Novel crossover and recombination hotspots massively spread across human genome

doi:10.21203/rs.3.rs-4394938/v1

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Novel crossover and recombination hotspots massively spread across human genome

https://doi.org/10.21203/rs.3.rs-4394938/v1

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published 21 Aug, 2024

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Background

The recombination landscape and subsequent natural selection have vast consequences in evolution and speciation. However, most of the recombination hotspots in the human genome are yet to be discovered. We previously reported hotspot colonies of CG-rich trinucleotide two-repeat units (CG-TTUs) across the human genome, several of which were shared, with extensive dynamicity, as phylogenetically distant as in mouse.

Results

Here we performed a whole-genome analysis of AT trinucleotide two-repeat units (AT-TTUs) in human and found that the majority (96%) resided in approximately 1.4 million colonies, spread throughout the genome. In comparison to the CG-TTU colonies, the AT-TTU colonies were significantly more abundant and larger in size. Pure units and overlapping units of the pure units were readily detectable in the same colonies, signifying that the units are the sites of unequal crossover. Subsequently, we analyzed several of the AT-TTU colonies in several primates and mouse. We discovered dynamic sharedness of several of the colonies across the primate species, which mainly reached maximum complexity and size in human.

Conclusions

We report novel crossover and recombination hotspots of the finest molecular resolution, and evolutionary relevance in human. In respect of crossover and recombination, the human genome is far more dynamic than previously envisioned.

Human

AT trinucleotide

Two-repeat

Unequal crossover

Recombination hotspot: Primate

Shared

Crossover and recombination, alongside mutation, generate the raw material of evolution and speciation [1, 2]. Recombination hotspots are regions in a genome that exhibit elevated rates of recombination relative to a neutral expectation. Studies on recombination hotspots are mainly founded on mapping crossover events through pedigree analysis and linkage disequilibrium [3, 4]. Identification of those hotspots paved the way for the discovery of PRDM9, a trimethyl transferase, which is associated with hotspot activity in both humans and mouse [5–7]. Using HapMap data, Myers et al. identified a 13-bp “core” motif “CCTCCCTNNCCAC” for PRDM9 binding, which is strongly correlated with hotspot activity when it occurs in both repeat and nonrepeat DNA. A close match to this motif was reported to occur in about 40% of the cross-over hotspots known to date [8], and degenerate versions of the motif, of variable binding activity for PRDM9, have been identified in the human genome on centimorgan (cM) scales [9, 10]. The 13-mer motif is the most characterized hotspot locus in human to date. However, the level of expression of PRDM9 should control for only the fraction of targets that are hotspots and the overall temperature of the genome [11].

Other indirect approaches, such as phylogenetic and integrated genetic versus physical map analyses performed by several groups have led to the idea that the local rates of recombination are positively correlated with GC content in the human genome [12–15] and a few other mammals [16]. Lined with the above, there are reports that meiotic recombination favors GC-rich alleles over AT-rich alleles, and facilitates local GC-content [17, 18]. When a meiotic recombination hotspot from a GC-rich isochore was inserted into an AT-rich isochore domain, the site adopted the lower recombination activity, characteristic of its new environment [19]. It is reported that programmed in vitro double strand break formation and loading of axial structure proteins are much more prominent in GC-rich isochores [9, 10, 12].

We previously reported that CG-rich trinucleotide two-repeat units (CG-TTUs) form colonies of exceeding significance across the human genome, based on Poisson distribution [20, 21]. Several of the large and medium size colonies that were further analyzed in other species, unveiled crossover and recombination hotspots, shared across primates, and in some instances, even in mouse.

Here, we investigated AT-rich trinucleotide two-repeat units (AT-TTUs) with a similar protocol, and discovered that the colonies formed by AT-TTUs were significantly more abundant and larger than the colonies formed by CG-TTUs. These novel crossover sites vastly spread across primate genomes, and mainly reach maximum complexity and size in human.

Whole-genome extraction of AT-TTUs in human.

