Genomic Characterization of IS6110 Insertions in Mycobacterium orygis

Abstract

Mycobacterium orygis, a subspecies of the Mycobacterium tuberculosis complex (MTBC), has emerged as a significant concern in the context of One Health, with implications for zoonosis or zooanthroponosis or both. MTBC strains are characterized by the unique insertion element IS6110, which is widely used as a diagnostic marker. IS6110 transposition drives genetic modifications in MTBC, imparting genome plasticity and profound biological consequences. While IS6110 insertions are customarily found in the MTBC genomes, the evolutionary trajectory of strains seems to correlate with the number of IS6110 copies, indicating enhanced adaptability with increasing copy numbers. Here, we present a comprehensive analysis of IS6110 insertions in the M. orygis genome, utilizing ISMapper, and elucidate their genetic consequences in promoting successful host adaptation. Our study encompasses a panel of 67 paired-end reads, comprising 11 isolates from our laboratory and 56 sequences downloaded from public databases. Among these sequences, 91% exhibited high-copy, 4.5% low-copy, and 4.5% lacked IS6110 insertions. We identified 255 insertion loci, including 141 intragenic and 114 intergenic insertions. Most of these loci were either unique or shared among a limited number of isolates, potentially influencing strain behavior. Furthermore, we conducted gene ontology and pathway analysis, using eggNOG-mapper 5.0, on the protein sequences disrupted by IS6110 insertions, revealing 63 genes involved in diverse functions of Gene Ontology and 45 genes participating in various KEGG pathways. Our findings offer novel insights into IS6110 insertions, their preferential insertion regions, and their impact on metabolic processes and pathways, providing valuable knowledge on the genetic changes underpinning IS6110 transposition in M. orygis.

Introduction

The insertion sequence IS6110 is found explicitly in Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB) in humans and other mammals, including wildlife and farm animals, which are responsible for zoonotic or zooanthroponotic TB transmission. This unique trait made IS6110 a historical epidemiological marker for diagnosing MTBC in biological samples, enabling the detection of TB outbreaks and transmission chains, although currently being replaced by robust sequencing methods.^1-3 The transposition of IS6110 nurtures various genetic modifications in MTBC strains, conferring genomic plasticity and significant biological implications. ⁴ Insertions of IS6110 amid the coding region might render the gene inactive or act as a mobile promoter controlling gene activation, leading to unusual consequences in the host-pathogen evolution.^5,6 The members of the MTBC are classified into high (>6) and low (<7) copy strains based on the content of IS6110, although no clear evidence accounts for its phenotypic consequences in bacterial physiology and pathogenesis. ⁷ However, M. tuberculosis Beijing/W lineage (L2) possessing a high content of 15 IS6110 insertions is linked with high virulence and colossal spread of drug-resistant strains. ⁸ It has been demonstrated that a reasonable number of IS6110 might render strain-specific phenotypes catering to selective advantages during its course of infection rather than excessive accumulation resulting in inactivation or deletion of essential genetic regions, being detrimental to the bacterium. ⁵ Among the animal-adapted MTBC, Mycobacterium bovis (M. bovis) is known to possess only 1 or few copies of IS6110, ⁴ whereas M. orygis possess high copy numbers ranging from 17 to 20, however, the exact position of insertions are yet unknown ⁹ and not much information is available on other members which possess a zoonotic risk. M. orygis, has been isolated from various species, including oryx, waterbucks, cattle, deer, rhinoceros, monkeys, and humans and the exact extent of its host range still remains unknown.^9-16 A recent study in India identified 7 M. orygis isolates among 940 clinical isolates from humans. ¹⁷ Our laboratory has frequently been isolating M. orygis from cattle and wild ungulates recently, and various reports on this species are also being reported in South Asia.^13,14

The advent of Whole Genome Sequencing (WGS) has facilitated to localize the IS6110 position in the genomes of MTBC and has been recently used to identify its role in bovine adaptation.^18,19 This study investigates the distribution of IS6110 among M. orygis strains and its effect on the genes by in silico characterization. We have manifested the use of WGS to localize the IS6110 copies and their chromosomal distribution using the sequences of 67 M. orygis strains, comprising 56 WGS paired reads downloaded from the National Centre for Biotechnology Information-Sequence Read Archive (NCBI-SRA) and 11 WGS paired reads sequenced from our lab and to evaluate the level of IS6110 sequence stability and their orthology evolving in a multi-host system. Ever since the publication of the complete genome of M. orygis 51145, ²⁰ it has been used well as a reference genome (GenBank accession No. CP063804) for analysis of M. orygis strains. ¹³ Although the complete genome has been sequenced, the genes are not functionally annotated, and the mycobrowser (https://mycobrowser.epfl.ch/) identifies these genes as unknown, thereby demanding the use of orthologous comparative methods to identify its function. Hence, to overcome this, we additionally used M. tuberculosis H37Rv (NC000962.3) as the reference genome.

