Have the cake and eat it: Optimizing nondestructive DNA metabarcoding of macroinvertebrate samples for freshwater biomonitoring

DNA metabarcoding can contribute to improving cost‐effectiveness and accuracy of biological assessments of aquatic ecosystems, but significant optimization and standardization efforts are still required to mainstream its application into biomonitoring programmes. In assessments based on freshwater macroinvertebrates, a key challenge is that DNA is often extracted from cleaned, sorted and homogenized bulk samples, which is time‐consuming and may be incompatible with sample preservation requirements of regulatory agencies. Here, we optimize and evaluate metabarcoding procedures based on DNA recovered from 96% ethanol used to preserve field samples and thus including potential PCR inhibitors and nontarget organisms. We sampled macroinvertebrates at five sites and subsampled the preservative ethanol at 1 to 14 days thereafter. DNA was extracted using column‐based enzymatic (TISSUE) or mechanic (SOIL) protocols, or with a new magnetic‐based enzymatic protocol (BEAD), and a 313‐bp COI fragment was amplified. Metabarcoding detected at least 200 macroinvertebrate taxa, including most taxa detected through morphology and for which there was a reference barcode. Better results were obtained with BEAD than SOIL or TISSUE, and with subsamples taken 7–14 than 1–7 days after sampling, in terms of DNA concentration and integrity, taxa diversity and matching between metabarcoding and morphology. Most variation in community composition was explained by differences among sites, with small but significant contributions of subsampling day and extraction method, and negligible contributions of extraction and PCR replication. Our methods enhance reliability of preservative ethanol as a potential source of DNA for macroinvertebrate metabarcoding, with a strong potential application in freshwater biomonitoring.     

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.     

Towards next-generation biodiversity assessment using DNA metabarcoding

Virtually all empirical ecological studies require species identification during data collection. DNA metabarcoding refers to the automated identification of multiple species from a single bulk sample containing entire organisms or from a single environmental sample containing degraded DNA (soil, water, faeces, etc.). It can be implemented for both modern and ancient environmental samples. The availability of next-generation sequencing platforms and the ecologists' need for high-throughput taxon identification have facilitated the emergence of DNA metabarcoding. The potential power of DNA metabarcoding as it is implemented today is limited mainly by its dependency on PCR and by the considerable investment needed to build comprehensive taxonomic reference libraries. Further developments associated with the impressive progress in DNA sequencing will eliminate the currently required DNA amplification step, and comprehensive taxonomic reference libraries composed of whole organellar genomes and repetitive ribosomal nuclear DNA can be built based on the well-curated DNA extract collections maintained by standardized barcoding initiatives. The near-term future of DNA metabarcoding has an enormous potential to boost data acquisition in biodiversity research.     

Detection of multiple mycetoma pathogens using fungal metabarcoding analysis of soil DNA in an endemic area of Sudan

Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis, and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii, in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.     

DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimized.     

Establishing arthropod community composition using metabarcoding: Surprising inconsistencies between soil samples and preservative ethanol and homogenate from Malaise trap catches

DNA metabarcoding allows the analysis of insect communities faster and more efficiently than ever before. However, metabarcoding can be conducted through several approaches, and the consistency of results across methods has rarely been studied. We compare the results obtained by DNA metabarcoding of the same communities using two different markers – COI and 16S – and three different sampling methods: (a) homogenized Malaise trap samples (homogenate), (b) preservative ethanol from the same samples, and (c) soil samples. Our results indicate that COI and 16S offer partly complementary information on Malaise trap samples, with each marker detecting a significant number of species not detected by the other. Different sampling methods offer highly divergent estimates of community composition. The community recovered from preservative ethanol of Malaise trap samples is significantly different from that recovered from homogenate. Small and weakly sclerotized insects tend to be overrepresented in ethanol while strong and large taxa are overrepresented in homogenate. For soil samples, highly degenerate COI primers pick up large amounts of nontarget DNA and only 16S provides adequate analyses of insect diversity. However, even with 16S, very little overlap in molecular operational taxonomic unit (MOTU) content was found between the trap and the soil samples. Our results demonstrate that none of the tested sampling approaches is satisfactory on its own. For instance, DNA extraction from preservative ethanol is not a valid replacement for destructive bulk extraction but a complement. In future metabarcoding studies, both should ideally be used together to achieve comprehensive representation of the target community.     

