Detection Of Short Tandem Repeats Research Articles

Microprobes for Label-Free Detection of Short Tandem Repeats: An Insight into Alleviating Secondary Structure Effects.

Overgrowth of short tandem repeat sequences in our genes can cause various neurodegenerative disorders. Such repeat sequences are ideal targets for the label-free electrochemical detection of such potential expansions. However, their length- and sequence-dependent secondary structures may interfere with the interfacial charge transfer of a detection platform, making them complex targets. In addition, the gene contains sporadic repeats that may result in false-positive signals. Therefore, it is necessary to design a platform capable of mitigating these effects and ultimately enhancing the specificity of tandem repeats. Here, we analyzed three different backbones of nucleic acid microprobes [DNA, peptide nucleic acid, and lock-nucleic acid (LNA)] to detect in vitro transcribed RNA carrying CAG repeats, which are associated with Huntington's disease, based on the charge-transfer resistance of the interface. We found that the LNA microprobe can distinguish lengths down to the attomolar concentration level and alleviate the effect of secondary structures and sporadic repeats in the sequence, thus distinguishing the "tandem repeats" specifically. Additionally, the control experiments conducted with and without Mg2+ demonstrated the LNA microprobe to perform better in the presence of the divalent cation. The results suggest that the LNA-based platform may eventually lead to the development of a reliable and straightforward biosensor for genetic neurodegenerative disorders.

Abstract 2081: Nano2NGS: A framework for converting nanopore sequencing data to NGS-liked sequencing data for data analysis

Abstract Background: Nanopore sequencing, also known as single molecule real-time sequencing, enables deciphering single DNA/RNA molecules without the polymerase chain reaction (PCR). Although nanopore sequencing has made significant progress in some areas, its application has been limited compared with next-generation sequencing (NGS) due to specific design principle and data characteristics, especially in hotspot mutation detection, metagenomic analysis, short tandem repeat (STR) detection and human leukocyte antigen (HLA) typing. Methods: The Nano2NGS framework included 4 modules, namely Nano2NGS-Muta, -Meta, -STR and -HLA. For hotspot mutation detection, we tested Nano2NGS-Muta + Freebayes using simulated data including 12 hotspot mutations (SNVs and InDels), and standard sample including 7 hotspot mutations with ~5% mutation frequency. For metagenomic analysis, we tested Nano2NGS-Meta + Kraken2 using simulated data including 20 bacterial species from the NCBI database, and standard sample including 7 bacteria with ~14% theoretical microbial composition. For STR detection, we tested Nano2NGS-STR using simulated data including 4 STR markers (DYS392, DYS438, DYS448, and DYS635), and standard sample including 44 STR markers from the intersection of two commercial products. For HLA typing, we tested Nano2NGS-HLA + Athlates using 1 simulated data and 1 standard sample. The GM12878 gDNA sample was used as negative control. All samples have 3 experimental repetitive. The nanopore sequencing was performed on the QNome-9604/3841 instrument according to the manufacturer’s instructions. Results: The performance of all 4 modules in simulated datasets was perfect. In standard sample datasets, for hotspot mutation detection, we found the repeated experiments detection sensitivity was 95.24%, 100.00% and 85.71%, respectively. The limit of detection (LoD) of Nano2NGS-Muta + Freebayes may be 2% or even 5% according to the negative dataset. For metagenomic analysis, we found the overall performance of Nano2NGS-Meta + Kraken2 was better, and R-square value compared with expected relative abundance was 0.9357. For STR detection, we found Nano2NGS-STR achieved the best performance compared with Tandem-Genotypes and TriCoLOR, with 71.97% (9948) and 53.03% (2800M) concordance, respectively. For HLA typing, we found the result of Nano2NGS-HLA + Athlates was HLA-A*02/HLA-A*11, HLA-B*35/HLA-B*39 and HLA-C*07/HLA-C*12, which were consistent with the expected genotypes of allele group (field 1). Conclusions: Nano2NGS framework converts nanopore sequencing data into NGS-liked short-read data and can be compatible with the NGS data analysis software. Importantly, Nano2NGS breaks the barriers of data analysis methods between short-read sequencing and long-read sequencing, and accelerates the application of nanopore sequencing technology in scientific research and clinical practice. Citation Format: Jidong Lang. Nano2NGS: A framework for converting nanopore sequencing data to NGS-liked sequencing data for data analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2081.