A Java software package was created (available at https://github.com/arabfard/Java_STR_Finder) to facilitate the extraction of specific type of DNA sequence units, known as AT-TTUs, including AATAAT, ATAATA, ATTATT, TTATTA, TATTAT, and TAATAA, along with their corresponding locations. To ensure the accuracy and reliability of the data obtained from the algorithm, a validation process was conducted. This involved randomly examining these units across the entire genome. By manually inspecting the identified units, we were able to detect any potential errors or false positives generated by the algorithm. Through this verification process, we confirmed that the algorithm functioned as intended, and produced reliable results. In order to extract all AT-TTUs, we utilized the latest version of the human genome assembly (GRCh38. p14) obtained from the UCSC genome browser (accessible at https://hgdownload.soe.ucsc.edu).

Comparison of the AT-TTU versus CG-TTU colonies in the human genome.

The AT-TTU colonies from the present study were compared with the CG-TTU colonies, yielded from our previous study [21], https://figshare.com/articles/dataset/All_possible_CG-rich_trinucleotides/23260562 .

The extraction algorithm

The developed Java program was used to extract all possible AT-TTUs, as follows: AATAAT, ATAATA, ATTATT, TTATTA, TATTAT, and TAATAA, from the human genome sequence. The program initiated its search from the first nucleotide of the genome, continuously scanning for duplicated occurrences of AT-rich trinucleotide cores. It employed a window frame consisting of 6 nucleotides to identify instances of the core sequence repeating twice. Upon discovering a unit, the program recorded the number and location of the two-repeat occurrences. It then proceeded to search for new AT-TTUs, starting from the next nucleotide.

To validate the results, the final list of identified AT-TTUs underwent manual evaluation, using the Ensembl genome browser 109 (https://asia.ensembl.org/index.html). The precise locations of the AT-TTUs were determined as follows: The output was organized and classified in an Excel file, where the start and end points of each unit were determined within the genome. By subtracting the start and end points of subsequent units, colonies were identified. If the resulting distance was less than 500 bp, the units were considered part of the same colony. Subsequently, a list of colonies consisting of two or more units was compiled, the total count of colonies was determined, and the output was saved in a readily available format (https://doi.org/10.6084/m9.figshare.24202461.v1).

Screening several of the large and medium-size human colonies in other species

Several of the large and medium-sized colonies in human were screened in several other species, using Genome Browser 109 (https://asia.ensembl.org/index.html) BLASTN program. The genome assemblies used were as follows: Chimpanzee: Pan_tro_3.0, Gorilla: gorGor4, Macaque:Mmul_10, Mouse lemur: Mmur_3.0, and Mouse: GRCm39.

Statistical analysis

The Poisson distribution was employed to determine the probability distribution of colonies. This model assumed that the occurrence of the units was random and independent of each other, and that their distribution across the genome was relatively even. This assumption was subsequent to our observations of the ubiquitous occurrence of the colonies (Table 1). Therefore, the probability of occurrence of various size colonies was calculated by the Poisson density function, using the following formula:

$${\lambda }=\frac{Colony Interval \left(bp\right) \text{*}\text{a}\text{l}\text{l} \text{p}\text{o}\text{s}\text{s}\text{i}\text{b}\text{l}\text{e} \text{u}\text{n}\text{i}\text{t}\text{s} \text{o}\text{f} \text{A}\text{T}-\text{r}\text{i}\text{c}\text{h} \text{t}\text{r}\text{i}\text{n}\text{u}\text{c}\text{l}\text{e}\text{o}\text{t}\text{i}\text{d}\text{e}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{g}\text{e}\text{n}\text{o}\text{m}\text{e} }{\text{G}\text{e}\text{n}\text{o}\text{m}\text{e} \text{S}\text{i}\text{z}\text{e}\left(3\text{g}\text{b}\right)}$$

For example, the largest colony, C718, spanned 21,859 bases. On the other hand, the total number of AT-TTUs in the human genome was about 10,330,879, resulting in λ = 75.27 for the C718 colony, meaning that based on the Poisson distribution, the average expected count of AT-TTUs in the 21,859 interval was 75.27. Table 1 presents values of λ for several colony sizes. The calculated probability value for the occurrence of those colonies was inherent zero.

