DNA Typing Methods Research Articles

Internal Validation of RapidHIT® ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†.

A new rapid DNA solution, the RapidHIT® ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low-throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost-saving, efficient design to generate a validated RapidHIT® ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120min.

CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method

This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9-sgRNA- or CRISPR-typing PCR. The technique can detect and type target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). This method was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. This method was also verified by detecting the L1 and E6-E7 genes of two high-risk HPVs (HPV16 and 18) in cervical carcinoma cells and many clinical samples. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). Based on these proof-of-concept experiments, this study provides a new CRISPR/Cas9-based DNA detection and typing method.

Development of a DNA methylation-based semen-specific SNP typing method: A new approach for genotyping from a mixture of body fluids

Genotyping from samples containing different types of body fluids is a major difficulty in forensic investigations. Recently, CpG sites that are specifically methylated or unmethylated in different types of body fluids have been reported as novel markers for body fluid identification. In this study, we hypothesized that the simultaneous analysis of CpGs and neighboring polymorphic sites on the same molecule could be useful for individual DNA typing from mixed samples. We performed a proof-of-concept study of this approach by searching the genome-wide methylation dataset deposited at the National Center for Biotechnology Information Gene Expression Omnibus repository for semen-specific CpG markers adjacent to common single nucleotide polymorphisms. From the identified candidates, we selected 5 regions on different chromosomes and validated the presence of semen-specific methylation or unmethylation in each region by pyrosequencing analyses. By combining methylation-specific polymerase chain reaction and pyrosequencing technology, we developed a semen-specific DNA typing method for two semen-specific methylated regions and one semen-specific unmethylated region. Finally, the method successfully identified semen-derived alleles from mixed stains, indicating that this methylation-based approach can be applicable to actual forensic samples. Since existing separation techniques physically isolate cells derived from each type of body fluid, this approach may be useful when existing methods cannot be performed due to the degradation of samples.

Massive parallel sequencing of short tandem repeats in the Korean population.

STR analysis using capillary electrophoresis has been the most widely used method for forensic DNA typing. Recently, massive parallel sequencing (MPS) technique has been emerging as an innovative tool to supplement or replace the conventional CE process. In this study, we evaluated the application of commercial MiSeqFGx™ forensic signature kit (Illumina Inc., San Diego, CA, USA) in the Korean population, including performance comparison with CE-based STR profiling kits. The genotyping results of 209 unrelated random Korean individuals were summarized according to the International Society for Forensic Genetics guideline. The study revealed that 26 novel sequence variations in autosomal STR were newly found that had not been previously reported in other forensic literature. This indicates that MPS may be an effective supplementary tool for forensic DNA typing and the database to increase the discriminatory power of individual identification.

Optimization of Allele Specific PCR for the Development of Human Mitochondrial DNA Typing Method

Optimization of Allele Specific PCR for the Development of Human Mitochondrial DNA Typing Method

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Systematic evaluation of the early access applied biosystems precision ID Globalfiler mixture ID and Globalfiler NGS STR panels for the ion S5 system

Human HLA-B27 typing using the BD™ HLA-B27 kit on the BD FACSVia™ system: A multicenter study.

The BD FACSVia™ system is a novel flow cytometer with improved workflow efficiencies. To evaluate the HLA‐B27 application developed on the BD FACSVia system utilizing the BD™ HLA‐B27 kit, we conducted a concordance study at three centers to compare with the BD FACSCalibur™ system. Prepared donor samples (n = 594) were analyzed on both the BD FACSVia and BD FACSCalibur for the HLA‐B27 assay. Adjudication of HLA‐B27 discordant results was performed using the reverse sequence‐specific oligonucleotide (rSSO) DNA typing method (LABType® SSO, One Lambda). On the BD FACSVia system 80 B27 positive, 499 B27 negative and 15 “Inconclusive” samples were observed. The corresponding BD FACSCalibur results were 73 B27 positive, 502 B27 negative and 19 “gray zone” samples. The overall concordance of HLA‐B27 determination was 98% between the two systems with seven more positives identified on BD FACSVia as compared to BD FACSCalibur. The equivocal zone between positive and negative on BD FACSVia (named “Inconclusive”) and on BD FACSCalibur (named “gray zone”) is due to antibody cross reactivity of HLA‐B27 clone GS145.2. One negative sample verified with the rSSO DNA method was reported as HLA‐B27 positive by the BD FACSVia system leading to a false positive result. Our study demonstrated concordance results between the BD FACSVia system and BD FACSCalibur. Intersite reproducibility of BD HLA‐B27 assay remained within the limits of acceptability. © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society

