Microsatellite Length Research Articles

Detecting microsatellite instability by length comparison of microsatellites in the 3' untranslated region with RNA-seq.

Microsatellite instability (MSI), a phenomenon caused by deoxyribonucleic acid (DNA) mismatch repair system deficiencies, is an important biomarker in cancer research and clinical diagnostics. MSI detection often involves next-generation sequencing data, with many studies focusing on DNA. Here, we introduce a novel approach by measuring microsatellite lengths directly from ribonucleic acid sequencing (RNA-seq) data and comparing its distribution to detect MSI. Our findings reveal distinct instability patterns between MSI-high (MSI-H) and microsatellite stable samples, indicating the efficacy of RNA-based MSI detection. Additionally, microsatellites in the 3'-untranslated regions showed the greatest predictive value for MSI detection. Notably, this efficacy extends to detecting MSI-H samples even in tumors not commonly associated with MSI. Our approach highlights the utility of RNA-seq data in MSI detection, facilitating more precise diagnostics through the integration of various biological data.

One-instrument, objective microsatellite instability analysis using high-resolution melt.

In recent years, immune checkpoint inhibitors have proved immense clinical progression in the treatment of certain cancers. The efficacy of immune checkpoint inhibitors is correlated with mismatch repair system deficiency and is exceptionally administered based exclusively on this biological mechanism independent of the cancer type. The promising effect of immune checkpoint inhibitors has left an increasing demand for analytical tools evaluating the mismatch repair status. The analysis of microsatellite instability (MSI), reflecting an indirect but objective manner the inactivation of the mismatch repair system, plays several roles in clinical practice and, therefore, its evaluation is of high relevance. Analysis of MSI by PCR followed by fragment analysis on capillary electrophoresis remains the gold standard method for detection of a deficient mismatch repair system and thereby treatment with immune checkpoint inhibitors. Novel technologies have been applied and concepts such as tumor mutation burden have been introduced. However, to date, most of these technologies require high costs or the need of matched non-tumor tissue as internal comparator. In this study, we present a novel, one-instrument, fast, and objective method for the detection of MSI (MicroSight® MSI 1-step HRM Analysis), which does not depend on the use of matched non-tumor tissue. The assay analyzes five well-described mononucleotide microsatellite sequences by real-time PCR followed by high-resolution melt and evaluates microsatellite length variations via PCR product melting profiles. The assay was evaluated using two different patient cohorts and evaluation included several DNA extraction methodologies, two different PCR platforms, and an inter-laboratory ring study. The MicroSight® MSI assay showed a high repeatability regardless of DNA extraction method and PCR platform, and a 100% agreement of the MSI status with PCR fragment analysis methods applied as clinical comparator.

Abstract 2943: Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS

Abstract MSI measurements are typically made on DNA, but it would be useful to make similar measurements from RNA or total nucleic acid inputs (TNA), so that MSI determinations could be made in parallel to interrogation of the RNA. Doing so requires characterizations of microsatellite lengths from those which are transcribed and untranscribed, because both will be detected following cDNA creation. Microsatellite instability (MSI) occurs when small unit repeats of DNA undergo incorrect replication resulting in the growth or contraction of the number of repeats in the newly synthesized DNA. This accumulation of errors at microsatellite DNA is usually mitigated by the mismatch repair (MMR) pathway which can correct these errors. MSI is of interest because it serves as a phenotypic readout of the status of the MMR machinery, and MSI status can correlate to tumor progression and response to agents that regulate these cellular mechanisms. We investigated the status of microsatellites using Anchored Multiplex PCR (AMP™) targeted panels (RUO) and compared results from TNA inputs using an AMP MSI module and both VARIANTPlex™ chemistry to interrogate DNA, and FUSIONPlex™ chemistry to interrogate both RNA + DNA. Our results indicate differences in the diversity of lengths of microsatellites present in RNA, and the resulting stability calls which would be made at those sites. Microsatellite length diversity was highly dependent on the genomic context of the microsatellite. Microsatellites located within transcripts showed increased length diversity when examining the RNA + DNA results compared to DNA alone. Microsatellites located in intergenic regions, which are unlikely to be transcribed, showed no increase in diversity of lengths when comparing the RNA + DNA results to DNA alone. While these results suggest that MSI determinations could be made from RNA or RNA + DNA NGS data, the unique behavior of microsatellites in RNA is likely to require additional investigations and unique data analysis techniques to correctly categorize microsatellites as stable or unstable. Confidential - Company Proprietary Citation Format: Ryan Rogge, Taylor Patterson, Alicia Navetta, Dhruti Legare. Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2943.

