PCR Artifacts Research Articles

Benchmarking UMI-aware and standard variant callers for low frequency ctDNA variant detection

BackgroundCirculating tumour DNA (ctDNA) is a subset of cell free DNA (cfDNA) released by tumour cells into the bloodstream. Circulating tumour DNA has shown great potential as a biomarker to inform treatment in cancer patients. Collecting ctDNA is minimally invasive and reflects the entire genetic makeup of a patient’s cancer. ctDNA variants in NGS data can be difficult to distinguish from sequencing and PCR artefacts due to low abundance, particularly in the early stages of cancer. Unique Molecular Identifiers (UMIs) are short sequences ligated to the sequencing library before amplification. These sequences are useful for filtering out low frequency artefacts. The utility of ctDNA as a cancer biomarker depends on accurate detection of cancer variants.ResultsIn this study, we benchmarked six variant calling tools, including two UMI-aware callers for their ability to call ctDNA variants. The standard variant callers tested included Mutect2, bcftools, LoFreq and FreeBayes. The UMI-aware variant callers benchmarked were UMI-VarCal and UMIErrorCorrect. We used both datasets with known variants spiked in at low frequencies, and datasets containing ctDNA, and generated synthetic UMI sequences for these datasets. Variant callers displayed different preferences for sensitivity and specificity. Mutect2 showed high sensitivity, while returning more privately called variants than any other caller in data without synthetic UMIs – an indicator of false positive variant discovery. In data encoded with synthetic UMIs, UMI-VarCal detected fewer putative false positive variants than all other callers in synthetic datasets. Mutect2 showed a balance between high sensitivity and specificity in data encoded with synthetic UMIs.ConclusionsOur results indicate UMI-aware variant callers have potential to improve sensitivity and specificity in calling low frequency ctDNA variants over standard variant calling tools. There is a growing need for further development of UMI-aware variant calling tools if effective early detection methods for cancer using ctDNA samples are to be realised.

DNA typing of cyanoacrylate fumed latent fingerprints using GlobalFiler™ and ForenSeq™ Signature Prep kits.

DNA typing of latent fingerprints is highly desirable to increase chances of individualization. We recovered DNA from Cyanoacrylate (CA) fumed fingerprints and used both GlobalFiler™ and ForenSeq™ DNA Signature Prep kits for DNA typing. For GlobalFiler™, samples were processed using a protocol modified for Low Template (LT)-DNA samples (half-volume reactions, 30 cycles) while for ForenSeq™ DNA Signature Prep, samples were processed using a standard protocol and fluorometer-based library quantitation. We evaluated genotyping success and quality of profiles in terms of completeness, Peak Height Ratio/Allele Coverage Ratio, presence of PCR artifacts and drop-in alleles. With GlobalFiler™, average autosomal STR (aSTR) profile completeness was 44.4% with 2-20 pg, 54.3% with 22-60 pg, and 95% with 64-250 pg DNA input. CODIS uploadable profiles were obtained in 2/10, 3/11, and 11/12 samples in these ranges. With ForenSeq™ DNA Signature Prep, average aSTR profile completeness was 19.7% with 1-20 pg and 45.2% with 22-47 pg but increased to 78.3% with 68-122 pg and 86.7% with 618-1000 pg DNA input. Uploadable profiles were obtained in 0/12, 4/11, 4/7, and 3/3 samples for these ranges. Results show very high sensitivity using both kits. Half-volume reactions and 30 cycles had minimal negative effect on Globalfiler™ profile quality, providing support for wider use after validation experiments to routinely improve results from LT samples. A standard protocol for the ForenSeq™ DNA Signature Prep kit was also highly successful with LT DNA obtained from CA-fumed fingerprints with additional information from isometric STR alleles and other markers.

Low cycle number multiplex PCR: A novel strategy for the construction of amplicon libraries for next-generation sequencing.