Visualization

The six pure AT-TTUs were visualized as: TTATTA, TATTAT, AATAAT, ATTATT, ATAATA, and TAATAA. The overlapping units were also highlighted, using various highlight and text colors.

The majority of the AT-TTUs resided in colonies.

In total, 10,330,879 AT-TTUs were detected genome-wide, of which the majority (9,936,861) (96.18%) were arranged in 1,390,055 colonies (Fig. 1). The AT-TTUs were spread across all chromosomes (Fig. 2).

AT-TTU colonies were significantly more abundant and larger than the CG-TTU colonies.

In comparison with the CG-TTU colonies (https://figshare.com/articles/dataset/All_possible_CGrich_trinucleotides/23260562), the colonies formed by AT-TTUs were significantly more abundant (Fig. 3). Large intervals of chromosomes were occupied by colony intervals in many chromosomes, for example in chromosome 4. Furthermore, the pattern of distribution of the AT-TTU colonies across human chromosomes was significantly different from the CG-TTU colonies. For example, whereas chromosome 1 had the highest percentage of CG-TTU colonies (Ohadi et al. 2023), AT-TTU colonies reached highest percentage on chromosome 4. Chromosome X was also particularly rich in AT-TTU colonies.

Several of the large and medium-size AT-TTU colonies coincided with extensive dynamicity in great apes

Several of the large and medium-size AT-TTU colonies in human were also detected in other great apes (Table 1). Exceedingly dynamic events were detected across those colonies, affecting the AT-TTUs and the flanking sequences to those units. Across the colonies, the AT-TTUs were either pure or overlaps of two or more pure units.