Materials and Methods

Data acquisition and analysis

To compare the sequence of our study isolates with existing sequences available in the database, NCBI SRA was searched with the term “Mycobacterium orygis.” All the sequences branded as M. orygis or M. tuberculosis var. orygis were downloaded from SRA with the fasterq-dump tool from the SRA toolkit (version 2.9.6) available at https://ncbi.github.io/sra-tools/. Only the sequences which downloaded as paired reads, and the sequences which possessed the species-specific Region of Difference (RDs) and single nucleotide polymorphisms (SNPs) were used for further analysis (Table S6). A total of 56 paired-end sequences of M. orygis strains isolated from human and animal sources were used, along with 11 study isolates. The raw sequences were filtered using trimmomatic (version 0.39) with a framework of minimum phred quality score and read length set to 30 and 80, respectively. The filtered sequences were further analyzed through vSNP https://github.com/USDA-VS/vSNP using M. orygis 51145 (CP063804) as the reference genome to generate SNPs. The phylogenetic tree was constructed based on the aligned whole-genome SNP sequences under a GTRCATI model of substitution and maximum-likelihood algorithm with a bootstrap replication of 1000 ²¹ and visualized using the interactive tree of life (iTOL). ²²

Analysis of IS6110 distribution

ISMapper (version 2.0) ²³ pipeline https://github.com/jhawkey/IS_mapper was used to localize IS6110 on short-read sequences with M. tuberulosis H37Rv (NC000962.3) and M. orygis 51145 (CP063804) as reference genomes. The presence of IS6110 was visualized manually with Integrative Genomics Viewer^24,25 using the bam file generated by ISMapper 2.0.

Analysis of orthologous genes

The genomic positions of the IS6110 were deduced through ISMapper analysis. The genes surrounding the insertion sites (intergenic) and genes possessing the insertion sites within them (intragenic) were identified based on the respective reference genomes to determine orthologous genomic sites of the IS.

Gene ontology enrichment and KEGG pathway analysis

The protein sequences of the upstream and downstream genes of IS6110 and the protein sequence of the genes which possessed the IS6110 insertions were used to perform functional annotation using eggNOG-mapper (version emapper—2.1.9) ²⁶ based on eggNOG orthology data. ²⁷ Sequence searches were performed using DIAMOND (version 2.0.11) ²⁸  to mine the orthologs.

Results

Distibution of IS6110 among M. orygis strains

In our analysis of IS6110 localization among the 67 sequences from both reference genomes, we observed that 4.5% (3/67) of the strains in the human cluster did not possess any IS6110 insertions and were classified as no-copy strains. We found that 91% (61/67) of the strains were high-copy, while 4.5% (3/67) were classified as low-copy. The number of IS6110 loci varied within the samples. In the human cluster, the loci ranged from 2 to 30 (with a median of 25) using the reference CP063804. In the animal cluster, the loci ranged from 20 to 42 (with a median of 26.5) using the same reference. However, when using the reference NC000962.3, the number of loci ranged from 2 to 43 (with a median of 32) in the human cluster and from 26 to 54 (with a median of 35) in the animal cluster. Overall, we identified a total of 24 common insertion loci. The specific regions of these insertion sites are tabulated and schematically represented, along with the phylogenetic tree generated using the maximum likelihood approach with all 67 M. orygis sequences (Figure 1 and Table 1).

Figure 1.

Contextual Phylogenetic tree combining the 11 study isolates along with 56 M. orygis isolates around the globe: Constructed using whole genome SNPs obtained after alignment with M. orygis reference genome CP063804. The heatmap shows the presence or absence of IS6110 in 67 M. orygis genomes. Red squares indicate the presence of IS6110 in a specific site. Descriptions of each IS6110 site are described in Table 1. The source and the geographic location of the isolates are represented in the respective bands. The study isolates are labeled in blue.