Nosocomial transmission of Aspergillus flavus in a neonatal intensive care unit: Long-term persistence in environment and interest of MALDI-ToF mass-spectrometry coupled with convolutional neural network for rapid clone recognition.

Aspergillosis of the newborn remains a rare but severe disease. We report four cases of primary cutaneous Aspergillus flavus infections in premature newborns linked to incubators contamination by putative clonal strains. Our objective was to evaluate the ability of matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) coupled to convolutional neural network (CNN) for clone recognition in a context where only a very small number of strains are available for machine learning. Clinical and environmental A. flavus isolates (n=64) were studied, 15 were epidemiologically related to the four cases. All strains were typed using microsatellite length polymorphism. We found a common genotype for 9/15 related strains. The isolates of this common genotype were selected to obtain a training dataset (6 clonal isolates/25 non-clonal) and a test dataset (3 clonal isolates/31 non-clonal), and spectra were analysed with a simple CNN model. On the test dataset using CNN model, all 31 non-clonal isolates were correctly classified, 2/3 clonal isolates were unambiguously correctly classified, whereas the third strain was undetermined (i.e., the CNN model was unable to discriminate between GT8 and non-GT8). Clonal strains of A. flavus have persisted in the neonatal intensive care unit for several years. Indeed, two strains of A. flavus isolated from incubators in September 2007 are identical to the strain responsible for the second case that occurred 3 years later. MALDI-TOF is a promising tool for detecting clonal isolates of A. flavus using CNN even with a limited training set for limited cost and handling time.

Analysis of Microsatellite Length Polymorphism for Clinical Isolates of Candida albicans from Animals

Background: Candida albicans has been shown as the most common species of Candida collected from different animals. Objectives: This study aimed to evaluate the genetic diversity and genetic relationships among C. albicans isolates collected from clinical specimens of animals suffering from candidiasis using microsatellite length polymorphism (MLP). Methods: We used MLP for a group of 60 C. albicans strains isolated from various animal species (dog: 16, cat: 10, horse: 10, cow: 14, chicken: 10), previously defined as animal clinical isolates. Three loci, including EF3, CDC3, and HIS3, were amplified, and the products ran onto an ABI XL 370 genetic analyzer, and fragment sizes were determined. Results: Of the 60 clinical strains illustrated, 49 different genotypes were identified with a discriminatory power index of 0.991. A total of 17 alleles and 26 different combinations were identified for EF3 locus, six alleles and 13 combinations for CDC3 locus, and 17 alleles and 27 combinations for HIS3 locus. The most common genotypes were GP9 (four strains) and GP1 and GP33 (three strains). Wright’s fixation index (FST) values were calculated to assess inter-group genetic diversity for all pairwise combinations of the five sub-populations of C. albicans isolated from the different animal hosts. The highest FST values related to C. albicans isolated from chicken to three sub-populations of cats (FST: 0.1397), cows (FST: 0.0639), and horses (FST: 0.0585). Conclusions: The results indicated a moderate genetic differentiation (0.05 < FST < 0.15) between C. albicans strains isolated from cats, cows, and horses as a mammal vs. chickens.

Enzymatic and antifungal susceptibility profiles of Candida glabrata isolates from paediatric patients and their genetic diversity based on microsatellite length polymorphism.