Table 1

Several large and medium-size AT-TTU colonies in human and their corresponding colonies in other primates.
Colony Formula^b					Chr. No.	Location (Colony interval)	λ value
Colony Size in Human^a	Human	Chimpanzee	Gorilla	Macaque	Chr. No.	Location (Colony interval)	λ value
C718	[(ATT)2]32 [(ATA)2]5 [(AAT)2]11 [(TAA)2]2 [(TTA)2]318 [(TAT)2]350	[(ATT)2]21 [(ATA)2]5 [(AAT)2]3 [(TAA)2]2 [(TTA)2]262 [(TAT)2]288	[(ATT)2]3 [(ATA)2]2 [(TAA)2]1 [(TTA)2]4 [(TAT)2]7		11	11:114603789–114625648	75.27
C457	[(ATT)2]13 [(ATA)2]160 [(AAT)2]40 [(TAA)2]43 [(TTA)2]8 [(TAT)2]193				22	22:18728881–18733753	16.78
C350	[(ATT)2]12 [(ATA)2]165 [(AAT)2]15 [(TAA)2]11 [(TTA)2]11 [(TAT)2]136				11	11:96561723–96568076	21.88
C317	[(ATT)2]115 [(ATA)2]59 [(AAT)2]8 [(TAA)2]4 [(TTA)2]14 [(TAT)2]117	[(ATT)2]102 [(ATA)2]47 [(AAT)2]5 [(TAA)2]3 [(TTA)2]12 [(TAT)2]103			12	12:79456025–79468112	41.62
C287	[(ATT)2]11 [(ATA)2]112 [(AAT)2]30 [(TAA)2]13 [(TTA)2]16 [(TAT)2]105	[(ATT)2]2 [(ATA)2]17 [(AAT)2]1 [(TAA)2]1 [(TTA)2]2 [(TAT)2]24		[(ATT)2]2 [(ATA)2]3 [(AAT)2]2 [(TAA)2]1 [(TTA)2]2 [(TAT)2]6	X	X:143653179–143659751	22.63
C275	[(ATT)2]6 [(ATA)2]135 [(AAT)2]13 [(TAA)2]10 [(TTA)2]6 [(TAT)2]105				22	22:18891229–18896934	19.65
C267	[(ATT)2]4 [(ATA)2]151 [(AAT)2]4 [(TAA)2]4 [(TTA)2]5 [(TAT)2]99				2	2:16146128–16148756	9.05
C255	[(ATA)2]4 [(AAT)2]247 [(TAA)2]2 [(TTA)2]1 [(TAT)2]1	[(ATA)2]2 [(AAT)2]16 [(TAA)2]1 [(TTA)2]1 [(TAT)2]1			2	2:231822812–231850574	95.60
C212	[(ATA)2]101 [(AAT)2]4 [(TAA)2]6 [(TTA)2]2 [(TAT)2]99	[(ATT)2]20 [(ATA)2]95 [(AAT)2]20 [(TAA)2]16 [(TTA)2]8 [(TAT)2]88		[(ATT)2]1 [(ATA)2]17 [(AAT)2]1 [(TAA)2]3 [(TTA)2]1 [(TAT)2]20	X	X: 108772450–108776693	14.61
C200	[(ATT)2]4 [(ATA)2]77 [(AAT)2]76 [(TAA)2]9 [(TTA)2]2 [(TAT)2]32	[(ATT)2]2 [(ATA)2]72 [(AAT)2]70 [(TAA)2]7 [(TTA)2]2 [(TAT)2]30		[(ATA)2]80 [(AAT)2]78 [(TAA)2]10 [(TTA)2]3 [(TAT)2]29	7	7:37785667–37794029	28.80
C198	[(ATT)2]9 [(ATA)2]62 [(AAT)2]14 [(TAA)2]15 [(TTA)2]5 [(TAT)2]93				22	22:18235269–18238724	11.90
C195	[(ATT)2]12 [(ATA)2]61 [(AAT)2]9 [(TAA)2]6 [(TTA)2]15 [(TAT)2]92	[(ATT)2]10 [(ATA)2]48 [(AAT)2]3 [(TAA)2]4 [(TTA)2]10 [(TAT)2]58	[(ATT)2]6 [(ATA)2]31 [(AAT)2]4 [(TAA)2]4 [(TTA)2]4 [(TAT)2]17		10	10:67724852–67731164	21.74
C190	[(ATT)2]2 [(ATA)2]38 [(AAT)2]5 [(TAA)2]6 [(TTA)2]3 [(TAT)2]136				2	2:194462444–194465074	9.06
C184	[(ATT)2]34 [(ATA)2]18 [(AAT)2]30 [(TAA)2]4 [(TTA)2]61 [(TAT)2]37	[(ATT)2]23 [(ATA)2]10 [(AAT)2]19 [(TAA)2]3 [(TTA)2]34 [(TAT)2]24	[(ATT)2]19 [(ATA)2]10 [(AAT)2]17 [(TAA)2]2 [(TTA)2]26 [(TAT)2]19	[(ATT)2]6 [(ATA)2]1 [(AAT)2]3 [(TAA)2]1 [(TTA)2]2 [(TAT)2]5	6	6:77601972–77604184	7.62
C182	[(ATT)2]13 [(ATA)2]51 [(AAT)2]17 [(TAA)2]16 [(TTA)2]12 [(TAT)2]73	[(ATT)2]6 [(ATA)2]65 [(AAT)2]12 [(TAA)2]11 [(TTA)2]9 [(TAT)2]38	[(ATT)2]4 [(ATA)2]31 [(AAT)2]2 [(TAA)2]2 [(TTA)2]6 [(TAT)2]22	[(ATT)2]3 [(ATA)2]11 [(AAT)2]2 [(TAA)2]3 [(TTA)2]7 [(TAT)2]15	14	14:52527096–52531874	16.45
C175	[(ATT)2]5 [(ATA)2]68 [(AAT)2]5 [(TAA)2]7 [(TTA)2]23 [(TAT)2]67				7	7:54395223–54397576	8.10
C173	[(ATA)2]69 [(AAT)2]64 [(TAA)2]18 [(TAT)2]22				16	16:18565943–18570325	15.09
C161	[(ATT)2]68 [(ATA)2]17 [(AAT)2]2 [(TAA)2]2 [(TTA)2]8 [(TAT)2]64	[(ATT)2]65 [(ATA)2]17 [(AAT)2]2 [(TAA)2]2 [(TTA)2]8 [(TAT)2]64	[(ATT)2]68 [(ATA)2]17 [(AAT)2]3 [(TAA)2]2 [(TTA)2]4 [(TAT)2]65		3	3:8693642–8702099	29.12