Multiplex PCR is a critical step when preparing amplicon library for next-generation sequencing. However, there are several challenges related to multiplex PCR including poor uniformity, nonspecific amplification, and primer-dimers. To address these issues, we propose a novel solution strategy that involves using a low cycle number (<10 cycles) in multiplex PCR and then employing carrier DNAs and magnetic beads for the selection of targeted products. This technique improves the amplicon uniformity while also reducing primer-dimers and PCR artifacts. To evaluate our technique, we initially utilized 120 DNA fragments from mouse genome containing single nucleotide polymorphism (SNP) sites. Sequencing results demonstrated that with only 7 cycles of multiplex PCR, 95.8% of the targeted SNP sites were mapped, with a coverage of at least 1×. The average sequencing depth of all amplicons was 1705.79±1205.30×; 87% of them reached a coverage depth that exceeded 0.2-fold of the average sequencing depth. Our method had a greater uniformity (87%) when compared to Hi-Plex PCR (53.3%). Furthermore, we validated our strategy by randomly selecting 90 primer pairs twice from the initial set of 120 primer-pairs. Next, we used the same protocol to prepare amplicon libraries. The two groups had an average sequencing depth of 1013.30±585.57× and 219.10±158.27×, respectively; over 84% of the amplicons had a sequencing depth that exceeded 0.2-fold of average depth. These results suggest that the use of a low cycle number in multiplex PCR is a cost-effective and efficient approach for the preparation of amplicon libraries.

Using unique molecular identifiers to improve allele calling in low-template mixtures

PCR artifacts are an ever-present challenge in sequencing applications. These artifacts can seriously limit the analysis and interpretation of low-template samples and mixtures, especially with respect to a minor contributor. In medicine, molecular barcoding techniques have been employed to decrease the impact of PCR error and to allow the examination of low-abundance somatic variation. In principle, it should be possible to apply the same techniques to the forensic analysis of mixtures. To that end, several short tandem repeat loci were selected for targeted sequencing, and a bioinformatic pipeline for analyzing the sequence data was developed. The pipeline notes the relevant unique molecular identifiers (UMIs) attached to each read and, using machine learning, filters the noise products out of the set of potential alleles. To evaluate this pipeline, DNA from pairs of individuals were mixed at different ratios (1−1, 1−9) and sequenced with different starting amounts of DNA (10, 1 and 0.1 ng). Naïvely using the information in the molecular barcodes led to increased performance, with the machine learning resulting in an additional benefit. In concrete terms, using the UMI data results in less noise for a given amount of drop out. For instance, if thresholds are selected that filter out a quarter of the true alleles, using read counts accepts 2381 noise alleles and using raw UMI counts accepts 1726 noise alleles, while the machine learning approach only accepts 307.

Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing.

Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3'-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts.

Peptide nucleic acid–dependent artifact can lead to false-positive triplex gene editing signals

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

The impact of DNA extract homogenization and replication on marine sediment metabarcoding diversity and heterogeneity

AbstractMetabarcoding of environmental DNA (eDNA) is an attractive complement to morphological methods for surveys and routine monitoring of marine sediment benthic communities. However, metabarcoding and other genetic techniques are heavily affected by choices made during sampling, processing, and analysis. Here, we investigated the effect of different eDNA extraction protocols on observed alpha‐ and beta diversity of replicates from the same grab. Specifically, we compared (A) homogenization intensity during sediment DNA extraction, (B) extraction replicates vs larger sediment extraction volume, and (C) pre‐ and post‐PCR extract pooling. Using the 18S V1‐V2 region marker, we show that a Precellys homogenizer protocol during DNA extraction can significantly improve sediment metabarcoding results in terms of captured diversity and inter‐replicate homogeneity compared to vortexing only. This effect superseded that of increased sediment extract volume. Pre‐PCR pooling of DNA extraction replicates increased observed rarefied richness compared to data from single extracts only, but not to the extent of sample extract replicates amplified individually before pooling. We argue that this discrepancy was due to a reduction both in recovered sample diversity, but also the number of PCR artifacts and PCR drift. Our results demonstrate that extraction replicates of smaller sediment volumes, in combination with moderate Precellys homogenization and pre‐PCR pooling, are a cost‐effective way to increase the amount of organism diversity that is recovered from sediment eDNA metabarcoding samples.

Use and Misuse of Cq in qPCR Data Analysis and Reporting.