Parallel sequencing of 87 STR and 294 SNP markers using the prototype of the SifaMPS panel on the MiSeq FGx™ system

Massively parallel sequencing (MPS), or next generation sequencing (NGS), is a promising methodology for the detection of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) in forensic genetics. Here, the prototype SifaMPS Panel is designed to simultaneously target 87 STRs and 294 SNPs with forensic interest in a single multiplex in conjunction with the TruSeq™ Custom Amplicon workflow and MiSeq FGx™ System. Two in-house python scripts are adopted for the fastq-to-genotype interpretation of MPS data concerning STR and SNP, respectively. In the present study, by sequencing 50 Chinese Hans and many other DNA samples involved in validation studies, system parameters including the depth of coverage (DoC), heterozygote balance (Hb) and sequence coverage ratios (SCRs), as well as different forensic parameters of STRs and SNPs in a population study, were calculated to evaluate the overall performance of this new panel and its practicality in forensic application. In general, except for two STRs (DYS505 and DYS449) and one SNP (rs4288409) that performed poorly, the other 85 STRs and 293 SNPs in our panel had good performance that could strengthen efficiency for human identification and paternity testing. In addition, discordant STR genotype results between those generated from capillary electrophoresis (CE) and from the MPS platform were clearly illustrated, and these results could be a useful reference for applying these particular non-CODIS STRs in forensic practice.

A Targeted Gene Panel That Covers Coding, Non-coding and Short Tandem Repeat Regions Improves the Diagnosis of Patients With Neurodegenerative Diseases.

Genetic testing for neurodegenerative diseases (NDs) is highly challenging because of genetic heterogeneity and overlapping manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington’s disease and spinocerebellar ataxias type 3 (SCA3). Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known or likely pathogenic variation was found in 54 patients; 27 patients demonstrated clinical profiles that matched the variants; and 16 patients whose original diagnosis were refined. A high concordance of variant calling were observed when comparing the results from TGP and whole-exome sequencing of four patients. Our in-house STR detection algorithm has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combined comprehensive TGPs-bioinformatics pipeline can improve the clinical diagnosis of NDs.

Detection of short tandem repeats at 5 loci and amelogenin with cell-free fetal DNA as a specimen in the development of prenatal paternity diagnostic tests

IntroductionA prenatal paternity test is one widely-used method of determining the paternity of an unborn child. Such tests using chorionic villus or amniocentesis may increase the risk of harm to both mother and foetus. In the present day, a prenatal paternity test using circulating cell-free fetal Deoxyribonucleic Acid (DNA) is one alternative method due to it being non-invasive and safe for both mother and foetus.AimThe aim of this study is to detect Short Tandem Repeat (STR) at 5 Loci (vWA, TH01, D13S317, D18S51, and D21S11) and amelogenin genes in circulating cell-free fetal DNA in paternity tests.Materials and methodsForty-one samples of maternal blood were obtained from pregnant woman. Circulating free fetal DNA was subsequently extracted. A paternity test was conducted using an STR test at loci vWA, TH01, D13S317, D18S51, D21S11 in circulating free fetal DNA. An analysis of the paternity test between loci > 200 bp and < 200 bp was also conducted to establish the sensitivity of the test.ResultsThere was a significant difference between maternal blood DNA and circulating cell-free fetal DNA (p = 0.000 D13S317; p = 0.000 D21S11; p = 0.000 D18S51; p = 0.000 vWA; p = 0.000 TH01; and p = 0.000 amelogenin genes). The locus < 200 bp also had a higher sensitivity than locus > 200 bp.ConclusionCirculating free fetal DNA can be used as an alternative sample for prenatal paternity tests because of its similarity with maternal DNA.

