Coronaviruses, first identified in the mid-1960s, are a group of RNA viruses that causes respiratory illness in mammals and birds; these constitute the subfamily Orthocoronavirinae in the family Coronaviridae [1,2]. They have club-shaped spikes projecting from their surface, so the name has been derived from the Latin word "Corona", meaning crown. The term was first coined by June Almeida and David Tyrrell, who first observed and studied human coronaviruses. Coronavirus was accepted as a genus name in 1971 [3]. As the number of new species increased, the genus was split into four genera: Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus. The seven commonly found coronaviruses, where the host is Homo sapiens are: 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), and HKU1 (beta coronavirus); the rest uncommon viruses found are MERS-CoV (Middle East respiratory syndrome coronavirus; the beta coronavirus that caused Middle East respiratory syndrome or MERS), SARS-CoV (severe acute respiratory syndrome coronavirus; the beta coronavirus that caused severe acute respiratory syndrome or SARS), and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2; the novel coronavirus that causes coronavirus disease 2019 or coronavirus disease 2019 [COVID-19]). People commonly get infected with human coronaviruses 229E, NL63, OC43, and HKU1. Although significant genetic diversity of coronaviruses was detected from Shenzhen in Mainland China and Hong Kong ports, all the strains had a high homology compared with the published strains; several novel mutations, including nucleotides substitution and the insertion of the spike of the glycoprotein on the viral surface, were discovered [4]. Sometimes coronaviruses that infect animals can evolve and make people sick and become a new human coronavirus. Three recent examples of these are SARS-CoV-2, SARS-CoV, and MERS-CoV. SARS-CoV-2 is a strain of coronavirus that causes COVID-19 and is responsible for respiratory illness in human beings; this was first identified in Wuhan city of, China, in January 2020 (NCBI GenBank No. MN908947.3). Coronavirus contains a positive-sense, single-stranded RNA genome; their genome size ranges from approximately 26 to 32 kb. The treatment of coronavirus is symptomatic; the transmission can be reduced by practising hygienic measures and getting the vaccination. Currently, three major approaches are being followed for designing vaccines: the whole microbe approach, the subunit approach, and the genetic approach. The mRNA and viral vector vaccines were rapidly developed using the genetic and whole microbe approaches. Also, at least nine different technology platforms are under research and development to design an effective vaccine against COVID-19.

The viral genome research will contribute to understanding and solving numerous problems, including their origin, evolution, infection mechanism, disease treatment, etc. [5]. The origin and evolution of viruses can be better understood by investigating them at the molecular level [6-9]. Accumulation of transposable elements [10,11] and tandem repeats [12] are considered for changes in genome size. Ninety-two genome sequences of severe acute respiratory syndrome coronavirus 2 have been uncovered with the SARS-CoV-2 reference genome (NC_045512.2) [13]. The hypervariability and the hotspots of mutations in coronavirus genome sequences can be discovered by studying simple sequence repeats (SSRs).

"Microsatellites" or SSRs [14] are short tandem repeats (motifs) of lengths 1–6 nucleotides [15] and are found in the genomes of both prokaryotes and eukaryotes [16]. SSRs can be categorized as perfect [without interruptions, or we can say a continuous repeat of a single motif; (AGA) ₁₅], imperfect [with interruptions by non-repeat nucleotide or with a base pair disruption between repeats; (AGA) ₇ A (AGA) ₈] and compound [two or more SSRs are found adjacent to one another; (GTG) ₈ (AT) ₁₆] [17], also known as compound simple sequence repeats (cSSR). For a microsatellite to be categorized as a compound, the maximum permissible distance between two adjacent microsatellites is known as dMAX [18]. The dMAX value can be set only from 0 to 50 for IMEx [19]. These are present in the genome's coding and non-coding regions [20,21]. The SSRs found in the coding region affect gene activation, resulting in protein expression and lesser polymorphism in the coding part [22]. SSRs present in the non-coding area affects gene regulation [23]. These repeats may be generated due to the slippage mechanism during replication [24]. These microsatellites promote the development of markers widely used by researchers in DNA‐based genetic analyses for the past 25 years, which show locus specificity, high reproducibility, co-dominance inheritance and hypervariability [25]. The flanking sequences of SSRs help select polymerase chain reaction primers that amplify the repeat sequence [26]. SSRs are essential in studying genetic variation, gene tagging, linkage mapping [27-29], and evolutionary studies [30]. Many researchers have reported the involvement of SSRs in transcription, translation, regulation of promoters [31,32] and certain neurodegenerative diseases [33].