Table 1.

Details of IS6110 insertions among all M. orygis isolates.

IS No.	Orientation	Loci	Position	No. of isolates
1	R	RJtmp_000364: RJtmp_000365	418095-419370	61
2	R	RJtmp_000420: RJtmp_000421	481559-482854	59
3	R	RJtmp_000782: RJtmp_000783	835711-837062	60
4	R	RJtmp_000841: Rjtmp_000842	892501-893790	52
5	R	RJtmp_001232: RJtmp_001233	1304672-1305974	60
6	F	RJtmp_001332: RJtmp_001333	1423001-1424355	55
7	R	RJtmp_001435: RJtmp_001436	1536637-1537898	44
8	R	RJtmp_001448: RJtmp_001449	1550641-1551955	58
9	F	RJtmp_001738: RJtmp_001739	1891878-1893148	56
10	R	RJtmp_001757: RJtmp_001758	1909881-1911200	59
11	F	RJtmp_001833: RJtmp_001834	198611-1987961	55
12	R	RJtmp_001837: RJtmp_001838	1994072-1995329	44
13	F	RJtmp_001872: RJtmp_001873	2035374-2036722	50
14	F	RJtmp_001988: RJtmp_001989	2151738-2152992	58
15	F	RJtmp_002433: RJtmp_002434	2602192-2603492	56
16	R	RJtmp_002654: RJtmp_002655	2842275-2843648	60
17	F	RJtmp_002901: RJtmp_002902	3068456-3069736	59
18	F	RJtmp_003056: RJtmp_003057	3264524-3265824	57
19	R	RJtmp_003106: RJtmp_003107	3314904-3316281	51
20	R	RJtmp_003217: RJtmp_003218	3429234-3430489	52
21	F	RJtmp_003255: RJtmp_003256	3470593-3471871	53
22	F	RJtmp_003278: RJtmp_003279	3493266-3494534	55
23	R	RJtmp_003430: RJtmp_003431	3653243-3654495	52
24	R	RJtmp_003855: RJtmp_003856	4137024-4138299	59

Unique and novel IS6110 insertions in M. orygis strains

In addition to the previously mentioned typical and known IS6110 insertions, we also identified novel IS6110 insertions using ISMapper. When mapped against CP063804, we identified 107 novel insertion loci, of which, 61.7% (66/107) were intragenic and 38.3% (41/107) were intergenic. Approximately 58% (62/107) of these insertion loci were unique or shared between 2 and 3 strains, while 15 loci were shared in at least 4 strains, with only one being intergenic. Of the 15 common insertion sites, 11 were exclusively found in the animal cluster, 3 in both the human and animal clusters, and 1 insertion site was found only in the human cluster. Almost 124 novel insertion loci were identified when mapped against NC000962.3, out of which 39.5% (49/124) were intergenic and 60.5% (75/124) intragenic. Only 14 insertion sites were identified in both clusters, of which only 2 were intergenic. Notably, we observed variations in the localization and orientation of IS6110 insertions among the strains despite being present in the same genomic region. These findings shed light on the diversity and distribution of novel IS6110 insertions, providing valuable insights into the genetic landscape of the studied clusters. The genes on either side of IS6110 and those that contained IS6110 in both the H37Rv and 51145 reference genomes were combined. We identified 227 genes, among which 85 were from M. tuberculosis, 64 from M. orygis and 79 genes were found to be common in both M. orygis and M. tuberculosis (Supplemental Table S1). Using eggNOG Mapper, the protein sequences of these 227 genes were subjected to functional annotation.