Establishment and characterization of a laryngeal squamous cell carcinoma cell line

Objective: To establish a laryngeal squamous cell carcinoma (LSCC) cell line through primary cell culture and observe its biological characteristics. Methods: Tissue block culture method was used for primary cell culture. After LSCC cells passed 25 times in vitro, the morphology of cells was observed, keratin was stained histochemically, cell cycle was tested by PI-FACS, and the specie of cells was detected by PCR and short tandem repeat(STR) typing. Results: This newly established LSCC cell line was named as TR-LCC-1, most of the cancer cells were polygonal shape, like the cobblestone, loss of contact inhibition and with overlapping growth. Cell size was large and cell pleomorphism was very obvious. Cytokeratin staining was positive. After 6 months of continuous culture in vitro, the TR-LCC-1 cells passed more than 30 times, and cell doubling time was 201.2h. Cell cycle assay indicated that G1 phase accounted for 51.71%, S phase was 44.56%, and G2 phase was 2.28%. Mycoplasma test showed no mycoplasma contamination. Cell species identification identified TR-LCC-1 was human-derived cells. STR detection showed P26 and P6 were same, and they were different from the STR typing of disclosed cells. Conclusion: The establish ment of the new laryngeal squamous carcinoma cell line TR-LCC-1 can be helpful to the research for laryngeal squamous cell cancer.

A comprehensive 16 loci-based DNA fingerprinting dataset of a broad tumor cell line panel for cancer research

Motivations. Tumor cell lines are widely used as in vitro models and as screening tools in cancer research. A significant portion of cell lines have been reported to be misidentified, potentially generating misleading data interpretation. Correct assessment of cell line genetic identity is therefore critical to cancer biology studies as well as to drug discovery. Several methods may be applied to authenticate cell lines, but DNA fingerprinting based on the detection of Short Tandem Repeats (STR) has emerged as the standard approach. STR profiles for several commercial cell lines can be retrieved from sparse literature reports or from vendor databases, however these are mainly based on the analysis of 8 loci, except for the NCI60 panel, on which 16 loci were profiled. Inconsistencies can be found in allele designation among the different authors, mainly when more than two alleles per locus are present, as is frequently found in tumor cell lines. A resource to facilitate literature interrogation is represented by the Cell Line Integrated Molecular Authentication database (CLIMA), but to date there are no reported comprehensive databases containing 16 loci profile sets generated in parallel. Here we disclose the in-house generation of a homogeneous 16 loci dataset, reporting the STR profiles for a panel of about 300 tumor cell lines, representative of diverse solid and circulating tumor types. Our intent was to facilitate the management of the internal cell bank, by assessing the identity and stability over time of the tumor cell lines used for research, with the highest accuracy in discrimination and with an optimized score for profile similarity checking. The STR database can be linked to Nerviano Medical Sciences (NMS) internal database, which contains information on cell line origin, morphology, growth conditions, as well as known somatic mutations (from the Wellcome Trust Sanger Institute Cosmic database), thus making it a comprehensive integrated platform for cell line utilization in drug discovery. Our plan is to make the STR database available to the scientific community. Methods. DNA fingerprinting is based on the simultaneous amplification of highly polymorphic STR sites, which are short DNA sequences with a varying number of repeats in each cell line. Genetic abnormalities in tumor cell lines can result in more than two alleles at each locus, which complicates the analysis. Most STR profiles reported so far are based on the analysis of a limited number of STR loci, usually 8. The probability for two individuals to share the same STR profile is estimated to range from ~10-8 for 8 loci to ~10-17 for 16 loci. A 16 loci profile can be beneficial in cancer research, due to the intrinsic genetic instability of tumor cells that may result in allele acquisition or loss. In the current study we performed a 16-loci STR fingerprinting analysis on a panel of about 300 commercially available cancer cell lines. DNA was prepared directly from frozen cells, using NucleoSpin Tissue (Macherey-Nagel), and then analysed using AmpFlSTR Identifiler Plus PCR Amplification kit (Applied Biosystems), that amplifies simultaneously 15 tetranucleotide repeat loci and the amelogenin gender marker. For a more accurate comparison of STR profiles in different cell lines, we have introduced a modification to the standard calculation of similarity scores to account for partial allele identity at each locus, allowing a more precise definition of the level of similarity, which is particularly relevant when multiple alleles are present. This is obtained by applying the following formula that calculates the number of identical alleles at each locus, divided by the total number of alleles: given a cell line A and a cell line B, the score is defined as the identified sum of similarities at each i-th locus s(i) normalized by the number of not empty loci, where s(i) = 2 x [alleles(A) = alleles(B)] / [#alleles(A) + #alleles(B)]. Based on a preliminary sensitivity analysis, the similarity score threshold to classify two cell lines as identical was set at 80%. Results. A DNA fingerprinting analysis based on 16 loci was performed on a panel of about 300 widely used tumor cell lines, purchased at NMS over several years from diverse providers. Using an established cut off of 80%, we found that all cell lines were correctly classified, suggesting that the cell line panel is genetically stable over time. As expected, an overall pairwise analysis of all the different cell line profiles revealed a generally low similarity degree (less than 50%), with no evidence for any similarity bias due to tumor type. The main outcome of this study is the availability of a large 16 loci STR dataset of tumor cell lines, which was integrated as a fundamental part of the NMS Cell Bank database. The STR dataset can be easily interrogated and distributed for use as a reliable reference for cancer research.