In the analysis of quantitative PCR (qPCR) data, the quantification cycle (Cq) indicates the position of the amplification curve with respect to the cycle axis. Because Cq is directly related to the starting concentration of the target, and the difference in Cq values is related to the starting concentration ratio, the only results of qPCR analysis reported are often Cq, ΔCq or ΔΔCq values. However, reporting of Cq values ignores the fact that Cq values may differ between runs and machines, and, therefore, cannot be compared between laboratories. Moreover, Cq values are highly dependent on the PCR efficiency, which differs between assays and may differ between samples. Interpreting reported Cq values, assuming a 100% efficient PCR, may lead to assumed gene expression ratios that are 100-fold off. This review describes how differences in quantification threshold setting, PCR efficiency, starting material, PCR artefacts, pipetting errors and sampling variation are at the origin of differences and variability in Cq values and discusses the limits to the interpretation of observed Cq values. These issues can be avoided by calculating efficiency-corrected starting concentrations per reaction. The reporting of gene expression ratios and fold difference between treatments can then easily be based on these starting concentrations.

Validated removal of nuclear pseudogenes and sequencing artefacts from mitochondrial metabarcode data.

Metabarcoding of Metazoa using mitochondrial genes may be confounded by both the accumulation of PCR and sequencing artefacts and the co-amplification of nuclear mitochondrial pseudogenes (NUMTs). The application of read abundance thresholds and denoising methods is efficient in reducing noise accompanying authentic mitochondrial amplicon sequence variants (ASVs). However, these procedures do not fully account for the complex nature of concomitant sequences and the highly variable DNA contribution of specimens in a metabarcoding sample. We propose, as a complement to denoising, the metabarcoding Multidimensional Abundance Threshold Evaluation (metaMATE) framework, a novel approach that allows comprehensive examination of multiple dimensions of abundance filtering and the evaluation of the prevalence of unwanted concomitant sequences in denoised metabarcoding datasets. metaMATE requires a denoised set of ASVs as input, and designates a subset of ASVs as being either authentic (mitochondrial DNA haplotypes) or nonauthentic ASVs (NUMTs and erroneous sequences) by comparison to external reference data and by analysing nucleotide substitution patterns. metaMATE (i) facilitates the application of read abundance filtering strategies, which are structured with regard to sequence library and phylogeny and applied for a range of increasing abundance threshold values, and (ii) evaluates their performance by quantifying the prevalence of nonauthentic ASVs and the collateral effects on the removal of authentic ASVs. The output from metaMATE facilitates decision-making about required filtering stringency and can be used to improve the reliability of intraspecific genetic information derived from metabarcode data. The framework is implemented in the metaMATE software (available at https://github.com/tjcreedy/metamate).

A novel workflow to improve genotyping of multigene families in wildlife species: An experimental set-up with a known model system.

Genotyping complex multigene families in novel systems is particularly challenging. Target primers frequently amplify simultaneously multiple loci leading to high PCR and sequencing artefacts such as chimeras and allele amplification bias. Most genotyping pipelines have been validated in nonmodel systems whereby the real genotype is unknown and the generation of artefacts may be highly repeatable. Further hindering accurate genotyping, the relationship between artefacts and genotype complexity (i.e. number of alleles per genotype) within a PCR remains poorly described. Here, we investigated the latter by experimentally combining multiple known major histocompatibility complex (MHC) haplotypes of a model organism (chicken, Gallus gallus, 43 artificial genotypes with 2-13 alleles per amplicon). In addition to well-defined 'optimal' primers, we simulated a nonmodel species situation by designing 'cross-species' primers based on sequence data from closely related Galliform species. We applied a novel open-source genotyping pipeline (ACACIA; https://gitlab.com/psc_santos/ACACIA), and compared its performance with another, previously published pipeline (AmpliSAS). Allele calling accuracy was higher when using ACACIA (98.5% versus 97% and 77.8% versus 75% for the 'optimal' and 'cross-species' data sets, respectively). Systematic allele dropout of three alleles owing to primer mismatch in the 'cross-species' data set explained high allele calling repeatability (100% when using ACACIA) despite low accuracy, demonstrating that repeatability can be misleading when evaluating genotyping workflows. Genotype complexity was positively associated with nonchimeric artefacts, chimeric artefacts (nonlinearly by levelling when amplifying more than 4-6 alleles) and allele amplification bias. Our study exemplifies and demonstrates pitfalls researchers should avoid to reliably genotype complex multigene families.