Genetic significances of IS6110 insertions

Approximately 204/227 genes were scanned by eggNOG 5.0, and 82 were identified to be functionally involved in Gene Ontology (GO) and KEGG pathway (KP) enrichment analysis. Almost 37/82 genes were exclusively linked to various processes in GO and 19/82 genes were mapped in numerous KP. Additionally, we also identified 26 genes that were involved in both GO and KP. A total of 63 genes were encompassed in 669 GOs comprising 455 Biological processes (BP), 161 Molecular Functions (MF) and 53 Cellular components (CC), and the distribution of the genes in various functions are represented in Figure 2. Among the 63 genes involved in GO, 32 were found only in the human cluster, 15 in the animal cluster, and 16 in both clusters. The intergenic insertion sequence upstream of Rv0742 (Hypothetical protein) and Rv2965c/RJtmp_003059 (kdtB/coaD) were found in almost 61 and 60 isolates, respectively. The intragenic insertion sequences present within the genes Rv1266/Rjtmp_001332 (pknH), Rv2565 (NTE family protein), Rv1753 (PPE24), Rv0402/Rjtmp_000420/Rjtmp_000422 (mmpL1) and Rv1367c/Rjtmp_001448 (Hypothetical protein) were also found in almost 55 to 61 isolates. All these genes were involved in various GO processes (Supplemental Tables S2 and S3). Other insertion regions in the human cluster were unique and found in only 1 strain or at least 2 strains. A similar pattern was observed in the animal cluster except for 1 intergenic region between Rv2338c/Rjtmp_002419 (moeW/thiF family adenyltransferase) and Rv2339/RJtmp_002420 (mmpL9) and an intragenic insertion in Rv2430/RJtmp_002513 (PPE41) which were found in at least 6 and 7 strains, respectively.

Figure 2.

Distribution of 63 genes in various GO process.

Almost 62 KPs were identified in which nearly 45 genes were involved, including 20 from the human cluster, 14 from the animal cluster and 11 genes from both human and animal clusters. Here too, we found that the genes from both clusters mainly were unique or shared between a couple of strains. The majority of the genes (23/45) were involved in metabolic pathways (map01100) and 13/45 genes were involved in the biosynthesis of secondary metabolites (map01110). In addition to metabolic pathways, genes such as Rv0742, Rv2964/Rjtmp_003056 (purU), Rv2965c/RJtmp_003059 (kdtB/coaD) were found in almost 60 strains and these genes were involved in glycerolipid metabolism (map00561), Glycolate and dicarboxylate metabolism (map00630) & Pantothenate and CoA biosynthesis (map00770) respectively. The gene Rv1368/RJtmp_001450 (lprF), found in 7 isolates (3 in human and 4 in animal cluster) was involved in the TB pathway (map05152), and RJtmp_000891 (pip) found only in 14 isolates from the animal cluster was involved in arginine and proline metabolism (map00330). Most of the genes dnaN, gap, icd, lpdB, proA, trpE, glgC, plcC, and Rjtmp_002367 were involved in at least 5 or more pathways but were found only in 1 or 2 isolates (Supplemental Tables S4 and S5).

IS6110 insertions and genetic significance among our study isolates

All our study isolates belonged to the animal cluster and were high-copy strains possessing more than 25 IS6110 insertions. In addition to the previously mentioned insertion regions, we also identified an IS6110 intergenic insertion between Rv1470/Rjtmp_001552 (trxA) and Rv1471/Rjtmp_001553 (trxB1) in 2 of our study isolates (KL013, KL017). TrxA was involved in seleno compound metabolism (map00450), NOD-like receptor signaling pathway (map04621), and Fluid sheer stress and atherosclerosis pathway (map05418). Another intragenic insertion within RJtmp_001745 was exclusively found in 6 of our isolates (KL012, KL013, KL017, KL115, KL036, and KL292). Strain KL012 and KL292 possess a unique insertion in the genes Rjtmp_000047 (marR transcriptional regulator) and Rjtmp_002096 (csm5), respectively and the gene csm5 was identified to be involved in 11 GOs. A total of 7 unique insertions were found in strain KL036, among which 3 were intergenic and 4 intragenic. The intragenic insertions were found in genes Rv1364/Rjtmp_001464 (carB), Rv1631/RJtmp_001705 (dephospho-coA kinase), Rv2379/RJtmp_002451 (mbtF), and Rv1609/RJtmp_001681 (anthranilate synthase component). Among these genes, carB was involved in almost 50 GOs and mbtF is involved in the Biosynthesis of siderophore group non-ribosomal peptides (map01053). Intergenic insertions were found between Rv1187/Rjtmp_001253 (rocA/pruA) & Rv1188/Rjtmp_001254 (proline dehydrogenase/putA), Rv1523/RJtmp_001610 (methyltransferase) and Rv1524/RJtmp_001611 (glycosyltransferase), and Rv3800/RJtmp_003912 (pks13) and Rv3801/RJtmp_003913 (fadD32). Among these genes, pruA and putA are involved in arginine & proline metabolism along with the pip gene and fadD32 is involved in nearly 47 different GOs.