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.     

Enhancing DNA metabarcoding performance and applicability with bait capture enrichment and DNA from conservative ethanol

Metabarcoding is often presented as an alternative identification tool to compensate for coarse taxonomic resolution and misidentification encountered with traditional morphological approaches. However, metabarcoding comes with two major impediments which slow down its adoption. First, the picking and destruction of organisms for DNA extraction are time and cost consuming and do not allow organism conservation for further evaluations. Second, current metabarcoding protocols include a PCR enrichment step which induces errors in the estimation of species diversity and relative biomasses. In this study, we first evaluated the capacity of capture enrichment to replace PCR enrichment using controlled freshwater macrozoobenthos mock communities. Then, we tested if DNA extracted from the fixative ethanol (etDNA) of the same mock communities can be used as an alternative to DNA extracted from pools of whole organisms (bulk DNA). We show that capture enrichment provides more reliable and accurate representation of species occurrences and relative biomasses in comparison with PCR enrichment for bulk DNA. While etDNA does not permit to estimate relative biomasses, etDNA and bulk DNA provide equivalent species detection rates. Thanks to its robustness to mismatches, capture enrichment is already an efficient alternative to PCR enrichment for metabarcoding and, if coupled to etDNA, is a time‐saver option in studies where presence information only is sufficient.     

A practical guide to DNA metabarcoding for entomological ecologists

1. DNA metabarcoding is a cost-effective species identification approach with great potential to assist entomological ecologists. This review presents a practical guide to help entomological ecologists design their own DNA metabarcoding studies and ensure that sound ecological conclusions can be obtained. 2. The review considers approaches to field sampling, laboratory work, and bioinformatic analyses, with the aim of providing the background knowledge needed to make decisions at each step of a DNA metabarcoding workflow. 3. Although most conventional sampling methods can be adapted to DNA metabarcoding, this review highlights techniques that will ensure suitable DNA preservation during field sampling and laboratory storage. The review also calls for a greater understanding of the occurrence, transportation, and deposition of environmental DNA when applying DNA metabarcoding approaches for different ecosystems. 4. Accurate species detection with DNA metabarcoding needs to consider biases introduced during DNA extraction and PCR amplification, cross-contamination resulting from inappropriate amplicon library preparation, and downstream bioinformatic analyses. Quantifying species abundance with DNA metabarcoding is in its infancy, yet recent studies demonstrate promise for estimating relative species abundance from DNA sequencing reads. 5. Given that bioinformatics is one of the biggest hurdles for researchers new to DNA metabarcoding, several useful graphical user interface programs are recommended for sequence data processing, and the application of emerging sequencing technologies is discussed.     

An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.     

DNA isolation from soil samples for cloning in different hosts

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.     

Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids

Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.     

基于环境DNA宏条形码技术的辽东湾典型围海养殖池塘内水母多样性研究

研究使用环境DNA宏条形码技术(eDNA metabarcoding)检测辽东湾东北部河口区围海养殖池塘水母种类多样性,探索适用于水母种类物种鉴定和监测的新方法。利用环境DNA宏条形码技术,分别基于18S rDNA和COI宏条形码检测了辽东湾东北部河口区围海养殖池塘水母种类多样性,通过水样采集、过滤、eDNA提取、遗传标记扩增、测序与生物信息分析的环境DNA宏条形码标准化分析流程,从围海养殖池塘7个采样点中获得可检测的采样点数据。结果显示,基于18S rDNA宏条形码检测出8种水母种类,其中钵水母纲大型水母2种、水螅水母总纲小型水母6种;基于COI宏条形码技术共检测出19种水母种类,其中钵水母纲大型水母5种、水螅水母总纲小型水母14种;两种DNA条形码标记都显示养殖种类海蜇(Rhopilema esculentum)为优势种。研究结果表明,环境DNA宏条形码技术作为一种新兴的生物多样性监测手段可用于快速检测水母种类多样性,在水母类物种鉴定、监测及早期预警中有较大的应用潜能。     