Detection of circulating fetal DNA in maternal plasma for non-invasive prenatal diagnosis

目的 寻找一种无创性产前基因检测孕妇血浆中胎儿游离DNA的新方法.方法 提取32名健康孕妇血浆标本中胎儿游离DNA,经套式聚合酶链反应(PCR)扩增其性别决定基因(sex-determining region Y,SRY),并引入内参照基因序列ATL1;并采用16个短串联重复序列(short tandem repeat,STR)多态性位点的多重荧光PCR方法对血浆标本中DNA进行STR等位基因扩增.同时,检测孕妇及其丈夫的外周血白细胞DNA,进行基因扫描及对照分析.结果 经SRY-PCR检测发现,17名妊娠男胎孕妇血浆中均出现基因扩增带,其余15名妊娠女胎孕妇中有2名出现假阳性,性别总符合率为94%(30/32).经STR-PCR检测发现,均从孕妇血浆中检测出父源性胎儿DNA的存在.结论 应用孕妇血浆中胎儿DNA作产前诊断准确性较高,是一种无创性产前基因诊断方法,具有广泛的临床应用前景。

毛根の形態とＳＴＲ型検出

For the purpose of investigating the relation between hair root morphology and STR (Short tandem repeat) typing, the root morphology of head hairs collected by sweeping, combing and pulling from twenty Japanese males was classified as follows. Hair roots swept or combed were classified into four types; A: with no root sheath, B: with little root sheath at the proximal end, C: with medial root sheath at the proximal end or little root sheath around the telogen club, and D: with much root sheath around the telogen club. Hair roots pulled were classified into three types; E: with no root sheath, F: with little root sheath, and G: with much translucent root sheath tissue around the anagen growth bulb.Histomorphological examination of hair roots showed that root sheaths were not observed around telogen clubs of type A, and root sheaths were abundantly observed around anagen growth bulbs of types F and G. In roots of type E, root sheaths were scarcely observed but nuclei were observed in the hair cortex of the anagen growth bulb. In roots of telogen or catagen phases such as types B and C, distinct root sheaths were not observed but translucent process-like structures with blue-stained nuclei were observed at the bottom part of the telogen club. When roots that adhered materials observed around the root of type D were opaque, nuclei were almost never observed.From the hair roots of eleven individuals arbitrarily selected from twenty Japanese males, STR typing was examined using the AmpFlSTR profiler kit. As a result, no STR loci were detected from all eleven individuals in roots of type A, but all STR loci were detected from them in roots of types E, F and G. In roots of types B, C and D, the STR loci of each hair showed a multifarious detection pattern. When roots adhered materials at the proximal end of the root were translucent, the possibility of STR detection was relatively high. But, when roots adhered materials not at the proximal end of the root and were opaque, the possibility of detection became low.These results suggest that the detection of STR loci from hair root samples is not influenced by the quantity but the quality of adhered materials observed in the hair roots.

