driving forces leading to such centromeric variation has been thought to be structural alterations such as unequal crossovers and/or gene conversions 26,27 .Previous studies have investigated centromeric sequence variations via traditional approaches such as restriction enzymes sensitive to alpha-satellite monomers, Southern blotting, or analysis of k-mers unique to centromeres in short reads obtained in the 1000 Genomes Project 28 , but their observations have remained indirect and were confined to specific types of variations due to technological limitations.Recently, the advent of long-rea...

The string decomposition problem and its applications to centromere analysis and assembly

“…Although no existing tool explicitly addresses the String Decomposition Problem, Minimap2 ( Li, 2018 ), PERCON ( Kazakov et al , 2003 ), TRF ( Benson, 1999 ), Alpha-CENTAURI ( Sevim et al , 2016 ) and NCRF ( Harris et al , 2019 ) tools address related problems. Suzuki et al (2019) also developed a custom script implementing a blastn-based string decomposition approach.…”

“…Assembling ETRs is important since variations in ETR have been linked to cancer and infertility ( Barra and Fachinetti, 2018 ; Black and Giunta, 2018 ; Ferreira et al , 2015 ; Giunta and Funabiki, 2017 ; Miga et al , 2019 ; Smurova and De Wulf, 2018 ; Zhu et al , 2018 ). ETR sequencing is also important for addressing open problems about centromere evolution ( Alkan et al , 2007 ; Henikoff et al , 2015 ; Lower et al , 2018 ; Shepelev et al , 2009 ; Suzuki et al , 2019 ).…”

“…Recent attempts to assemble ETRs ( Bzikadze and Pevzner, 2019 ; Jain et al , 2018 ; Miga et al , 2019 ), and recent evolutionary studies of centromeres ( Suzuki et al , 2019 ; Uralsky et al , 2019 ) revealed the importance of partitioning an ETR (or an error-prone long read sampled from an ETR) into repetitive units forming these repeats. We refer to this problem as the String Decomposition Problem .…”

“…Although different HOR units on cenX are highly similar (95–100% sequence identity), the 12 monomers forming each HOR are rather diverged (50–90% sequence identity). In addition to standard 12-monomer HORs, some units on cenX have non-canonical monomer structure: 35 out of 1510 units are formed by a smaller or larger number of monomers than the canonical 12-monomer unit ( Bzikadze and Pevzner, 2019 ; Suzuki et al , 2019 ). The tandem repeat structure of human centromeres may be interrupted by retrotransposon insertions (e.g.…”

“…Partitioning of long error-prone reads into units and monomers is critically important for centromere assembly ( Bzikadze and Pevzner, 2019 ; Miga et al , 2019 ) and evolutionary studies of centromeres ( Suzuki et al , 2019 ). For example, centroFlye ( Bzikadze and Pevzner, 2019 ) requires a translation of each read into a monoread in the monomer alphabet.…”