Due to the importance of microsatellite applications in genomic research, various studies have been made to identify and characterise them in the laboratory. However, developing microsatellite markers in vitro is intensive and time-consuming [34]. The increasing availability of next-generation sequencing tools and genome sequences of various organisms in biological databases are providing a simple, fast and inexpensive way for in silico mining of SSRs [35].

In the present study, seven complete genome sequences of human coronavirus were mined and analyzed for perfect and compound SSRs occurrence and abundance by an in-house Java pipeline. Similar strains with sequence identity above 99.97% from different regions indicative of very recent emergence were not considered.

Because of the pandemic outbreak and loss to human health and the economy, it is essential to explore the virus genome to study and analyze the SSRs pattern to help establish an evolutionary relationship, genetic diversity, and genetic similarity/dissimilarity. Furthermore, analysis of perfect repeats would also help study the polymorphic nature and suitability for marker developments by using computing methods in less time and at no cost within the two genera Alphacoronvirus and Betacoronavirus.

The incidence of SSRs and cSSR distribution exhibits a similar pattern as reported in earlier studies in genomes of the Filoviridae family [34]. Of the total SSRs identified, dinucleotide repeat motifs (51 to 73) were predominant as found in Flavivirus genomes, and Mycobacteriophage genomes of the Siphoviridae family [35,36] may be unstable due to higher slippages rate [37]. The presence of poly (T/A) is in line with the prokaryotic and eukaryotic genomes having abundant poly (T/A) tracts [14,39]. Mononucleotide A was plentiful, and in plant viroids, it tends to form loops in secondary structure and a possibly higher number of repeats, making it more difficult to form stable base pairs [40]. The cSSR percentage increases with dMAX size nonlinearly; this conversion of SSRs to cSSR in approximate similar size genomes suggests a differential role of repeat sequences [39]. At least one cSSR in each human coronavirus genome may be responsible for variation and evolution [41]. Di and trinucleotide repeats were mainly present in the coding region [39,42]. As far as motif types and their distribution in the coding and non-coding area is concerned, the reference sequence of SARS-CoV-2 (accession No. NC_045512.2) is close to the SARS virus genome (accession No. NC_004718.3), levels of the genetic relationship were also suggested among Bat coronavirus RaTG13 and the prototype strain of SARS-CoV-2 [43].

SSRs are insignificantly but positively correlated to genome size; the longer the genome size, the greater the number of SSRs [5,12,44] and repeat length. The study of the relationship between genome size and tandem repeat length in CoV HKU1 strains, a beta coronavirus, also provides evidence of a similar pattern to our findings [45].

The Insignificant correlation between genome size to relative abundance and density has also been found in the case of Escherichia coli and human Immunodeficiency virus type 1 (HIV-1) genome. These results reflect a slight effect of genome size on the relative abundance and density of SSRs in viral genomes [5,46]. As observed, the negative correlation of GC content with SSRs, relative abundance, relative density, and cSSR was also reported [39,41,42].

A literature survey observed that limited research had been done on identifying and analysing microsatellites in virus genomes. The study conducted on eukaryotic and prokaryotic genome sequences, including in-depth analysis of Flavivirus, Dengue virus, HIV, plant viroids, Ebolavirus, Filoviridae, and Siphoviridae family genomes, revealed the role of genome size in accumulation of numbers and length of SSRs also to particular extent host are also found responsible for variances as they may participate in recombination and integration [47,48]. An increase in SSRs numbers may be due to the combination of partial sequences of the host during the infection [44]. All parameters under study were relevant and matched with previous research [5]. The maximum primers were designed in MERS-CoV/THA/CU/17_06_2015 with accession No. KT225476.2 followed by accession No. NC_004718.3 which is a SARS-CoV and least in human coronavirus HKU1 with accession No. NC_006577.2. Overall, in our study, it has been observed that HKU1 is showing a slightly different pattern in SSRs and cSSR abundance per kb and consequently in relative abundance, density and GC % content; such a pattern has also been highlighted in earlier studies in screening microsatellites in 55 Coronaviridae genomes [38], and it is among the top four strains found to be infecting human beings.

This study revealed the microsatellite identification, distribution, and analysis in seven genomic sequences of human coronavirus strains, including the reference sequence of SARS-CoV-2. From computational and statistical data, it was observed that the greater the genome size more is the SSRs number/length of repeats. The presence of a minimum of one compound SSRs, poly T and A mononucleotides, and abundant presence of AT/TA dinucleotides may be responsible for variation, instability, and evolution of the genome. It may contribute to understanding the genetic diversity and polymorphic nature of the genomes among alpha and beta-coronavirus genera. However, further study can elaborate on the mutable hotspots.