This study aimed to detect different genotypes of Candida glabrata isolates in paediatric patients with and without neutropenia utilizing microsatellite length polymorphism (MLP) and its correlation with drug resistance and enzymatic activity were assessed. Samples from neutropenic and non-neutropenic patients were collected from November 2020 to November 2021. Thirty-six C. glabrata strains were isolated and identified using classical and molecular methods. Then, C. glabrata isolates were genotyped by the MLP technique, and their antifungal susceptibility was performed based on the CLSI M27 guideline. Eighteen different multi-loci genotypes (G1-G18) were detected based on MLP analysis. Analysis of molecular variance revealed high genetic variation within populations (94%) and low genetic differentiation amongst populations (6%). Also, 40% (n = 4) of isolates from neutropenic patients were non-wild-type for posaconazole, and 30% (n = 3) were resistant to caspofungin. Very strong hemolytic and proteinase activity were seen in 97·2 and 86·1% of isolates. Candida glabrata strains from neutropenic patients were genetically divergent from other populations. The minimum spanning tree shows that observed genotypes were mainly related to previously reported genotypes from Iran, Spain, and China.

Accurate measurement of microsatellite length by disrupting its tandem repeat structure.

Tandem repeats of simple sequence motifs, also known as microsatellites, are abundant in the genome. Because their repeat structure makes replication error-prone, variant microsatellite lengths are often generated during germline and other somatic expansions. As such, microsatellite length variations can serve as markers for cancer. However, accurate error-free measurement of microsatellite lengths is difficult with current methods precisely because of this high error rate during amplification. We have solved this problem by using partial mutagenesis to disrupt enough of the repeat structure of initial templates so that their sequence lengths replicate faithfully. In this work, we use bisulfite mutagenesis to convert a C to a U, later read as T. Compared to untreated templates, we achieve three orders of magnitude reduction in the error rate per round of replication. By requiring agreement from two independent first copies of an initial template, we reach error rates below one in a million. We apply this method to a thousand microsatellite loci from the human genome, revealing microsatellite length distributions not observable without mutagenesis.

Orthologous microsatellites, transposable elements, and DNA deletions correlate with generation time and body mass in neoavian birds.

The rate of mutation accumulation in germline cells can be affected by cell replication and/or DNA damage, which are further related to life history traits such as generation time and body mass. Leveraging the existing datasets of 233 neoavian bird species, here, we investigated whether generation time and body mass contribute to the interspecific variation of orthologous microsatellite length, transposable element (TE) length, and deletion length and how these genomic attributes affect genome sizes. In nonpasserines, we found that generation time is correlated to both orthologous microsatellite length and TE length, and body mass is negatively correlated to DNA deletions. These patterns are less pronounced in passerines. In all species, we found that DNA deletions relate to genome size similarly as TE length, suggesting a role of body mass dynamics in genome evolution. Our results indicate that generation time and body mass shape the evolution of genomic attributes in neoavian birds.

Single-Cell Mononucleotide Microsatellite Analysis Reveals Differential Insertion-Deletion Dynamics in Mouse T Cells.

Microsatellite sequences are particularly prone to slippage during DNA replication, forming insertion-deletion loops that, if left unrepaired, result in de novo mutations (expansions or contractions of the repeat array). Mismatch repair (MMR) is a critical DNA repair mechanism that corrects these insertion-deletion loops, thereby maintaining microsatellite stability. MMR deficiency gives rise to the molecular phenotype known as microsatellite instability (MSI). By sequencing MMR-proficient and -deficient (Mlh1 +/+ and Mlh1 −/− ) single-cell exomes from mouse T cells, we reveal here several previously unrecognized features of in vivo MSI. Specifically, mutational dynamics of insertions and deletions were different on multiple levels. Factors that associated with propensity of mononucleotide microsatellites to insertions versus deletions were: microsatellite length, nucleotide composition of the mononucleotide tract, gene length and transcriptional status, as well replication timing. Here, we show on a single-cell level that deletions — the predominant MSI type in MMR-deficient cells — are preferentially associated with longer A/T tracts, long or transcribed genes and later-replicating genes.