An effective protocol to solve the problem in genomic DNA isolation of tung tree

Tung tree (Vernicia fordii) is a native and oil-producing woody plant in China. The oil is industrially important and promising biodiesel raw material. However, until recently the lack of effective protocols for the extraction of genomic DNA had made DNA-based molecular studies of tung tree difficult. Here, four conventional protocols and one novel protocol were compared for their capacity in isolating DNA from tung tree leaves of different age. Our results showed that all the four conventional protocols could isolate DNA from old leaves, two from matured leaves, but none from young leaves. However, the detectable DNA samples contained many contaminations, leading to overestimation of DNA concentration measured by ultraviolet spectrophotometer, also interfering with the downstream PCR reaction. The novel protocol could produce high-yield and good-quality DNA from tung tree leaves regardless of leaf age. Its key steps were that a single leaf tissue sample could be recycled for DNA extraction for up to four times, and correspondingly four DNA precipitations (termed as the 1st, 2nd, 3rd and 4th DNA sample, respectively) were conducted. All the four DNA samples of a single tissue were good template for PCR reaction. The novel protocol is an effective method for genomic DNA isolation of tung tree.     

Optimization and validation of a cost‐effective protocol for biosurveillance of invasive alien species

Environmental DNA (eDNA) metabarcoding has revolutionized biodiversity monitoring and invasive pest biosurveillance programs. The introduction of insect pests considered invasive alien species (IAS) into a non‐native range poses a threat to native plant health. The early detection of IAS can allow for prompt actions by regulating authorities, thereby mitigating their impacts. In the present study, we optimized and validated a fast and cost‐effective eDNA metabarcoding protocol for biosurveillance of IAS and characterization of insect and microorganism diversity. Forty‐eight traps were placed, following the CFIA''s annual forest insect trapping survey, at four locations in southern Ontario that are high risk for forest IAS. We collected insects and eDNA samples using Lindgren funnel traps that contained a saturated salt (NaCl) solution in the collection jar. Using cytochrome c oxidase I (COI) as a molecular marker, a modified Illumina protocol effectively identified 2,535 Barcode Index Numbers (BINs). BINs were distributed among 57 Orders and 304 Families, with the vast majority being arthropods. Two IAS (Agrilus planipennis and Lymantria dispar) are regulated by the Canadian Food Inspection Agency (CFIA) as plant health pests, are known to occur in the study area, and were identified through eDNA in collected traps. Similarly, using 16S ribosomal RNA and nuclear ribosomal internal transcribed spacer (ITS), five bacterial and three fungal genera, which contain species of regulatory concern across several Canadian jurisdictions, were recovered from all sampling locations. Our study results reaffirm the effectiveness and importance of integrating eDNA metabarcoding as part of identification protocols in biosurveillance programs.     

Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils

The presence of certain metabolites has been observed to interfere with DNA isolation procedures and downstream reactions such as DNA restriction, amplification and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yields with a single protocol, and thus, even closely related species may require different isolation protocols. Here we describe the essential steps of a rapid DNA isolation protocol that can be used for diverse medicinal and aromatic plants, which produce essential oils and secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides, terpenes and quinones. The procedure is applicable to dry as well as fresh plant tissues. This protocol, in our experiments, permitted isolation of DNA from tissues of diverse plant species and produced fairly good yields. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

Testing multiple substrates for terrestrial biodiversity monitoring using environmental DNA metabarcoding

Biological surveys based on visual identification of the biota are challenging, expensive and time consuming, yet crucial for effective biomonitoring. DNA metabarcoding is a rapidly developing technology that can also facilitate biological surveys. This method involves the use of next generation sequencing technology to determine the community composition of a sample. However, it is uncertain as to what biological substrate should be the primary focus of metabarcoding surveys. This study aims to test multiple sample substrates (soil, scat, plant material and bulk arthropods) to determine what organisms can be detected from each and where they overlap. Samples (n = 200) were collected in the Pilbara (hot desert climate) and Swan Coastal Plain (hot Mediterranean climate) regions of Western Australia. Soil samples yielded little plant or animal DNA, especially in the Pilbara, probably due to conditions not conducive to long‐term preservation. In contrast, scat samples contained the highest overall diversity with 131 plant, vertebrate and invertebrate families detected. Invertebrate and plant sequences were detected in the plant (86 families), pitfall (127 families) and vane trap (126 families) samples. In total, 278 families were recovered from the survey, 217 in the Swan Coastal Plain and 156 in the Pilbara. Aside from soil, 22%–43% of the families detected were unique to the particular substrate, and community composition varied significantly between substrates. These results demonstrate the importance of selecting appropriate metabarcoding substrates when undertaking terrestrial surveys. If the aim is to broadly capture all biota then multiple substrates will be required.     

Tracking earthworm communities from soil DNA

Earthworms are known for their important role within the functioning of an ecosystem, and their diversity can be used as an indicator of ecosystem health. To date, earthworm diversity has been investigated through conventional extraction methods such as handsorting, soil washing or the application of a mustard solution. Such techniques are time consuming and often difficult to apply. We showed that combining DNA metabarcoding and next-generation sequencing facilitates the identification of earthworm species from soil samples. The first step of our experiments was to create a reference database of mitochondrial DNA (mtDNA) 16S gene for 14 earthworm species found in the French Alps. Using this database, we designed two new primer pairs targeting very short and informative DNA sequences (about 30 and 70 bp) that allow unambiguous species identification. Finally, we analysed extracellular DNA taken from soil samples in two localities (two plots per locality and eight samples per plot). The two short metabarcode regions led to the identification of a total of eight earthworm species. The earthworm communities identified by the DNA-based approach appeared to be well differentiated between the two localities and are consistent with results derived from inventories collected using the handsorting method. The possibility of assessing earthworm communities from hundreds or even thousands of localities through the use of extracellular soil DNA will undoubtedly stimulate further ecological research on these organisms. Using the same DNA extracts, our study also illustrates the potential of environmental DNA as a tool to assess the diversity of other soil-dwelling animal taxa.     

DNA extraction from soil: comparison of different methods using spore-forming bacteria and theswrAA gene as indicators

Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of theswrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodium-dodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L) expressing theStSy gene for resveratrol production and thebar gene for Basta® tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genusBacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.     

Reliable noninvasive genotyping based on excremental PCR of nuclear DNA purified with a magnetic bead protocol

A new protocol for extraction of DNA from faeces is presented. The protocol involves gentle washing of the surface of the faeces followed by a very simple DNA extraction utilizing the wash supernatant as the source of DNA. Unlike most other protocols, it does not involve the use of proteinase K and/or organic extraction, but is instead based on adsorption of the DNA to magnetic beads. The protocol was tested by microsatellite genotyping across six loci for sheep and reindeer faeces. Comparison with DNA extracted from blood demonstrated that the protocol was very reliable, even when used on material stored for a long time. The protocol was compared with another simple, solid-phase DNA-binding protocol, with the result that the bead-based protocol gave a slightly better amplification success and a lower frequency of allelic drop-outs. Furthermore, our experiments showed that the surface wash prior to DNA extraction is a crucial step, not only for our protocol, but for other solid-phase protocols as well.