Discussion

The fact that members of the MTBC exhibit non-specific host preferences have now been well established, as demonstrated by the isolation of M. tuberculosis from cattle and the identification of M. bovis and M. orygis in humans. The concept of host tropism within MTBC species reflects the interplay between host-driven and pathogen-driven processes, where variations in the host immune response and alterations in MTBC phenotypes contribute to differences in infection, persistence, disease development, and transmission. ²⁹ The adaptation of the pathogen to the host is a highly complex process, as the mechanisms underlying the transition from latent TB infection (LTBI) to active TB disease are still not fully understood. In this context, the MTBC species possess a distinctive insertion sequence called IS6110, which belongs to the IS3 family and plays a significant role in transposition. The transposition of IS6110 follows a copy-out-paste-in mechanism.^30-32 This implicit trait offers an outstanding chromosomal polymorphism to study TB outbreaks and its unquestionable role as a clinical epidemiological marker.

The perseverance of this insertion element in MTBC might also steer phenotypic changes and affect the fitness of the strain. Various reports claim these transposition events are instigated by external stress conditions leading to genetic variability.^33,34 IS6110 insertions are well known to disturb gene expression by intruding protein-coding genes, by facilitating recombination events resulting in deletions and inversions, or by up-regulating the expression of adjacent genes due to its presence within the promoter region.^18,33,35,36 Recently Charles et al ¹⁹ have made use of the WGS approach to study the abundance and chromosomal distribution of IS6110 copy on M. bovis genomic data of French animal field strains and stated that these insertions seem to be stable within specific genotypes over time and between host species, signifying that the transposition of IS6110 is not an evolutionary driver for modern French M. bovis strains at least over a 15-year period. The effect of IS6110 on genome function prompted us to take a deeper look at the distribution and patterns of IS6110 insertions among the recently circulating strain M. orygis, the emerging pathogen in India.

M. tuberculosis as a reference genome has been well utilized and is also well known to possess 16 IS6110 insertions and all the possible regions of insertions have been vastly explored. ³⁷ Meanwhile, in the case of M. orygis, van Ingen et al had previously identified these strains as high copy strains possessing 17 to 20 IS6110 copies. However, the exact regions of insertions remained unknown. ⁹ Here, we identified 25 IS6110 insertions in the reference strain and any additional insertions other than the ones found in the reference were identified as novel insertions.

We found that most of the strains in this study were high copy strains, but also identified strains with no copy (SRR10321152, SRR21691066, SRR21842108) and low copy (SRR10321141, SRR10321142, SRR10321138) IS6110 insertions although insignificant. No copy and low copy strains were found only in the human cluster, whereas the animal cluster consisted of only high copy strains. Among all the isolates, KL036 possessed the highest number of IS6110 insertions with 42 insertions against CP063804 and 54 against NC000962.3. Most novel insertions were highly confined to a single strain or found in 2 or 3 strains in both human and animal clusters, indicating the potential role of IS6110 transposition.

The 25 known IS6110 were present in all the high-copy strains but were found to be localized in different positions and orientations within the same region and these insertions were mostly intergenic. Nearly all the high copy strains possessed IS6110 insertions downstream to the genes mosR, lipx, pknH, EccA5, MgtC/sapB family protein, and ExeA family protein. A few other genes include oxidoreductase, phospholipase, transposase and hypothetical proteins. We found 2 genes for each of mmpL1 (RJtmp_000420 (912 bp), RJtmp_000422 (1971 bp)), lipX (RJtmp_001232 (177 bp), RJtmp_001234 (129 bp)) and mmpL10 (RJtmp_001247 (1743 bp), RJtmp_001249 (1269 bp)) in M. orygis genome compared to Rv0402c (2877 bp), Rv1169c (303 bp), and Rv1183 (3009 bp), respectively in M.tuberculosis genome. A closer observation showed the presence of IS6110 insertion sequence RJtmp_000421, RJtmp_001233, and RJtmp_001248 between these genes, which leads to the speculation that the existence of 2 genes might be due to the transposition of the insertion element. Most novel and unique insertion sequences were found within the hypothetical protein, PPE/PE family proteins or PPE-domain-containing proteins. Few transcriptional regulators, such as marR, gntR, LuxR, and TetR had IS6110 insertions upstream of the gene but were confined to a single strain. We found no insertion sequences upstream of the previously reported genes PhoP, esxS, or ctpD. However, we found an insertion element in the upstream region of dnaN in 1 strain from the human cluster. ¹⁸

