Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification

Kieser, Rachel; Buś, Magdalena M.; King, Jonathan L.; Vliet, Walter van der; Theelen, Joop; Budowle, Bruce

doi:10.1016/j.fsigen.2019.102201

Cited by 20 publications

(12 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Single nucleotide polymorphisms (SNPs) are being studied and applied for identifying degraded DNA due to the low mutation rate and short amplicon [8] and lots of interesting work have been reported [9][10][11][12][13]. Rachel et al [14] focus on investigating highly degraded forensic samples, provided a 27 identity-testing SNPs panel with amplicon lengths smaller than 50bp. In addition, a new genetic marker named microhaplotype was proposed by Kidd et al, which consists of two or more closely linked SNPs and produces three or more allelic combinations [15].…”

Section: Introductionmentioning

confidence: 99%

An 18 Multi‐InDels panel for analysis of highly degraded forensic biological samples

Lin

Jiang

et al. 2021

Electrophoresis

View full text Add to dashboard Cite

DNA genotyping from trace and highly degraded biological samples is one of the most significant challenges of forensic DNA identification. There is a lack of simple and effective methods for genotyping highly degraded samples. In this study, a multiple loci insertion/deletion polymorphisms (Multi-InDels) panel was designed for detecting 18 autosomal Multi-InDels through capillary electrophoresis (CE) with amplicon sizes no longer than 125 bp. Studies of sensitivity, degradation, and species specificity were performed and a population study was carried out using 192 samples from Han populations in Hunan province in the south of China. The combined random match probability (CMP) of these 18 Multi-InDels was 3.23 × 10 -12 and the cumulative probability of exclusion (CPE) was 0.9989, suggesting this panel could be used independently for human identification and could provide efficient supporting information for parentage testing. Complete profiles were obtained from as low as 62.5 pg of total input DNA after increasing the number of PCR cycles. Moreover, all alleles were detected from artificially highly degraded DNA after 80 min of boiling water bath treatment. This 18 Multi-InDels panel is simple, fast, and effective for the forensic analysis of highly degraded DNA.

show abstract

Section: Introductionmentioning

confidence: 99%

An 18 Multi‐InDels panel for analysis of highly degraded forensic biological samples

Lin

Jiang

et al. 2021

Electrophoresis

View full text Add to dashboard Cite

show abstract

“…cDNA was amplified using the EasySeq SARS-CoV-2 WGS Library Prep Kit (Nimagen B.V.; Nijmegen, Netherlands), hereafter called Nimagen, using Reverse Complement PCR (RC-PCR) technology that produces short amplicons (around 200 bp). This technology allows amplification of cDNA using two types of primers: target-specific primers with a universal tail, and universal primers coupled to Illumina adapter sequences and to index sequences i5 and i7 for sample identification 27 . Two RC-PCRs (A and B) were performed for each sample with two primer pools that cover the whole SARS-CoV-2 genome.…”

Section: Methodsmentioning

confidence: 99%

Minimal requirements for ISO15189 validation and accreditation of three next generation sequencing procedures for SARS-CoV-2 surveillance in clinical setting

Maschietto

Otto

Rouzè

et al. 2023

Sci Rep

View full text Add to dashboard Cite

Rapid and recurrent breakthroughs of new SARS-CoV-2 strains (variants) have prompted public health authorities worldwide to set up surveillance networks to monitor the circulation of variants of concern. The use of next-generation sequencing technologies has raised the need for quality control assessment as required in clinical laboratories. The present study is the first to propose a validation guide for SARS-CoV-2 typing using three different NGS methods fulfilling ISO15189 standards. These include the assessment of the risk, specificity, accuracy, reproducibility, and repeatability of the methods. Among the three methods used, two are amplicon-based involving reverse transcription polymerase chain reaction (Artic v3 and Midnight v1) on Oxford Nanopore Technologies while the third one is amplicon-based using reverse complement polymerase chain reaction (Nimagen) on Illumina technology. We found that all methods met the quality requirement (e.g., 100% concordant typing results for accuracy, reproducibility, and repeatability) for SARS-CoV-2 typing in clinical setting. Additionally, the typing results emerging from each of the three sequencing methods were compared using three widely known nomenclatures (WHO, Pangolineage, and Nextclade). They were also compared regarding single nucleotide variations. The outcomes showed that Artic v3 and Nimagen should be privileged for outbreak investigation as they provide higher quality results for samples that do not meet inclusion criteria for analysis in a clinical setting. This study is a first step towards validation of laboratory developed NGS tests in the context of the new European regulation for medical devices and in vitro diagnostics.

show abstract

“…While SNPs are the most abundant genetic variation in the human genome, generating a robust Benchmark multiplex of short amplicons is challenging. Our first attempt to design reduced 50 bp-long SNP amplicons resulted in only 27 markers meeting the criterion to be included in the multiplex [15]. A requirement for short amplicons leaves limited flexibility to design primers for PCR, due to molecular and thermodynamic constraints.…”

mentioning

confidence: 99%

“…Two advances now may make it possible to analyze highly degraded DNA: reverse complement PCR (RC-PCR) and massively parallel sequencing (MPS). RC-PCR is a novel target enrichment and library preparation method for MPS [15]. Target enrichment and indexing are performed in a single closed-tube system.…”

mentioning

confidence: 99%

“…In a proof-of-concept study, Kieser and colleagues [15] demonstrated that RC-PCR technology could successfully amplify, with high fidelity, 27 SNPs with the targets contained within 50 bases in length. The method revealed a high sensitivity with a majority of SNP alleles detected with a limited DNA input of 60 pg (i.e., <10 cells of human genome equivalents) and successful analyses in the presence of PCR inhibitors (and, to a lesser extent, hematin and humic acid) and the potential for detecting mixed DNA profiles.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Reverse Complement-PCR, an Innovative and Effective method for Multiplexing Forensically Relevant Single Nucleotide Polymorphism Marker Systems

Buś

Jong²,

King

et al. 2021

BioTechniques

Self Cite

View full text Add to dashboard Cite

DNA analyses from challenging samples such as touch evidence, hairs and skeletal remains push the limits of the current forensic DNA typing technologies. Reverse complement PCR (RC-PCR) is a novel, single-step PCR target enrichment method adapted to amplify degraded DNA. The sample preparation process involves a limited number of steps, decreasing the labor required for library preparation and reducing the possibility of contamination due to less sample manipulation. These features of the RC-PCR make the technology a unique application to successfully target single nucleotide polymorphisms (SNPs) in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods. The developed RC-PCR short amplicon 85 SNP-plex panel is a substantial improvement over the previously reported 27-plex RC-PCR multiplex that will provide higher discrimination power for challenging DNA sample analyses.

show abstract

Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification

Cited by 20 publications

References 19 publications

An 18 Multi‐InDels panel for analysis of highly degraded forensic biological samples

An 18 Multi‐InDels panel for analysis of highly degraded forensic biological samples

Minimal requirements for ISO15189 validation and accreditation of three next generation sequencing procedures for SARS-CoV-2 surveillance in clinical setting

Reverse Complement-PCR, an Innovative and Effective method for Multiplexing Forensically Relevant Single Nucleotide Polymorphism Marker Systems

Contact Info

Product

Resources

About