Detection of Trisomy 21 by Quantitative Mass Spectrometric Analysis of Single-Nucleotide Polymorphisms

Down syndrome is the most common chromosomal abnormality of live-born babies. Currently, cytogenetic analysis of fetal cells such as full chromosome karyotyping (1) and fluorescence in situ hybridization (2) are the most widely used techniques for prenatal diagnosis of this chromosomal aneuploidy. Recently, however, alternative molecular strategies have been developed. Quantitative fluorescence PCR involves the detection of short tandem repeats on chromosome 21 and is by far the most thoroughly evaluated molecular technique. Additional reported molecular detection methodologies include real-time quantitative PCR (3)(4), multiplex probe ligation assays (5), and paralogous sequence quantification (6). Using melting curve analysis of single-nucleotide polymorphisms (SNPs), Pont-Kingdon et al. (7) have measured the relative allele copy number in fixed cells and amniocyte cell cultures of trisomy 21 samples. Compared with short tandem repeats, SNPs occur much more frequently in the human genome (8) and thus would potentially provide more information on chromosomal abnormalities. In the present study, we investigated the feasibility of measuring the allelic ratios of the chromosome-21 SNPs to identify trisomy 21 in various clinical samples. We detected and quantified the allelic ratios by PCR followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (9). Theoretically, normal euploid samples would give an allelic ratio of 1, whereas trisomy 21 samples would give allelic ratios of ratios of 0.5 or 2.0. Placenta, amniotic fluid, and chorionic villus sampling (CVS) from normal and trisomic 21 cases were collected from the Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong, and the King’s College Hospital, London, United Kingdom. Normal or trisomic status was confirmed by karyotyping. Ethics approval was obtained from the corresponding Institutional Review Boards. DNA extraction from the placentas and CVS was performed with a QIAamp DNA Mini Kit (Qiagen) with the “tissue protocol”. For the amniotic fluid, DNA …

Multiplex Amplification and Fluorimetric Detection of Short Tandem Repeats for Mixed Chimerism After Bone Marrow Transplant

We present the clinical application of a simple method for mixed chimerism analysis after allogeneic bone marrow (BM) or peripheral blood stem cell (PBSC) transplant. A commercial kit which enables multiplex amplification of 9 highly polymorphic short tandem repeats in a single reaction tube was used. Molecular size and relative quantities of each amplified fragment are subsequently measured using an automated fluorimeter. The sensitivity and linearity were tested using whole blood or genomic DNA from two subjects, mixed in various proportions from 0.25% to 99.75%. In 70 donor-recipient pairs the median number of informative alleles for the assessment of relapse was 5.9 (range 3-11). Results showed that the linearity between the measured and expected relative quantities of amplified DNA showed a regression coefficient of 0.99 in the interval 10-90%. The mean sensitivity was 1.5% (range 0.5-2.5), greater than previously reported. A total of 70 cases of allogeneic transplant (49 with family and 21 with unrelated donors) were monitored before transplant and after 1, 2, 4, 6 or 12 months thereafter and then at 6 months intervals (range 6-36). In 18 patients mixed chimerism was observed 1 month from transplant, with donor allele percentage ranging from 1 to 6%. Fragment dimension reproducibility (CV 0.34, range 0.52-0.66) was confirmed by an internal DNA control and by amelogenin fragment length repeatability on all patients. In conclusion, the proposed method is sufficiently simple, rapid, sensible, specific and cost effective for the evaluation of mixed chimerism after BM or PBSC transplant in a clinical setting.

