Efficient extraction of adventitious virus nucleic acid using commercially available methods.

An essential step in pharmaceutical product development is screening for contamination with adventitious agents, and there is desire to develop highly sensitive assays to detect adventitious viral nucleic acid. This study sought to examine the nucleic acid extraction efficiency of three viral candidates in relevant background matrices using four different extraction methods. Three model adventitious viruses, Minute virus of Mice, Porcine Circovirus, and Feline Leukemia Virus, were diluted within a variety of background matrices relevant to pharmaceutical production methods. Upon extraction, the nucleic acid was quantified using droplet digital PCR methods. Four nucleic acid extraction methods were assessed, including commercially available kits and manual extraction methods. Each method recovered nucleic acid post-extraction for each of the model viruses within the tested background matrices. The silica-column based method recovered a greater amount of viral nucleic acid, compared to the other methods tested. Similar trends were observed when model virus was diluted in bioreactor supernatant, which replicates industry testing conditions and provides details on which extraction methods might be used in Next Generation Sequencing and PCR methods for detecting contamination within pharmaceutical products.

Reducing Bias and Quantifying Uncertainty in Fluorescence Produced by PCR.

We present a new approach for relating nucleic-acid content to fluorescence in a real-time Polymerase Chain Reaction (PCR) assay. By coupling a two-type branching process for PCR with a fluorescence analog of Beer's Law, the approach reduces bias and quantifies uncertainty in fluorescence. As the two-type branching process distinguishes between complementary strands of DNA, it allows for a stoichiometric description of reactions between fluorescent probes and DNA and can capture the initial conditions encountered in assays targeting RNA. Analysis of the expected copy-number identifies additional dynamics that occur at short times (or, equivalently, low cycle numbers), while investigation of the variance reveals the contributions from liquid volume transfer, imperfect amplification, and strand-specific amplification (i.e., if one strand is synthesized more efficiently than its complement). Linking the branching process to fluorescence by the Beer's Law analog allows for an a priori description of background fluorescence. It also enables uncertainty quantification (UQ) in fluorescence which, in turn, leads to analytical relationships between amplification efficiency (probability) and limit of detection. This work sets the stage for UQ-PCR, where both the input copy-number and its uncertainty are quantified from fluorescence kinetics.

Rapid production and free distribution of a synthetic RNA material to support SARS-CoV-2 molecular diagnostic testing.

In response to the COVID-19 pandemic, the National Institute of Standards and Technology released a synthetic RNA material for SARS-CoV-2 in June 2020. The goal was to rapidly produce a material to support molecular diagnostic testing applications. This material, referred to as Research Grade Test Material 10169, was shipped free of charge to laboratories across the globe to provide a non-hazardous material for assay development and assay calibration. The material consisted of two unique regions of the SARS-CoV-2 genome approximately 4 kb nucleotides in length. The concentration of each synthetic fragment was measured using RT-dPCR methods and confirmed to be compatible with RT-qPCR methods. In this report, the preparation, stability, and limitations of this material are described.

Evidence for multi-copy Mega-NUMTs in the human genome.

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.

A history of adventitious agent contamination and the current methods to detect and remove them from pharmaceutical products.

Preventing adventitious agents from contaminating pharmaceutical products has been an important goal of regulatory agencies and industry for decades. Contamination of these products does not only erode consumer trust but also can have potentially serious health consequences. There are a wide variety of adventitious agents that can contaminate many different classifications of products, with each combination requiring different techniques for prevention or detection of adventitious agent contamination. This review seeks to give a brief overview of adventitious agents that have contaminated released pharmaceutical products, explain the different products that are at risk of contamination, then describe the methods commonly used for the prevention and detection of adventitious agent contamination.

One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation.

PURPOSE: To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. METHODS: An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)-based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. RESULTS: In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. CONCLUSION: The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

Affine analysis for quantitative PCR measurements.