Genetic bases of tomatо marker-assisted selection in Belarus

Abstract To develop tomato hybrids and varieties with a high fruit quality, we gradually solved the following tasks: development of DNA-marking methods for long shelf life genes; the genes modifying the biosynthesis of carotenoids and their composition; testing of the elaborated methods on the developed breeding material; the selection of samples with different allelic composition of fruit quality genes; the development of F 1 hybrids using the method of successive crosses and their study; the selection of tomato forms by DNA-typing methods with target genes in F 2 populations to develop valuable breeding samples; the study of carotenoids’ accumulation peculiarities and their inheritance. We used DNA-identification methods for fruit quality genes: nor, rin, norA (long shelf life), B, og c , hp2 dg , gf-3 (carotenoid content). The tomato hybrids, combining two pigment content genes and one long shelf life gene and the model forms with different combinations of fruit quality genes (B/rin/gf-3, B/rin/hp2 dg ; B /nor/gf-3, B /nor/hp2 dg ; оg c /rin/gf-3, оg c /rin/hp2 dg ; оg c /nor/gf-3, оg c /nor/hp2 dg ) in a homozygous state were developed. Use of the developed accessions with carotenoid content genes (og c /hp2 dg , B/hp2 dg ) as maternal forms and the accessions with complex fruit quality genes (og c /hp2 dg /nor, ogc/hp2 dg /rin, B/hp2 dg /nor, B/hp2 dg /rin, og c /gf-3/nor, og c /gf-3/rin, B/gf-3/rin, B/gf-3/nor) as paternal forms for hybridization contributes to high accumulation of carotenoids and a lond period of fruit storability.

Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique.

This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection. Graphical abstract CRISPR-associated reverse PCR (CARP) can be used to detect and type target DNA in a simple three-step procedure, cutting, ligation, and amplification.

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa , causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca . In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

Super High Resolution for Single Molecule-Sequence-Based Typing of Classical HLA Loci Using Ion Torrent PGM.

Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method to the field 4 level of allelic resolution (formerly known as 8-digit typing) to efficiently detect novel and null alleles without phase ambiguity by combination of long ranged PCR amplification and next-generation sequencing (NGS) technologies. In this chapter, we describe three basic steps, long ranged PCR, NGS, and allele assignment.

Genome Target Evaluator (GTEvaluator): A workflow exploiting genome dataset to measure the sensitivity and specificity of genetic markers.

Most of the bacterial typing methods used to discriminate isolates in medical or food safety microbiology are based on genetic markers used as targets in PCR or hybridization experiments. These DNA typing methods are important tools for studying prevalence and epidemiology, for conducting surveillance, investigations and control of biological hazard sources. In that perspective, it is crucial to insure that the chosen genetic markers have the greatest specificity and sensitivity. The wealth of whole-genome sequences available for many bacterial species offers the opportunity to evaluate the performance of these genetic markers. In the present study, we have developed GTEvaluator, a bioinformatics workflow which ranks genetic markers depending on their sensitivity and specificity towards groups of well-defined genomes. GTEvaluator identifies the most performant genetic markers to target individuals among a population. The individuals (i.e. a group of genomes within a collection) are defined by any kind of particular phenotypic or biological properties inside a related population (i.e. collection of genomes). The performance of the genetic markers is computed by a distance value which takes into account both sensitivity and specificity. In this study we report two examples of GTEvaluator application. In the first example Bacillus phenotypic markers were evaluated for their capacity to distinguish B. cereus from B. thuringiensis. In the second experiment, GTEvaluator measured the performance of genetic markers dedicated to the molecular serotyping of Salmonella enterica. In one in silico experiment it was possible to test 64 markers onto 134 genomes corresponding to 14 different serotypes.