Evocation of DNA from the Blood Sample Mixed with Road Concrete- A Forensic Review

Several types of biological samples are recovered in different conditions in various types of crimes. Sometimes, due to mishandling of the evidence they do not produce good results. Blood is a common type of biological sample found at crime scene involving hitand-run, murder and various sexual assaults, etc. In the cases such as hit and run, the blood gets mixed up with the soil and act as resistance in obtaining proper DNA profiling of a victim or a suspect. The advanced technologies for DNA profiling like STR analysis help in the identification of a criminal even if there is not much quantity of biological sample from the crime scene. But non-scientific procedure of blood collection and preservation reduces the chances of amplification of DNA. Additionally, in many outdoor cases blood sample contaminated with soil becomes problematic because of the presence of humic acid in soil. Humic acid inhibits the amplification of DNA and leads to unsuccessful profiling of DNA. The inhibitors in the samples act as obstacle in the cases where blood is lifted from the surface of earth and lead to the unsuccessful DNA analysis. PCR artifacts like partial profiles, multi-peaks, or complete failure of DNA profile can be seen in STR profiles obtained from contaminated samples. In this study, we reviewed the blood samples recovered from different surfaces (wall of plaster, cemented floor pieces, black road concrete) over a period of 5 to 36 months. This study concludes that the sample containing less soil particles yield DNA higher than the samples containing high amount of soil particles.

High throughput error corrected Nanopore single cell transcriptome sequencing

Droplet-based high throughput single cell sequencing techniques tremendously advanced our insight into cell-to-cell heterogeneity. However, those approaches only allow analysis of one extremity of the transcript after short read sequencing. In consequence, information on splicing and sequence heterogeneity is lost. To overcome this limitation, several approaches that use long-read sequencing were introduced recently. Yet, those techniques are limited by low sequencing depth and/or lacking or inaccurate assignment of unique molecular identifiers (UMIs), which are critical for elimination of PCR bias and artifacts. We introduce ScNaUmi-seq, an approach that combines the high throughput of Oxford Nanopore sequencing with an accurate cell barcode and UMI assignment strategy. UMI guided error correction allows to generate high accuracy full length sequence information with the 10x Genomics single cell isolation system at high sequencing depths. We analyzed transcript isoform diversity in embryonic mouse brain and show that ScNaUmi-seq allows defining splicing and SNVs (RNA editing) at a single cell level.

Compatibility of the ForenSeq™ DNA Signature Prep Kit with laser microdissected cells: An exploration of issues that arise with samples containing low cell numbers

Massively parallel sequencing is rapidly emerging as a valuable tool in forensic DNA analyses. As part of our validation of this technology, we established its compatibility with a laser microdissection cell collection method including a one-tube DNA extraction process. We also used the laser microdissector to explore the number of cells required to generate informative DNA sequence profiles and establish the limitations of the technology. Using the ForenSeq™ DNA Signature Prep Kit (Primer Mix B) and a MiSeq FGx™ sequencer, we successfully demonstrated the compatibility of MPS with a laser microdissection one-tube extraction method, with minor alterations made to the manufacturer’s recommended library preparation protocol, including the addition of magnesium chloride to counteract the effect of dithiothreitol on amplification efficacy.This work highlighted several quality issues that may be encountered when preparing sequencing libraries from low quantity DNA samples, particularly that libraries prepared from low cell numbers showed high levels of adapter dimer compared to those prepared from more cells. To remediate this, we replaced the bead normalisation step with a qPCR normalisation method, whereby sequencing libraries are diluted based on their molarity as determined after library purification.The work presented here focuses on the results from the autosomal and Y STR markers as these could be directly compared to results obtained from traditional capillary electrophoresis techniques. Full autosomal STR DNA sequence profiles (27 loci) could be obtained from 50 epithelial cells and 100 spermatozoa (sperm cells). The limit of detection for the ForenSeq™ system was determined to be 25 epithelial and 25 sperm cells for both autosomal and Y STRs. Cells were dissected from both single source samples and mixtures of semen and saliva. There was no apparent difference in sensitivity, presence of contamination or PCR artefacts between libraries prepared from single source samples and libraries prepared from mixed source samples.