Functional annotation by Gene ontology and KEGG pathway analysis identified numerous functions and pathways in which the genes affected by IS6110 were involved. However, only 7 insertion sequences were found in all these isolates, whereas the rest were only present in a single or a couple of strains. The effect of the insertion sequence between pknH and embR is unknown. However, the genes pknH and embR are non-essential regulatory proteins found to be involved in various processes, including biological processes, cellular components and molecular function by GO analysis. Similarly, another insertion sequence existing between purU and coaD/kdtB was also observed in all the isolates, and the gene kdtB is involved in the cell wall and cell process.

In the case of the novel and unique insertions, none of the loci were found among our isolates except for Rv2357 (glyS) and Rv1368/RJtmp_001450 (lprF). The former was found in 6 isolates, 3 from each cluster including 2 study isolates (KL043 and KL036) and the latter was found in 7 isolates in the animal cluster, including 3 study isolates (KL004, KL115, and AH144). Another insertion loci within Rv0804/RJtmp_000891 (pip) was found in all 11 of our study isolates and 3 more in the animal cluster. The gene glyS known as glycyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into protein. ³⁸ The role of LprF in the tuberculosis pathway is unclear, and we speculate its possibility to display TLR2 agonist activity as most of the Lipoproteins like LprG, LprA and LpqH are well known for the same.^39-41 Similarly, in the arginine and proline metabolism, proline iminopeptidase (Pip) explicitly catalyzes the removal of N-terminal proline residues from peptides. ⁴² Gamma-glutamyl phosphate reductase (proA) catalyzes the NADPH-specific reduction of L-gamma-glutamyl phosphate into L-glutamate-5 semialdehyde, which spontaneously cyclizes to form pyrroline-5-carboxylate. ⁴³ This intermediate product of proline biosynthesis is converted into glutamate by pyrroline-5-carboxylate dehydrogenase (PruA/RocA). ⁴⁴

To the best of our knowledge, this is the first study to report the identification and localization of IS6110 insertion sites in the M. orygis genome. Some of the insertion sites identified in this study have previously been recognized as insertion hotspots of MTBC. Only a few isolates in this study had IS6110 insertions in the hotspots region like Rv1758 (cut1), Rv1777 (cyp144), Rv3183, Rv3327, Rv1371, Rv1765c.^45,46 Nearly all the isolates had an insertion element in Rv1682 and Rv1169, the hotspot regions for L5 and L6 lineages of MTBC, respectively. ⁴⁶ Gene interruption or regulation of genes in these hotspot regions might differ depending on the orientations and/or insertion sites of IS6110 in the same genomic region. Fewer studies have suggested that the location of IS6110 in the same transcriptional orientation and not more than 400 bp upstream of a gene may lead to upregulation of the gene.^6,45 Although we have also detected similar insertions in our analysis, it warrants an additional in vivo or in vitro study to assess their role in virulence and other survival traits. The study is the beginning, and we presume that the genetic changes observed due to IS6110 transposition could be partly responsible for the fitness of M. orygis in establishing infection in various hosts. At the same time, other explanations could also support this success. The sequences used in this study were limited to fewer numbers and most of the sequences obtained were from India. An in-depth analysis involving more sequences from various other geographic locations will provide a clear understanding regarding the fitness of the organism. Further studies are warranted to correlate the effect of IS6110 transposition events to the successful adaptation of the pathogen.

Conclusion

In conclusion, our findings provide novel information about IS6110 insertions and the preferential insertion regions in the M. orygis genome. We have also elucidated the genomic impact of these insertions and their effect on various metabolic processes and pathways suggestive of their pertinence. Henceforward, as and when MTBC strains are being vastly sequenced, it is ideal to locate the IS6110 insertion sites, which will provide a better understanding regarding its biological role in the organism’s adaptation, evolution, and fitness.