A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis.

A five-capillary system has been developed for DNA sequencing and analysis. The post-column fluorescence detector is based on a sheath-flow cuvette. The instrument provides uniform and continuous illumination of the samples. The cuvette virtually eliminates cross-talk in the fluorescence signal between capillaries. Discrete single-photon counting avalanche photodiodes provide high efficiency light detection. The instrument has detection limits (3sigma) of 130 +/- 30 fluorescein molecules injected onto each capillary. Over 650 bases of sequence at 98.8% accuracy were generated in 100 min at 50 degrees C from M13mp18. Separation and detection of short tandem repeats proved efficient and accurate with the use of internal standards for direct comparison of migration times between capillaries.

Analysis and Interpretation of Short Tandem Repeat Microvariants and Three-Banded Allele Patterns Using Multiple Allele Detection Systems

The Palm Beach County Sheriffs Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented analysis of short tandem repeat (STR) sequences on casework using silver staining kit and SYBR Green I detection systems and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO 100 instrument. Concurrently, the Broward County Sheriff's Office (BSO) Crime Laboratory is validating the ABI Prism310 Genetic Analyzer capillary electrophoresis STR detection system (ABI CE310) from Perkin Elmer Applied BioSystems. During the course of analyzing over 10,000 individuals for the STR loci CSF1PO, TPOX and THO1 (CTT) using silver staining for allele detection, 42 samples demonstrated alleles that were "off ladder," contained three-banded patterns at a single locus, or exhibited an apparent THO1 "9.3,10" allele pattern. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the allele patterns observed in these 42 samples using the following allele detection systems: (1) manual silver staining, (2) SYBR Green I staining, and/or (3) fluorescently tagged amplified products separated by polyacrylamide gel electrophoresis or capillary electrophoresis followed by laser detection. Regardless of the CTT allele detection system utilized, concordant results were obtained for 41 of the 42 samples. The only exception was a sample in which a wide band within the THO1 locus was identified as a THO1 "9.3, 10" genotype by silver staining kit and SYBR Green I staining but was verified to be a THO1 "9.3" homozygote by all other allele detection systems. Manual allele detection could readily identify microvariants, as a visual assessment of stained gels clearly shows that alleles do not migrate coincident with well-characterized allele size standards. As would be predicted, however, the manual detection systems did not provide adequate resolution to approximate the basepair size for off-ladder variants. All fluorescent software program systems were consistent in designating alleles "not in range" or "off ladder," thereby indicating true microvariants. All single-locus three-banded patterns were detected using all of the STR multiplex systems. In addition, individual locus-specific primers verified multiplexed amplified products were specific for the locus in question.

A rapid scanning strip for tri- and dinucleotide short tandem repeats.

Oligonucleotides representing 60 trinucleotide (21mers) and four dinucleotide (20mers) tandem repeats were directly synthesized and arrayed onto an aminated polypropylene substrate. DNA samples of different complexities (a CAG-containing 21mer oligonucleotide, PCR fragments of 200 to 3,000 bp, and cosmids with 31 to 35 kb inserts) were radiolabelled and hybridized to the oligonucleotide array at various temperatures. When compared to sequence data available from the test DNAs, the reverse blot system specifically identified various tri- and dinucleotide short tandem repeats (STRs) in every case. Moreover, there was no random or cross hybridization to nonspecific sequences. It was possible to detect as few as three repeated units in a particular location, as shown for (CCT)n, (GCC)n and (CAC)n triplets in cosmid DNA. Varying the hybridization stringency can enhance the detection of STRs. This single-step reverse blot system therefore allows the rapid, specific and sensitive identification of various STRs in DNA sources of different complexity.