Using DNA typing methods to identify dental specimens exposed to different environmental conditions

Using DNA typing methods to identify dental specimens exposed to different environmental conditions

Evaluation of the Illumina® Beta Version ForenSeq™ DNA Signature Prep Kit for use in genetic profiling

While capillary electrophoresis-based technologies have been the mainstay for human identity typing applications, there are limitations with this methodology’s resolution, scalability, and throughput. Massively parallel sequencing (MPS) offers the capability to multiplex multiple types of forensically-relevant markers and multiple samples together in one run all at an overall lower cost per nucleotide than traditional capillary electrophoresis-based methods; thus, addressing some of these limitations. MPS also is poised to expand forensic typing capabilities by providing new strategies for mixture deconvolution with the identification of intra-STR allele sequence variants and the potential to generate new types of investigative leads with an increase in the overall number and types of genetic markers being analyzed. The beta version of the Illumina ForenSeq DNA Signature Prep Kit is a MPS library preparation method with a streamlined workflow that allows for targeted amplification and sequencing of 63 STRs and 95 identity SNPs, with the option to include an additional 56 ancestry SNPs and 22 phenotypic SNPs depending on the primer mix chosen for amplification, on the MiSeq desktop sequencer (Illumina). This study was divided into a series of experiments that evaluated reliability, sensitivity of detection, mixture analysis, concordance, and the ability to analyze challenged samples. Genotype accuracy, depth of coverage, and allele balance were used as informative metrics for the quality of the data produced. The ForenSeq DNA Signature Prep Kit produced reliable, reproducible results and obtained full profiles with DNA input amounts of 1ng. Data were found to be concordant with current capillary electrophoresis methods, and mixtures at a 1:19 ratio were resolved accurately. Data from the challenged samples showed concordant results with current DNA typing methods with markers in common and minimal allele drop out from the large number of markers typed on these samples. This set of experiments indicates the beta version of the ForenSeq DNA Signature Prep Kit is a valid tool for forensic DNA typing and warrants full validation studies of this MPS technology.

Robust and reliable DNA typing of soils

Soil is encountered commonly in forensic investigations yet rarely forms part of the examination process. This may be due to a lack of a robust and reliable DNA typing method preventing comparison of data from material taken from the clothing of a suspect or victim, or from a crime scene, reference or alibi location. We have developed a method that provides reproducible metagenomic profiles from soil samples taken from three distant sites at four time points throughout the year. The whole biota of the soil samples, as small as 50mg, was targeted in the proposed approach highlighting the advantages of DNA typing based on High-Throughput DNA Sequencing. Three distinct clusters of microbial assemblages were formed that relates to the collection site of the samples regardless of the time of year collected, thus allowing reliable conclusions to be drawn.

Ultrafast STR Separations on Short-Channel Microfluidic Systems for Forensic Screening and Genotyping.

There are situations in which it is important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. DNA typing methods provide the best biometric information yielding identity, kinship, and geographical origin, but they are not portable and rapid. This study details the development of a portable short-channel microfluidic device based on a modified Agilent 2100 bioanalyzer for applications in forensic genomics. The system utilizes a denaturing polymer matrix with dual-channel laser-induced fluorescence and is capable of producing a genotype in 80sec. The device was tested for precision and resolution using an allelic ladder created from 6 short tandem repeat (STR) loci and a sex marker (amelogenin). The results demonstrated a precision of 0.09-0.21bp over the entire size range and resolution values from 2.5 to 4.1bp. Overall, the results demonstrate the chip provides a portable, rapid, and precise method for screening amplified short tandem repeats and human identification screening.

Industrial Food Animal Production and Community Health.

Industrial food animal production (IFAP) is a source of environmental microbial and chemical hazards. A growing body of literature suggests that populations living near these operations and manure-applied crop fields are at elevated risk for several health outcomes. We reviewed the literature published since 2000 and identified four health outcomes consistently and positively associated with living near IFAP: respiratory outcomes, methicillin-resistant Staphylococcus aureus (MRSA), Q fever, and stress/mood. We found moderate evidence of an association of IFAP with quality of life and limited evidence of an association with cognitive impairment, Clostridium difficile, Enterococcus, birth outcomes, and hypertension. Distance-based exposure metrics were used by 17/33 studies reviewed. Future work should investigate exposure through drinking water and must improve exposure assessment with direct environmental sampling, modeling, and high-resolution DNA typing methods. Investigators should not limit study to high-profile pathogens like MRSA but include a broader range of pathogens, as well as other disease outcomes.