GGAA-Microsatellites of NR0B1 Promoter Region in Ewing’s Sarcoma Patients and Healthy Individuals of a Southern Brazilian Population

Introduction: The very aggressive soft tissue and bone pediatric tumor Ewing’s sarcoma (ES) is caused in most cases by the chromosomal translocation t(11;22)(q24;q12), which encodes an aberrant chimeric transcription factor (EWS-FLI1) that regulates target genes, including the critical oncogene NR0B1 (Xp21.2), via GGAA microsatellites. Objective: Our objective was to analyze the GGAA microsatellites of NR0B1 promoter region of ES patients and healthy subjects in our population. Methodology: Ten male ES patients and 71 adult healthy males from Rio Grande do Sul state, Brazil, were included in this study. Leukocyte DNA was extracted, amplified by PCR, sequenced by the Sanger method and analyzed by capillary electrophoresis. Total number of GGAA motifs, length of microsatellite in base pairs, number of segments separated by "A" insertions and greatest number of consecutive GGAA motifs were analyzed. Statistical analyses were performed in the SPSS® statistical software and p-value <0,05 was considered significant. Results: A total of 21 different alleles was identified in the 81 subjects, with 24.2 allele [(GGAA)7A(GGAA)7A(GGAA)10 sequence] being the most frequent, but when comparing the data between the two groups, no significant difference was found. Conclusion: We concluded that the studied sample was highly variable in terms microsatellite structure, including the presence of rare alleles, giving us the opportunity to describe our population which is a fundamental step on identifying genetic implications in ES tumorigenesis.

Tumor board presentation of a woman with metastatic, hormone receptor‐positive, mismatch repair‐deficient endometrial cancer

Tumor board presentation of a woman with metastatic, hormone receptor‐positive, mismatch repair‐deficient endometrial cancer

Genetic discrimination of wild versus farmed gilthead sea bream Sparus aurata using microsatellite markers associated with candidate genes

Farm escapees and their offspring impose a significant impact on the environment and may therefore alter the future evolutionary trajectories of wild populations. To date, there is no management plan in place in Mediterranean countries to prevent fish escapes. Here, we investigate microsatellite length variations in three candidate genes, including prolactin (PRL), growth hormone (GH), and the receptor activity modifying protein 3 gene (RAMP3), to study the genetic structure of the main fish species farmed in the Mediterranean, gilthead seabream (Sparus aurata). We also evaluate the performance of microsatellites in discriminating fish origin (wild or farmed). Results from 298 individuals, including farmed, wild adult and juvenile fish were compared with results from 19 neutral markers used in a previous study. All loci were polymorphic, selectively neutral, and had the statistical power to detect significant population differentiation. Global FST was similar to that estimated using 19 loci (0.019 and 0.023, respectively), while pairwise comparisons identified farmed populations as the main drivers of genetic divergence, with a much higher magnitude of overall genetic differentiation within farmed populations (0.076) than that estimated using the 19 neutral microsatellite loci (0.041). Bayesian structural analysis showed that the PRL, GH, and RAMP3 markers were able to distinguish farmed from wild populations, but were not able to distinguish different wild groups as 19 neutral microsatellite markers did. Farmed populations of different origins were assigned to a separate cluster with a high individual assignment score (>88%). It appears that the candidate markers are more influenced by artificial selection compared to neutral markers. Further validation of their efficiency in discriminating wild, farmed, and mixed fish origins using a more robust sample size is needed to ensure their potential use in an escaped fish monitoring programme.

Evidence for association of vasopressin receptor 1A promoter region repeat with childhood onset aggression

ObjectiveChildhood onset aggression can cause major suffering to affected families and is associated with many negative outcomes in the child's later life, including poor academic performance, adolescent delinquency, drug abuse, depression and antisocial personality disorder. Currently available prevention and intervention strategies have limited efficacy, but a better understanding of underlying genetic and neurobiological factors can lead to more effective prevention and treatment strategies, through genetic screening programs and novel therapies. MethodThis study examined the RS1 (n = 299 aggression, n = 192 controls) and RS3 (n = 291 aggression, n = 189 controls) microsatellite repeats within the promoter region of the vasopressin receptor 1A gene (AVPR1A) and their association with extreme childhood aggression, as assessed by the Child Behavior Checklist (CBCL), as well as the Teacher Report Form (TRF) and Youth Self Report (YSR). Binary logistic regression was used to model the relationship between microsatellite length and childhood aggression. Age and sex were used as covariates. ResultsLogistic regression revealed a nominally significant association between one specific RS3 repeat and non-aggressive status. No association was found for any of the RS1 repeats. In a separate model, grouping repeats into short and long, carriers of long RS3 repeats were nominally significantly associated with non-aggressive status. ConclusionsThese findings suggest a role for AVPR1A and its RS3 microsatellite in extreme childhood aggression and could lead to a better understanding of the biological pathways of aggressive behavior. However, independent replication and further research into the functionality of studied genetic variants is required.