^aColony size, chromosomal location, colony interval, and λ value are based on the human genome, as reference. The corresponding colonies in other species were identified, using BLASTN. Instances in which the colonies were partially sequenced (such as C287, C212, and C200 in gorilla), or lacked the corresponding colony in a species were left blank. Poisson probability for all the colony sizes in this table is inherent zero. None of the colonies in this table were detected in mouse lemur or mouse.

^bFormulas represent absolute numbers of units, regardless of being pure or overlapping.

Poisson probability values calculated for the occurrence of the colonies was inherent zero.

The largest AT-TTU colony in human was a compound colony of 718 units (C718), located on chromosome 11, which was detected with exceeding dynamicity in human and chimpanzee, and at a far lesser extent in gorilla. This colony reached maximum complexity and size in human (Fig. 4).

The absolute number of the AT-TTUs and the distribution of the units in the pure and overlapping compartments were exceedingly dynamic across those species, adding multiple layers of complexity of the events, and leading to massively divergent compositions. Most of the units in C718 and its orthologous colonies were in the overlapping compartment (Figs. 4 and 5A). The immediate flanking sequences of the overlapping units conformed to the flanking sequences of the involved pure units, and were significantly dynamic with respect to mutations (Fig. 5B).

Models proposed for the evolution of pure and overlapping units.

The pure units were the inverted or palindromic sequences of one another, and probably emerged, and resulted in DNA breakage and recombination events inherent to inverted and palindromic sequences, for example, two pure units of TTATTA and ATTATT (inversion), and TTATTA and TAATAA (palindrome).

Overlapping units were a consequence of unequal crossovers among the pure units. For example, in C718, the most prevalent overlapping unit, TTATTAT, was the consequence of unequal crossovers between pure units, TTATTA and TATTAT (Fig. 5A). In another example in C718, the overlapping unit, AATAATTATTAT, was the consequence of several unequal crossovers across units (Fig. 5A). It is conceivable that reverse processes leading to the overlapping units resulted in the re-emergence of the pure units.

The flanking sequences of the units were also highly dynamic (Fig. 5B), signifying the occurrence of crossovers at the sites of the AT-TTUs, and coupled breakage and repair at, and around these sites.

Coincidence of some of the colonies beyond great apes.

Several colonies, such as C212, C200, and C184 coincided beyond great apes, and included macaque (Table 1). As an example, in C184, the colonies were shared dynamically in human, chimpanzee, gorilla, and macaque, and there was a directional incremented trend of complexity of the events and units in human (Fig. 6). Pure and overlapping units were also detected across this colony in human and other primates. For example, TATTATTA, was the consequence of unequal crossovers between TATTAT, ATTATT, and TTATTA pure units.

Colonies that were detected in human and not the other five species studied.

We also detected many colonies that were found in human only (Table 1), examples of which are visualized for C457 (Fig. 7A) and C190 (Fig. 7B). Consecutive pure units recombining with each other, or pure and overlapping units recombining with each other were detected in those colonies.

AT-TTUs are a mechanism for the emergence of AT short tandem repeats (STRs).