High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach.

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR. Unfortunately, this approach is very often coupled with PCR artifacts which compromise overall efficiency of site-directed mutagenesis. To reduce the failure rate due to the PCR artifacts, we have set up a high-throughput mutagenesis protocol based on a two-fragment PCR approach. To this end, each mutation is introduced in two separate PCRs resulting in two linear fragments of the mutated plasmid. In the next steps, the PCR template is digested and the two matching plasmid fragments are joined together using Gibson assembly. Separating the corresponding mutagenic primers into two different PCRs decreases a number of artifacts and thus increases overall cloning efficiency. Furthermore, free software that we developed facilitates both high-throughput primer design and analysis of sequencing results. Overall, this protocol enabled us to efficiently produce several alanine-scanning libraries of 400 single-point mutations with complete coverage of the protein sequence.

UMI-Gen: A UMI-based read simulator for variant calling evaluation in paired-end sequencing NGS libraries

MotivationWith Next Generation Sequencing becoming more affordable every year, NGS technologies asserted themselves as the fastest and most reliable way to detect Single Nucleotide Variants (SNV) and Copy Number Variations (CNV) in cancer patients. These technologies can be used to sequence DNA at very high depths thus allowing to detect abnormalities in tumor cells with very low frequencies. Multiple variant callers are publicly available and are usually efficient at calling out variants. However, when frequencies begin to drop under 1%, the specificity of these tools suffers greatly as true variants at very low frequencies can be easily confused with sequencing or PCR artifacts. The recent use of Unique Molecular Identifiers (UMI) in NGS experiments has offered a way to accurately separate true variants from artifacts. UMI-based variant callers are slowly replacing raw-read based variant callers as the standard method for an accurate detection of variants at very low frequencies. However, benchmarking done in the tools publication are usually realized on real biological data in which real variants are not known, making it difficult to assess their accuracy. ResultsWe present UMI-Gen, a UMI-based read simulator for targeted sequencing paired-end data. UMI-Gen generates reference reads covering the targeted regions at a user customizable depth. After that, using a number of control files, it estimates the background error rate at each position and then modifies the generated reads to mimic real biological data. Finally, it will insert real variants in the reads from a list provided by the user. AvailabilityThe entire pipeline is available at https://gitlab.com/vincent-sater/umigen under MIT license.

Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish

Homology-directed (HD) genome modification offers an opportunity to precisely modify the genome. Despite reported successful cases, for many loci, precise genome editing remains challenging and inefficient in vivo. Here we report an effort to precisely knock-in a GFP reporter into gad locus mediated by CRISPR/Cas9 system in the zebrafish Danio rerio. PCR artifact was detected in testing for homologous recombination (HR), but was mitigated by optimizing PCR condition and decreasing the injected targeting plasmid concentration. Under this optimized condition, time course analysis revealed a decline of the HR-positive embryos at embryogenesis progressed. GFP signals also diminished at later developmental stages. The GFP signals were consistent with PCR detection, both of which suggested the loss of targeted insertion events at later stages. Such loss of insertion might be one underlying reason for the inability to obtain germ-line transgenic lines with GFP knocked into the gad locus. Our results suggest that the low HR efficiency associated with CRISPR-mediated knock-in is in part due to loss of insertion after targeted integration into the gad locus.