MSIFinder: A python package for detecting MSI status using random forest classifier.

2601 Background: Microsatellite instability (MSI) is a common genomic alteration in several tumors, such as colorectal cancer, endometrial carcinoma, and stomach, which is characterized as microsatellite instability-high (MSI-H) and microsatellite stable (MSS) based on a high degree of polymorphism in microsatellite lengths. MSI is a predictive biomarker for immunotherapy efficacy in advanced/metastatic solid tumors, especially in colorectal cancer (CRC) patients. Several computational approaches based on target panel sequencing data have been used to detect MSI; However, they are considerably affected by the sequencing depth and panel size. Methods: We developed MSIFinder, a python package for automatic MSI classification, using random forest classifier (RFC)-based genome sequencing, which is a machine learning technology. We included 19 MSI-H and 25 MSS samples as training sets. First, RFC model were built by 54 feature markers from the training sets. Second. The software was validated the classifier using a test set comprising 21 MSI-H and 379 MSS samples. Results: With this test set, MSIFinder achieved a sensitivity (recall) of 0.997, a specificity of 1, an accuracy of 0.998, a positive predictive value (PPV) of 0.954, an F1 score of 0.977, and an area under curve (AUC) of 0.999. We discovered that MSIFinder is less affected by low sequencing depth and can achieve a concordance of 0.993, while exhibiting a sequencing depth of 100×. Furthermore, we realized that MSIFinder is less affected by the panel size and can achieve a concordance of 0.99 when the panel size is 0.5 m (million base). Conclusions: These results indicated that MSIFinder is a robust MSI classification tool and not affected by the panel size and sequencing depth. Furthermore, MSIFinder can provide reliable MSI detection for scientific and clinical purposes.[Table: see text]

Genetic Analysis of Candida glabrata from Candiduric Patients Using Microsatellite Length Polymorphism, Antifungal Susceptibility, and Enzymatic Profiles

Background: Candida glabrata is the second agent of candiduria with increased resistance to antifungals. Microsatellite length polymorphism (MLP) is one of the genotyping techniques used in the epidemiological investigation to improve clinical management. Objectives: We aimed to detect different genotypes of C. glabrata isolates using six microsatellite markers and the MLP technique. Moreover, our genotypes' association with other countries' genotypes was illustrated using a minimum spanning tree. We investigated in vitro antifungal susceptibility and enzymatic activity profiles of the isolates. Methods: Six microsatellite markers were amplified using multiplex-PCR for 22 C. glabrata strains isolated from urine in pediatric patients admitted to the Abuzar Children's Hospital in Ahvaz, Iran. The PCR products were presented for fragment analysis, and the size of the alleles was determined. Antifungal susceptibility tests and extracellular enzyme activities were also performed. Results: Nineteen multilocus genotypes were detected so that 22.7% of the strains had identical genotypes. The isolates were wild-type for amphotericin B (0.0625 - 2 µg/mL), itraconazole (0.125 - 2 µg/mL), and voriconazole (0.0078 - 0.00625 µg/mL). All the isolates were sensitive to fluconazole at the minimum inhibitory concentration (MIC) range (0.0312 - 16 μg/mL), and three of them were resistant to caspofungin (MIC ≥ 0.5 μg/mL). Moreover, 72.7 and 68.2% of the isolates had no phospholipase and esterase activities. The highest potency of enzymatic activity was obtained in hemolysin and proteinase enzymes. A high genetic diversity (19 genotypes of the 22 isolates) existed among the urinary C. glabrata isolates. Based on the minimum spanning tree, two clusters of our genotypes were related to C. glabrata genotypes in a previous study in Iran, and the third cluster was entirely connected with Chinese genotypes. Conclusions: Most of the isolates were the non-wild type for posaconazole but were rarely resistant to other antifungals. Hemolysin and proteinase secreted as the main virulence factors among the urinary C. glabrata isolates.

