Comparative analysis of short tandem repeat data obtained by automated and gel electrophoresis techniques

Pagel, Uonis Raasch; Reis, Ruth; Carvalho, Vanessa; Santos, Eldamária V. W. dos; Zandonade, Eliana; Louro, Iúri Drumond; Paula, Fabiana Martins de

doi:10.4238/gmr.15038436

Cited by 5 publications

(5 citation statements)

References 8 publications

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“…Compared to the CE method, however, the PAGE method failed to provide similar allele frequency spectra in two out of the five microsatellite loci used, a phenomenon mainly attributed to the misinterpretation of allele banding patterns [ 12 , 20 ]. Since two previous studies with the PAGE method have provided allele frequency data that agree with that obtained using the CE method, by as much as 99% [ 16 , 21 ], methodological issues in this study can also not be ruled out as an error source. These findings highlight the limitations of non-HTP methods for accurate allele scoring and present avenues for improved error reduction by adjusting the data collection process.…”

Section: Discussionmentioning

confidence: 76%

“…These findings highlight the limitations of non-HTP methods for accurate allele scoring and present avenues for improved error reduction by adjusting the data collection process. For example, Pagel et al [ 21 ] indicate that to increase the power of resolution and avoid doubtful alleles, the PAGE’s electrophoretic conditions can be improved by decreasing the voltage and increasing the gel concentration and running time, conditions that even though make the protocols more time consuming and labor-intensive, ultimately allow the visualization of one-base differences.…”

Section: Discussionmentioning

confidence: 99%

“…Although the discrepancies in allele frequency between PAGE- and CE-derived genomic datasets did not affect genetic diversity estimation, it is considerable enough to bear in mind that they did affect more complex analyses such as kinship assignation, in which a single erroneously identified allele leads to misinterpretations [ 20 , 21 ]. Indeed, we obtained different patterns of relatedness while inferring relatedness from each dataset.…”

Section: Discussionmentioning

confidence: 99%

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Performance comparison of gel and capillary electrophoresis-based microsatellite genotyping strategies in a population research and kinship testing framework

et al. 2021

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Objective The advancement of molecular techniques in an era in which high-throughput sequencing has revolutionized biology renders old-fashioned alternatives to high-throughput methods obsolete. Such advanced molecular techniques, however, are not yet accessible to economically disadvantaged region-based laboratories that still obtain DNA profiles using gel-based techniques. To explore whether cost-efficient techniques can produce results that are as robust as those obtained using high-throughput methods, we compared the performance of polyacrylamide gel electrophoresis (PAGE)- and capillary electrophoresis (CE)-derived genomic data in estimating genetic diversity and inferring relatedness using 70 individuals of fine flounder (Paralichthys adspersus) selected from a hatchery population and genotyped for five microsatellite loci. Results Here, we show that PAGE- and CE-derived genomic datasets yield comparable genetic diversity levels regarding allelic diversity measures and heterozygosity. However, relatedness inferred from each dataset showed that the categorization of dyads in the different relationship categories strongly differed. This suggests that while scientists can reliably use PAGE-derived genomic data to estimate genetic diversity, they cannot use the same for parentage testing. The findings could help laboratories committed to population research not be discouraged from using the PAGE system if high-throughput technologies are unavailable and the method is adequate to address the biological question.

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Performance comparison of gel and capillary electrophoresis-based microsatellite genotyping strategies in a population research and kinship testing framework

et al. 2021

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“…The last portion of the comprehensive workflow is massively parallel sequencing (MPS), also known as nextgeneration sequencing (NGS), which was assessed for ability to relieve limitations of amplicon length-based STR allele calling after capillary electrophoresis (CE). These limitations include the inability to analyze different types of genetic polymorphisms in a single reaction and workflow, low-resolution genotyping, loss of data in degraded DNA samples, and incomplete resolution of complex DNA mixtures [10].…”

Section: Introductionmentioning

confidence: 99%

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Carrasco

Inostroza

Didier³

et al. 2019

Int J Legal Med

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Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFK MR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twentythree STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples. P. Carrasco, C. Inostroza and M. Didier contributed equally to this work.

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“…Denaturing PAGE does not require expensive instruments and fluorescent labels (Pagel et al, 2016). Comparatively, CE can increase the speed of electrophoresis and provide more accurate data.…”

Section: Introductionmentioning

confidence: 99%

Characterization and development of 56 EST-SSR markers derived from the transcriptome of Odontobutis potamophila

Wang¹,

Ren²,

Chen³

et al. 2017

Genet. Mol. Res.

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ABSTRACT. Expressed sequence tags (ETSs) are the sources of microsatellite development. In this study, we isolated and characterized microsatellite markers for Odontobutis potamophila by using Illumina RNA-sequencing. We sequenced a large number of ESTs and screened 200 potential microsatellites. Consequently, a total of 56 novel polymorphic microsatellite repeat markers were identified in thirty-two individuals from a wild population area (Jiande, Zhejiang Province, China). The number of alleles per locus varied from two to eight, the observed heterozygosity (H O ) ranged from 0.03571 to 0.9375, and the expected heterozygosity (H E ) ranged from 0.14326 to 0.81549. The average number of alleles, H O , and H E were 5.0, 0.4467, and 0.5518, respectively. By the calculation, the range of polymorphism information content (PIC) was 0.1177-0.8492. Most of the loci showed moderate or high polymorphism. These newly developed EST-simple sequence repeat (EST-SSR) markers would serve as an efficient tool for analyzing population connectivity and provide sufficient information for genetic diversity research, parentage, and molecular breeding of O. potamophila and other fishes with similar genetic relationship.

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Comparative analysis of short tandem repeat data obtained by automated and gel electrophoresis techniques

Cited by 5 publications

References 8 publications

Performance comparison of gel and capillary electrophoresis-based microsatellite genotyping strategies in a population research and kinship testing framework

Performance comparison of gel and capillary electrophoresis-based microsatellite genotyping strategies in a population research and kinship testing framework

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Characterization and development of 56 EST-SSR markers derived from the transcriptome of Odontobutis potamophila

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