Motivated by the current COVID-19 health crisis, we consider data analysis for quantitative polymerase chain-reaction (qPCR) measurements. We derive a theoretical result specifying the conditions under which all qPCR amplification curves (including their plateau phases) are identical up to an affine transformation, i.e. a multiplicative factor and horizontal shift. We use this result to develop a data analysis procedure for determining when an amplification curve exhibits characteristics of a true signal. The main idea behind this approach is to invoke a criterion based on constrained optimization that assesses when a measurement signal can be mapped to a master reference curve. We demonstrate that this approach: (i) can decrease the fluorescence detection threshold by up to a decade; and (ii) simultaneously improve confidence in interpretations of late-cycle amplification curves. Moreover, we demonstrate that the master curve is transferable reference data that can harmonize analyses between different labs and across several years. Application to reverse-transcriptase qPCR measurements of a SARS-CoV-2 RNA construct points to the usefulness of this approach for improving confidence and reducing limits of detection in diagnostic testing of emerging diseases. Graphical Abstract Left: a collection of qPCR amplification curves. Right: Example of data collapse after affine transformation.

Sequence-based US population data for the SE33 locus.

A set of 1036 U.S. Population Samples were sequenced using the Illumina ForenSeq DNA Signature Prep Kit. This sample set has been highly characterized using a variety of marker systems for human identification. The FASTQ files obtained from a ForenSeq DNA Signature Prep Kit experiment include several STR loci that are not reported in the associated software. These include SE33, DXS8377, DXS10148, DYS456, and DYS461. The sequence variation within the autosomal STR marker SE33 was evaluated using a customized bioinformatic approach to identify and characterize the locus in the 1036 data set. The analysis identified 53 unique alleles by length and 264 by sequence. An additional 10 alleles were detected when selected extended flanking regions were examined to resolve discordances. Allele frequencies and SE33 sequence motif patterns are reported for the 1036 data set. The comparison of numerical allele calls derived from sequence data to the allele calls obtained from commercial capillary electrophoresis-based STR typing kits resulted in 100% concordance, after manual data review and confirmation sequencing of three flanking region deletions. The analysis of this data set involved significant manual sequence curation and information support from length-based genotypes to ensure high confidence in the sequence-based allele calls. The challenges of interpreting the sequence data for SE33 consisted of high sequence noise, allele-size dependent variance in coverage, and heterozygote imbalance. As allele length increased, sequence depth of coverage and quality decreased at the terminal end. Accordingly, heterozygous genotype imbalance increased in proportion to increased distance between alleles.

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms.

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

A collaborative study on the precision of the Markov chain Monte Carlo algorithms used for DNA profile interpretation.

Several fully continuous probabilistic genotyping software (PGS) use Markov chain Monte Carlo algorithms (MCMC) to assign weights to different proposed genotype combinations at a locus. Replicate interpretations of the same profile in these software are expected not to produce identical weights and likelihood ratio (LR) values due to the Monte Carlo aspect. This paper reports a detailed precision study under reproducibility conditions conducted as a collaborative exercise across the National Institute of Standards and Technology (NIST), Federal Bureau of Investigation (FBI), and Institute of Environmental Science and Research (ESR). Replicate interpretations generated across the three laboratories used the same input files, software version, and settings but different random number seed and different computers. This work demonstrates that using different computers to analyze replicate interpretations does not contribute to any variations in LR values. The study quantifies the magnitude of differences in the assigned LRs that is only due to run-to-run MCMC variability and addresses the potential explanations for the observed differences.

Digital PCR for the characterization of reference materials.

Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the absolute concentration of a specific nucleic acid sequence in a background of non-target sequences, making it ideal for the characterization of nucleic acid-based reference materials. National Metrology Institutes are increasingly using dPCR to characterize and certify their reference materials, as it offers several advantages over indirect methods, such as UV-spectroscopy. While dPCR is gaining widespread adoption, it requires optimization and has certain limitations and considerations that users should be aware of when characterizing reference materials. This review highlights the technical considerations of dPCR, as well as its role when developing and characterizing nucleic acid-based reference materials.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method.

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1-15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

DNA purification from crude samples for human identification using gradient elution isotachophoresis.

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.

Allele frequencies for 40 autosomal SNP loci typed for US population samples using electrospray ionization mass spectrometry.