MSIFinder: a python package for detecting MSI status using random forest classifier

BackgroundMicrosatellite instability (MSI) is a common genomic alteration in colorectal cancer, endometrial carcinoma, and other solid tumors. MSI is characterized by a high degree of polymorphism in microsatellite lengths owing to the deficiency in the mismatch repair system. Based on the degree, MSI can be classified as microsatellite instability-high (MSI-H) and microsatellite stable (MSS). MSI is a predictive biomarker for immunotherapy efficacy in advanced/metastatic solid tumors, especially in colorectal cancer patients. Several computational approaches based on target panel sequencing data have been used to detect MSI; however, they are considerably affected by the sequencing depth and panel size.ResultsWe developed MSIFinder, a python package for automatic MSI classification, using random forest classifier (RFC)-based genome sequencing, which is a machine learning technology. We included 19 MSI-H and 25 MSS samples as training sets. First, we selected 54 feature markers from the training sets, built an RFC model, and validated the classifier using a test set comprising 21 MSI-H and 379 MSS samples. With this test set, MSIFinder achieved a sensitivity (recall) of 1.0, a specificity of 0.997, an accuracy of 0.998, a positive predictive value of 0.954, an F1 score of 0.977, and an area under the curve of 0.999. To further verify the robustness and effectiveness of the model, we used a prospective cohort consisting of 18 MSI-H samples and 122 MSS samples. MSIFinder achieved a sensitivity (recall) of 1.0 and a specificity of 1.0. We discovered that MSIFinder is less affected by a low sequencing depth and can achieve a concordance of 0.993 while exhibiting a sequencing depth of 100×. Furthermore, we realized that MSIFinder is less affected by the panel size and can achieve a concordance of 0.99 when the panel size is 0.5 M (million bases).ConclusionThese results indicate that MSIFinder is a robust and effective MSI classification tool that can provide reliable MSI detection for scientific and clinical purposes.

Occurrence and distribution of compound microsatellites in the genomes of three economically important virus families

Microsatellites are nonrandom hypervariable iterations of one to six nucleotides, existing across the coding as well as noncoding regions of virtually all known genomes, arising primarily due to polymerase slippage and unequal crossing over during replication events. Two or more perfect microsatellites located in close proximity form compound microsatellites. We studied the distribution of compound microsatellites in 118 ssDNA virus genomes belonging to three economically important virus families, namely Anelloviridae, Circoviridae, and Parvoviridae, known to predominantly infect livestock and humans. Among these virus families, 0-58.49% of perfect microsatellites were involved in the formation of compound microsatellites, the majority being located in the coding regions. No clear relationship existed between the genomic features (genome size and GC%) and compound microsatellite characteristics (relative abundance and relative density). The majority of the compound microsatellites resulted from di-SSR couples. A strong positive relationship was observed between the maximum distance value and length of compound microsatellite, percentage of microsatellites involved in the compound microsatellite formation, and relative microsatellite density. The degree of variability among microsatellite characteristics studied was largely a species-specific phenomenon. A major proportion of compound microsatellites was represented by similar motif combinations. The findings of the present study will help in better understanding of the structural, functional, and evolutionary role of compound microsatellites prevailing in the smaller genomes.

Bioinformatics analysis of microsatellite sites in the RNA-sequencing of <italic>Meretrix meretrix</italic>