The AT-TTUs and coupled unequal crossovers and recombination at these sites result in the emergence of STRs (repeats of ≥ 3). For example, in C184, the (TTA)3 STR could be a consequence of unequal crossovers through various paths (Fig. 8A and B). In other examples, in C457 and C190, unequal crossovers gave rise to overlapping units for the emergence of several (ATA)3 STRs (Fig. 8C, D, and E). We detected the pure units and intermediate overlapping units necessary for the emergence of STRs in the same (or orthologous) colonies that the STRs were detected.

The bulk of literature is dominated by reports of the preference of CG- over AT-rich sequences at the recombination hotspots [15, 22–27]. Limited reports of the involvement of AT-rich sequences in recombination and consequent translocations are available in the literature, which primarily concern AT-rich palindromic or inverted sequences. Those events are mainly involved in chromosomal translocations and deletions, for example in chromosomes 11, 17, and 22 [28–30].

Our methodology of including palindromes and inversions in the context of pure units, enabled detecting colonies that were enriched for those sequences, and detection of a phenomenon, in which AT-TTUs colonize across the genome with exceedingly significant Poisson probability values. In fact, the majority of AT-TTUs in the human genome reside in colonies, and those colonies span significant intervals of several chromosomes. Remarkably, chromosome X is also enriched with colonies.

The AT-TTU colonies were significantly larger and more complex than the CG-rich colonies that we reported previously [20, 21]. These findings support a more significant role of AT-rich sequences in comparison with CG-rich sequences, as crossover and recombination hotspots. While the AT-TTU crossover hotspots were largely ubiquitous, the most refined maps of recombination identified to date are on the scales of cM [31, 32].

The presence of pure units and overlapping units of those pure units, signify that the main reason for the hotspot events in those colonies is the AT-TTUs, and not their flanking sequences. The inversions and palindromes as a result of the pure and overlapping units increase the rate of various genetic rearrangement events and recombination across the colonies. Palindromes and inversions are known to be recombinogenic in the genomes, and a risk to instability [33, 34].

The flanking sequences of the AT-TTUs were also extensively dynamic. The very high dynamicity of the flanking sequences was in line with the previous reports that flanking sequences to the recombination sites are prone to mutations [35].

Some of the identified colonies, which were further studied in other primates, were shared in those primates with exceeding dynamicity of the events. These findings challenge the literature on the rarity of shared recombination hotspots between human and closely related species [36–39]. An isolate report of shared hotspot loci between human and chimpanzee was at β-globin and HLA regions on chromosome 21, which was based on high Bayes factors of shared hotspots at locations within both regions[40]. Our data extend crossover and recombination hotspot sharedness across primates, and envision a new perspective in the field, with respect to mechanistic, evolutionary, and magnitude of crossovers and recombination.

It is reasonable to consider the AT-TTUs a novel genomic entity, as although they are repeats, they do not conform to the conventional definition of repetitive DNA sequences, such as microsatellites and minisatellites [41]. It is also expected that novel proteins are recruited to these loci, as neither the well-characterized recombination hotspot 13-mer, nor the degenerate sequences of this sequence conform to the identified units and colonies, and the extent of the events occurring across those colonies.

It is possible that some of the colonies are a result of “non-crossover” recombination, which includes exchange of DNA fragments, without exchanging the flanking chromosome arm. In fact, the majority of recombination interactions in meiosis are of the non-crossover type, as opposed to crossovers, which include the flanking chromosomal arm as well. Similar to crossovers, knowledge on the sites and biological implications of non-crossovers are also limited (if not less) at this time, and they are more difficult to detect. Evidence indicates that there probably is no non-crossover-specific pathway, and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation[42]. PRDM9 recruitment, CG-bias at the sites of recombination, and nearby conversions are also inherent to non-crossovers[43, 44]. The AT-rich trinucleotide two-repeats identified here, unveil recombination hotspots and evolutionary implications (sharedness across primates, which can be of relevance in both crossover and non-crossover contexts. Throughout this paper, we used the term “crossover” as its general application (see Glossary). That term was not intended to differentiate between crossover and non-crossover events defined above.