Performance of a next generation sequencing SNP assay on degraded DNA

Forensic DNA casework samples are often of insufficient quantity or quality to generate full profiles by conventional DNA typing methods. Polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci is inherently limited in samples containing degraded DNA, as the cumulative size of repeat regions, primer binding regions, and flanking sequence is necessarily larger than the PCR template. Additionally, traditional capillary electrophoresis (CE) assay design further inherently limits shortening amplicons because the markers must be separated by size. Non-traditional markers, such as single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (InDels), may yield more information from challenging samples due to their smaller amplicon size. In this study, the performance of a next generation sequencing (NGS) SNP assay and CE-based STR, mini-STR, and InDel assays was evaluated with a series of fragmented, size-selected samples. Information obtained from the NGS SNP assay exhibited higher overall inverse random match probability (1/RMP) values compared to the CE-based typing assays, with particular benefit for fragment sizes ≤150 base pairs (bp). The InDel, mini-STR, and NGS SNP assays all had similar percentages of loci with reportable alleles at this level of degradation; however, the relatively fewer number of loci in the InDel and mini-STR assays results in the NGS SNP assay having at least nine orders of magnitude higher 1/RMP values. In addition, the NGS SNP assay and three CE-based assays (two STR and one InDel assay) were tested using a dilution series consisting of 0.5ng, 0.1ng, and 0.05ng non-degraded DNA. All tested assays showed similar percentages of loci with reportable alleles at these levels of input DNA; however, due to the larger number of loci, the NGS SNP assay and the larger of the two tested CE-based STR assays both resulted in considerably higher 1/RMP values than the other assays. These results indicate the potential advantage of NGS SNP assays for forensic analysis of degraded DNA samples.

A reliable, high-resolution and high-throughput genotyping method for HLA-DRB1

Human leukocyte antigen (HLA) DNA typing is an essential part to identify a donor who may be a good match for the patients who need haematopoietic stem cells from bone marrow. Thus, fetching quickly high-resolution genotype is very important at present. However, current HLA DNA typing methods especially for HLA-DRB1 generally yielded ambiguous typing results because of high degree of heterozygosity on exome region and primer pairs design limitations, which generally amplified only exon2 of HLA-DRB1 and the position of its primer pairs is on exome region. To solve this problem, we developed a reliable, high-resolution and high-throughput (RHH) sequence based typing approach for HLA-DRB1 through massively parallel paired-end sequencing. Several primer pairs were designed to amplify three exon regions, which are part of exon1, the whole region of exon2 and exon3 of HLA-DRB1, to genotyping for HLA-DRB1 by synthesis. 94 samples were included to analyze and the highly successful genotyping ratio (98.94%) and no ambiguous genotyping result demonstrated the accurate performance of our method for HLA-DRB1 genotyping.

LBP04: APPLICATION OF SINGLE MOLECULE REAL-TIME (SMRT) SEQUENCING TECHNOLOGY FOR THE FIELD 4 LEVEL GENOTYPING OF CLASSICAL HLA LOCI

Aim Super high resolution-single molecule-sequence-based typing (SS-SBT) is an HLA DNA typing method for the field 4 level of allelic resolution to efficiently detect new and null alleles without phase ambiguity by combination of long ranged PCR amplification and next generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method such as long ranged PCR system from the promoter-enhancer region to 3’UTR for 11 classical HLA loci, A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1 using NGS platforms such as Ion PGM, GS Junior and MiSeq. However, HLA alleles could not be assigned at the field 4 level in some HLA loci such as HLA-DQA1, -DPA1 and -DPB1, because SNP and indel densities to separate both of the phases were much lower than in other HLA loci. In this respect, third generation sequencers such as single molecule, real-time (SMRT®) DNA sequencer PacBio RS II (2.5∼10 kb) are expected to solve this current genotyping problem to improve the SS-SBT method. Here we report an accuracy of genotyping results obtained from SMRT® technology. Method Ten genomic DNAs (TU1∼TU10) obtained from Japanese were provided for long ranged PCR amplification using eight HLA loci specific primer pairs (A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1). Consensus sequences were obtained via HlaTools/Long Amplicon analysis such as overlapping, clustering of filtered subreads, phasing and removing PCR artifacts. Results HLA sequences obtained from 10 pooled amplicon samples applied to SMRT® technology with HlaTools/Long Amplicon analysis were compared to published reference sequences for consensus alleles. A total of 153 amplicon sequences were compared, 117 with the field 4 level sequences and 36 with the field 3 level sequences. At the field 3 level 100% of sequences were identical to reference ones while ∼70% (80/117) of sequences were identical at the field 4 level. Error sequences were mostly localized to intronic single- and di-nucleotide homopolymer regions. Conclusion The SMRT® technology is useful for highly accurate DNA typing for all the classical HLA loci.