<italic>Meretrix meretrix</italic> is one of the most important economic shellfish species in China due to rich nutrition, delicious meat and high economic value. In this study, <italic>Meretrix meretrix</italic> DNA was transcribed and sequenced by using the second generation high-throughput sequencing technique. Through the statistical analysis of different types of microsatellites in <italic>Meretrix meretrix</italic>, the distribution frequency and number of microsatellites were understood, and the SSR composition characteristics of <italic>Meretrix meretrix</italic> were clarified at the transcriptome level. It is beneficial to the research of individual identification, germplasm resource identification, biological genetic mapping and so on, and has important reference value for further using microsatellite marker technology to carry out genetic marker-assisted breeding and population genetics research. The bioinformatics method was used to analyze the distribution of microsatellite sequences in the transcriptome of <italic>Meretrix meretrix</italic>. After searching the SSR loci of <italic>Meretrix meretrix</italic>, 8 856 complete SSR loci were found. The total length of SSR was 162 416 bp, with an average length of 18.34 bp, accounting for about 0.15% of the total sequence size, including 3 001 single-base repeat sequences, 2 254 three-base repeat sequences, 2 200 four-base repeats sequence, 1 052 two-base repeats sequences, 332 five-base repeat sequences and 13 six-base repeat sequences. The SSR of <italic>Meretrix meretrix</italic> transcriptome contained 26 types of repeats, among which the most was single-base repeat sequence A / T (2 809). The second most was triple-base repeat sequence AAC / TTG (907). Four-base repeat sequence AAAC / TTTG and two-base repeat sequence AT / TA was 588 and 561, respectively, indicating that the distribution of microsatellites in the RNA-sequencing of <italic>Meretrix meretrix</italic> had a preference for A / T. The average length of SSR (integrity) of <italic>Meretrix meretrix</italic> was 18.34 bp, and the main length of microsatellite was 12-20 bp, accounting for 41%. Most of the 8 856 SSR loci in the transcriptome had moderate polymorphism potential, which meant certain value for the future development of microsatellite molecular markers. The results of this study will provide a theoretical basis for the development of microsatellite markers, population genetic diversity, genetic linkage map, identification of germplasm resources and molecular genetics and breeding of shellfish. It also has important reference value for further study of genetic marker-assisted breeding and population genetics by using microsatellite marker technology.

Characterization of Clonal Evolution in Microsatellite Unstable Metastatic Cancers through Multiregional Tumor Sequencing.

Microsatellites are short, repetitive segments of DNA, which are dysregulated in mismatch repair-deficient (MMRd) tumors resulting in microsatellite instability (MSI). MSI has been identified in many human cancer types with varying incidence, and microsatellite instability-high (MSI-H) tumors often exhibit increased sensitivity to immune-enhancing therapies such as PD-1/PD-L1 inhibition. Next-generation sequencing (NGS) has permitted advancements in MSI detection, and recent computational advances have enabled characterization of tumor heterogeneity via NGS. However, the evolution and heterogeneity of microsatellite changes in MSI-positive tumors remains poorly described. We determined MSI status in 6 patients using our previously published algorithm, MANTIS, and inferred subclonal composition and phylogeny with Canopy and SuperFreq. We developed a simulated annealing-based method to characterize microsatellite length distributions in specific subclones and assessed the evolution of MSI in the context of tumor heterogeneity. We identified three to eight tumor subclones per patient, and each subclone exhibited MMRd-associated base substitution signatures. We noted that microsatellites tend to shorten over time, and that MMRd fosters heterogeneity by introducing novel mutations throughout the disease course. Some microsatellites are altered among all subclones in a patient, whereas other loci are only altered in particular subclones corresponding to subclonal phylogenetic relationships. Overall, our results indicate that MMRd is a substantial driver of heterogeneity, leading to both MSI and subclonal divergence. IMPLICATIONS: We leveraged subclonal inference to assess clonal evolution based on somatic mutations and microsatellites, which provides insight into MMRd as a dynamic mutagenic process in MSI-H malignancies.

Genetic diversity assessed using PFGE, MLP and MLST in Candida spp. candidemia isolates obtained from a Brazilian hospital.

Candida spp. are the main causative agents of invasive fungal infections in immunocompromised patients. Candidemia has attributable mortality rates of 15 to 35% and increases hospitalisation time and costs, thus making this disease a public health concern. This study aimed to use pulsed-field gel electrophoresis (PFGE), microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST) to analyse the genetic relationships among 65 Candida spp. bloodstream isolates, including 35 Candida albicans, 15 Candida glabrata and 15 Candida tropicalis isolates, all of which were obtained from patients in a Brazilian hospital. Moreover, patient clinical data were assessed. All techniques resulted in high discriminatory indexes. C. albicans and C. tropicalis isolates showed high genetic variability, while C. glabrata isolates had relatively low genetic variability. Moreover, a cluster of C. glabrata isolates was identified in a hospital unit. New MLST sequence types, diploid sequence types and alleles are described. Relationships were not observed between the molecular typing results and clinical characteristics. The molecular typing of clinical strains increases our understanding of candidemia epidemiology and promotes the development of strategies that can reduce the incidence of this disease. Moreover, this study is the first to combine these techniques to genotype these three species in Brazil.