In comparison with CG-TTU colonies, the AT-TTU colonies (at least the colonies that were further analyzed in additional primates), were mainly more complex in human, at a directional trend. Furthermore, the rate of detecting those colonies in human and not in other species that were studied, was higher than the CG-TTU colonies[21]. One explanation may be that the mechanisms involved in the AT-TTU colonies have evolved more recently than the CG-TTU colonies. This is also supported by our observations that AT-TTU orthologous colonies were not detected in mouse lemur and mouse, whereas in the instance of CG-TTU colonies, several of the colonies were identified in those species [21].

The AT-TTUs and the crossovers coupled with those units are a novel mechanism for the emergence of STRs. This follows from our observations that all the pure and intermediate overlapping units necessary for the birth and maturation of a given STR were detectable in the same (or orthologous) colonies. The evolutionary, biological, and pathological implications of STRs are an emerging topic of research [45–48].

In summary, in view of the events and mechanisms associated with the identified units, their abundance and ubiquity throughout the genomes studied, and their exceedingly significant colonization based on Poisson distribution, we predict these units of phenomenal evolutionary and biological consequences. This is the tip of the iceberg, and the evolutionary purpose of this phenomenon is yet to be discovered in the future studies.

In conclusion, our findings unveil massive AT-TTU crossover and recombination hotspots of evolutionary relevance in human. These findings also signify preference of AT- over CG-rich sequences at the crossover and recombination hotspots, and conservation of these hotspots, at least across great apes and Old-World monkeys. AT-TTUs are a novel mechanism for the emergence of STRs across species. We propose that the human genome is far more dynamic than previously imagined.

AT-TTU AT Trinucleotide Two-repeat Unit

C: Colony

CG-TTU CG Trinucleotide Two-repeat Unit

STR Short Tandem Repeat

Unit Two-repeats of any AT trinucleotides, such as TTATTA.

Colony A group of two or more units that were located within a distance of less than 500 bp from each other, on the genomic DNA. Throughout the text, specific colonies are identified by adding "C" prefixes when necessary. The designation of colonies is based on their size in the human genome. For example, C718 was a colony of 718 units in human.

Compound Colony A colony that consists of more than one type of two-repeat units of AT trinucleotides. For example, a compound colony could include (TTA)2 and (TAT)2 units.

Pure unit A unit that contains only one type of AT trinucleotide, for example, ATTATT .

Overlapping Unit A unit that consists of two or more pure units that overlap. For example, the sequence "TTATTATT" consists of three pure units of TTATTA, TATTAT, and ATTATT, which overlap with each other.

Absolute number Number of units regardless of being pure or overlapping.

Crossover The exchange of DNA between paired homologous chromosomes (one from each parent) that occurs during the development of egg and sperm cells (meiosis).

Unequal crossover Unequal crossing-over, also referred to as illegitimate recombination, refers to crossover events that occur between nonequivalent sequences.

Non-crossover Recombination interactions, which include DNA fragments, without exchange of flanking chromosome arms.

Recombination hotspot A genomic region (typically in ~kb ranges) that experience intensely high levels of recombination compared to the genomic background.

Short tandem repeat Repeats of ≥3.

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

Funding:

Not applicable

Competing interests:

None to be declared.

Data and materials availability:

Raw data for AT-TTUs are available at the following link:

https://figshare.com/articles/dataset/AT-rich_trinucleotides/24202461

Raw data for CG-TTUs are available at the following link:

https://figshare.com/articles/dataset/All_possible_CG-rich_trinucleotides/23260562

Author contributions:

Conceptualization: MO

Methodology: MA, AMAM

Investigation: MA, SKh, SA, SV, HB, NT

Visualization: MA, SA, SKh

Project administration: MO, AD, HRKh

Supervision: MO

Writing – original draft: MO, MA

Writing – review & editing: MO, MA

Acknowledgments:

Not applicable.

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Novel crossover and recombination hotspots massively spread across human genome

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