Statistical modeling of STR capillary electrophoresis signal

BackgroundIn order to isolate an individual’s genotype from a sample of biological material, most laboratories use PCR and Capillary Electrophoresis (CE) to construct a genetic profile based on polymorphic loci known as Short Tandem Repeats (STRs). The resulting profile consists of CE signal which contains information about the length and number of STR units amplified. For samples collected from the environment, interpretation of the signal can be challenging given that information regarding the quality and quantity of the DNA is often limited. The signal can be further compounded by the presence of noise and PCR artifacts such as stutter which can mask or mimic biological alleles. Because manual interpretation methods cannot comprehensively account for such nuances, it would be valuable to develop a signal model that can effectively characterize the various components of STR signal independent of a priori knowledge of the quantity or quality of DNA.ResultsFirst, we seek to mathematically characterize the quality of the profile by measuring changes in the signal with respect to amplicon size. Next, we examine the noise, allele, and stutter components of the signal and develop distinct models for each. Using cross-validation and model selection, we identify a model that can be effectively utilized for downstream interpretation. Finally, we show an implementation of the model in NOCIt, a software system that calculates the a posteriori probability distribution on the number of contributors.ConclusionThe model was selected using a large, diverse set of DNA samples obtained from 144 different laboratory conditions; with DNA amounts ranging from a single copy of DNA to hundreds of copies, and the quality of the profiles ranging from pristine to highly degraded. Implemented in NOCIt, the model enables a probabilisitc approach to estimating the number of contributors to complex, environmental samples.

Estimation of genotyping errors of STR markers in dogs and wolves

This article will discuss the estimation of genotyping errors for Mini-DogFiler panel A and B in group of wolfs (n = 39) where the source material for DNA isolation were faeces and control group of dogs (n = 170), represented by Czechoslovakian wolfdog breed (CSW), where DNA was obtained from buccal swabs.DNA samples isolated from buccal swabs had a very low frequency of genotyping error estimated as mean error rate per allele (ea = 0.09 %), mean error per locus (el = 0.18 %) and error rate per multilocus genotype (eobs = 2.17 %). Completely different situation was observed for DNA samples isolated from faeces and affirms how complicated is to get reliable results from such material. Total performance was very poor where only 7 out of 39 samples replicated (∼18 %) at least 2 times out of 4 replicates. In such case would be eobs = 86.5 %. For this type of samples is typical very fragmented DNA with low concentration. This fact leads to the occurrence of many of negative effects like an allelic dropout, null allele or presence of PCR artefacts. Results confirm necessity of multiple DNA isolates from one sample and a multiple amplification of these samples for creating a consensus for each genotype.Moreover, both types of samples suffered by relatively high frequency of allelic dropout occurring in locus VGL2136, but it did not increase error rate because no signal was detected in all cases. The cause of such behaviour remained unknown but it raised the question whether is this locus appropriate for analysis of wolf and CSW samples.

Forensic analysis of MPS mtDNA data using QIAGEN biomedical genomics workbench and AQME tool – preliminary results

Massively Parallel Sequencing (MPS) allows to sequence the total mitochondrial DNA (mtDNA) through specific panels. Human Mitochondria Panel is one of the QIAseq Targeted DNA panels developed by QIAGEN for MPS, which have an optimized chemistry based on the integration of unique molecular indices (UMIs). However, the major problem is the analysis of the MPS data and, therefore, it became necessary to create bioinformatics tools that would enable mtDNA analysis such as the AQME tool to be used with the QIAGEN Biomedical Genomics Workbench software. In this study, a coverage comparison workflow was tested to verify the coverage differences between two mtDNA panels, QIAseq Human Mitochondria Panel and Precision ID mtDNA Whole Genome Panel. The preliminary results showed a significant decrease in the Precision ID coverage, after the bioinformatics removal of PCR artifacts. For the QIAseq panel, this coverage variation was lower, since the UMIs enable to know more accurately the initial number of molecules in the original mtDNA sequence.

MPS reveals isometric PCR artefacts in degraded samples

It is well known that DNA damage promotes PCR artefacts. In addition, as MPS provides the most accurate typing of PCR products, this technology should be able to highlight more PCR artefacts than the conventional CE approach. To test this hypothesis, a DNA sample was degraded by heating at 70 °C for 8, 16 and 24 h. The three resulting samples #1, #2 and #3 and the untreated control sample were then analysed by the Precision ID Globalfiler NGS STR Panel v2, in duplicate tests. The data analysis was performed by the Converge v2.1 software using the default setting parameters.Artefacts were identified only in the degraded samples #2 and #3. Few well known artefacts (allelic imbalance, stutter products and allelic drop out) and several drop in phenomena were flagged by the software. The majority (18 out 22) of these drop ins were sequenced as “isometric artefacts”, e.g. PCR artefacts which cannot be identified by the employment of CE. The molecular features of these PCR artefacts, as well as their risk to be confused with a DNA mixture, are described.