AIM: To type a set of 194 US African American, Caucasian, and Hispanic samples (self-declared ancestry) for 40 autosomal single nucleotide polymorphism (SNP) markers intended for human identification purposes. METHODS: Genotyping was performed on an automated commercial electrospray ionization time-of-flight mass spectrometer, the PLEX-ID. The 40 SNP markers were amplified in eight unique 5plex PCRs, desalted, and resolved based on amplicon mass. For each of the three US sample groups statistical analyses were performed on the resulting genotypes. RESULTS: The assay was found to be robust and capable of genotyping the 40 SNP markers consuming approximately 4 nanograms of template per sample. The combined random match probabilities for the 40 SNP assay ranged from 10-16 to 10-21. CONCLUSION: The multiplex PLEX-ID SNP-40 assay is the first fully automated genotyping method capable of typing a panel of 40 forensically relevant autosomal SNP markers on a mass spectrometry platform. The data produced provided the first allele frequencies estimates for these 40 SNPs in a National Institute of Standards and Technology US population sample set. No population bias was detected although one locus deviated from its expected level of heterozygosity.

US Population Data for 94 Identity-Informative SNP Loci.

The US National Institute of Standards and Technology (NIST) analyzed a set of 1036 samples representing four major US population groups (African American, Asian American, Caucasian, and Hispanic) with 94 single nucleotide polymorphisms (SNPs) used for individual identification (iiSNPs). The compact size of iiSNP amplicons compared to short tandem repeat (STR) markers increases the likelihood of successful amplification with degraded DNA samples. Allele frequencies and relevant forensic statistics were calculated for each population group as well as the aggregate population sample. Examination of sequence data in the regions flanking the targeted SNPs identified additional variants, which can be combined with the target SNPs to form microhaplotypes (multiple phased SNPs within a short-read sequence). Comparison of iiSNP performance with and without flanking SNP variation identified four amplicons containing microhaplotypes with observed heterozygosity increases of greater than 15% over the targeted SNP alone. For this set of 1036 samples, comparison of average match probabilities from iiSNPs with the 20 CODIS core STR markers yielded an estimate of 1.7 × 10-38 for iiSNPs (assuming independence between all 94 SNPs), which was four orders of magnitude lower (more discriminating) than STRs where internal sequence variation was considered, and 10 orders of magnitude lower than STRs using established capillary electrophoresis length-based genotypes.

Sequence-based allelic variations and frequencies for 22 autosomal STR loci in the Lebanese population.

This is the first study that characterizes the sequence-based allelic variations of 22 autosomal Short Tandem Repeat (aSTR) loci in a population dataset collected from Lebanon. Genomic DNA extracts from 195 unrelated Lebanese individuals were amplified with PowerSeq 46GY System Prototype. Targeted amplicons were subjected to DNA library preparation and sequenced on the Verogen MiSeq FGx Sequencing System. Raw FASTQ data files were processed by STRait Razor v3. Sequence strings were annotated according to the considerations of the DNA Commission of the International Society for Forensic Genetics (ISFG) and tabulated herein with their respective allelic frequencies and GeneBank accession and version numbers. The sequenced Lebanese dataset resulted in 429 distinct allelic sequences as compared to the 236 alleles identified by length only. The increase in the number of alleles was observed at 18 out of 22 aSTR loci and was attributed to the sequence variations residing in both the STR repeat motifs and flanking regions. The study uncovered 25 novel aSTR allelic sequences across 12 loci for which GenBank records did not previously exist in the STRSeq BioProject, PRJNA380127. For a concordance check, the length-based allelic calls derived from the full sequences were compared to those genotyped using capillary electrophoresis (CE) methods. Population genetic parameters relevant to the evaluation of forensic DNA evidence were assessed for the sequence-based data and compared to the parameters generated from the length-based information. Using the sequence-based data, Analysis of MOlecular VAriance (AMOVA), genetic distances, and population genetic structure were evaluated for 1231 individuals sampled from the Lebanese and four U.S. populations (African American, Asian, Caucasian, and Hispanic). The results were tabulated and visualized in a population tree, multidimensional scaling scatter plots, and bar plots. This newly established sequence-based database for the Lebanese population can be beneficial for extending NGS applicability to casework or paternity testing and assessing the strength of evidence for NGS-STR profiles. The described novel sequence variants at certain loci can further help in the effort to characterize the sequence diversity of STR markers from different populations around the world.