An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection.

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.

Development of tetra-primer ARMS-PCR protocol to genotype the prolactin receptor SNP 39136666 and assessment of this SNP in Brazilian locally adapted cattle breeds / [Desenvolvimento de protocolo de tetra-primer ARMS- PCR para genotipagem do SNP 39136666 do receptor da prolactina e avaliação desse SNP em raças bovinas brasileiras localmente adaptadas]

As raças taurinas de origem ibérica Limonero e Carora (Bos primigenius taurus) possuem o fenótipo de pelo curto, liso e com baixa densidade folicular, o que confere a esses animais maior tolerância térmica e melhor produtividade em regiões quentes. Diferentes mutações associadas a esse fenótipo foram descritas no gene do receptor de prolactina PRLR, localizado no cromossomo bovino BTA20. Uma mutação recentemente encontrada é a substituição do nucleotídeo C por T, SNP 39136666 (p. R497*), no exon 11, que gera um códon de parada e, consequentemente, uma menor isoforma desse receptor. Neste trabalho, desenvolveu-se um protocolo rápido e de baixo custo para detecção desse SNP, utilizando-se a técnica de tetra-primer ARMS-PCR. Assim, foi possível detectar essa mutação nas raças brasileiras de origem ibérica localmente adaptadas: Caracu, Crioulo Lageano, Mocho Nacional e Pantaneiro. O alelo T foi mais frequente na raça Caracu (80%), enquanto o alelo C foi mais frequente na raça Crioulo Lageano (84%). Essa simples metodologia pode ser usada para genotipar esse SNP e ajudar na aplicação dessas informações moleculares em programas de melhoramento focados na tolerância térmica em bovinos taurinos e seus mestiços.(AU)

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems.IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini).

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

PCR primers designed for new world Leishmania: A systematic review.

Studies of the primers that were designed to detect New World Leishmania were systematically reviewed to report the characteristics of each target, detection limit, specificity of the primers designed and diagnostic sensibility. The papers identified in the databases PubMed and Web of Science involved 50 studies. Minicircle is the most applied target in molecular research for diagnosis, due to its high sensitivity in detecting Leishmania in different clinical samples, a characteristic that can be partially attributed to the higher number of copies of the minicircle per cell. The other molecular targets shown in this review were less sensitive to diagnostic use because of the lower number of copies of the target gene per cell, but more specific for identification of the subgenus and/or species. The choice of the best target is an important step towards the result of the research. The target allows the design of primers that are specific to the genus, subgenus or a particular species and also imparts sensitivity to the method for diagnosis. The findings of this systematic review provide the advantages and disadvantages of the main molecular targets and primers designed for New World Leishmania, offering information so that the researcher can choose the PCR system best suited to their research need. This is a timely and extremely thorough review of the primers designed for New World Leishmania.

Development of conventional and real time PCR assays for rapid species authentication of mammalian cell lines commonly used in veterinary diagnostic laboratories.

Mammalian cell lines are valuable tools in biomedical fields, with applications ranging from disease diagnosis to the production of biological reagents and vaccines. Here we report the development of new conventional (cPCR) and real time PCR (qPCR) assays for species identification of several mammalian kidney cell lines originated from swine, green monkey, hamster and bovine tissues that are extensively used in veterinary diagnostic laboratories. The PCR primers and probes were selected from highly conserved mitochondrial genes and analyzed in silico by nucleotide BLAST in the National Center for Biotechnology Information (NCBI) website to ensure target specificity. The assays were highly species-specific and had no cross-reactivity against other tested cell lines originated from different mammalian species. Assay sensitivity (limit of detection; LOD) was determined using serial dilutions of cell line DNA as template. The estimated LODs were between 2.95 and 48 pg (picogram) DNA/assay for cPCR, and between 1.5 × 10-3 and 4.8 × 10-2 pg DNA/assay for qPCR. Multiplex qPCR assays were developed for simultaneous detection of up to three species in a single assay. The multiplex qPCR assays exhibited the same sensitivity as the corresponding singleplex assays with the exception of the green monkey species that demonstrated a 10-100 fold decline in the sensitivity. Contamination of swine cells was detected in one of the rabbit cell lines. The contamination was further confirmed by Sanger and Next-Generation sequencing.

Robust reference gene design and validation for expression studies in the large milkweed bug, Oncopeltus fasciatus, upon cardiac glycoside stress.

Despite its history as a developmental and evolutionary model organism, gene expression analysis in the large milkweed bug, Oncopeltus fasciatus, has rarely been explored using quantitative real-time PCR. The strength of this method depends greatly on the endogenous controls used for normalization, which are lacking for the milkweed bug system. Here, to fill in this gap in our knowledge, we validated the stability of a set of ten candidate reference genes identified from the O. fasciatus transcriptome, and did so upon exposure to a dietary toxin, a cardiac glycoside, and across four different exposure periods. To increase robustness against gDNA contaminants, genome resources were used to design intron-bridging primers. A comprehensive stability validation by the Bestkeeper, Normfinder, geNorm and comparative ΔCt methods identified ef1a and tubulin as the most stable genes across treatments and time points, whereas 18S rRNA was the most unstable. However, accounting for the temporal scale indicated that time point confined normalizers might enable higher quantification accuracy for treatment comparison. Overall this study demonstrates: (i) a robust RT-qPCR primer design approach is possible for non-model organisms where genome annotation is often incomplete, and (ii) the importance of detailed reference gene stability exploration in multifactorial experimental designs.

Screening nested-PCR primer for 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing and application in Hunan, China.

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated "Candidatus Liberibacter spp". "Candidatus Liberibacter spp" that infect Citrus includes "Candidatus Liberibacter asiaticus" (Las), "Candidatus Liberibacter africanus" (Laf) and "Candidatus Liberibacter americanus" (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.

Application of whole genome data for in silico evaluation of primers and probes routinely employed for the detection of viral species by RT-qPCR using dengue virus as a case study.

BACKGROUND: Viral infection by dengue virus is a major public health problem in tropical countries. Early diagnosis and detection are increasingly based on quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) directed against genomic regions conserved between different isolates. Genetic variation can however result in mismatches of primers and probes with their targeted nucleic acid regions. Whole genome sequencing allows to characterize and track such changes, which in turn enables to evaluate, optimize, and (re-)design novel and existing RT-qPCR methods. The immense amount of available sequence data renders this however a labour-intensive and complex task. RESULTS: We present a bioinformatics approach that enables in silico evaluation of primers and probes intended for routinely employed RT-qPCR methods. This approach is based on analysing large amounts of publically available whole genome data, by first employing BLASTN to mine the genomic regions targeted by the RT-qPCR method(s), and afterwards using BLASTN-SHORT to evaluate whether primers and probes will anneal based on a set of simple in silico criteria. Using dengue virus as a case study, we evaluated 18 published RT-qPCR methods using more than 3000 publically available genomes in the NCBI Virus Variation Resource, and provide a systematic overview of method performance based on in silico sensitivity and specificity. CONCLUSIONS: We provide a comprehensive overview of dengue virus RT-qPCR method performance that will aid appropriate method selection allowing to take specific measures that aim to contain and prevent viral spread in afflicted regions. Notably, we find that primer-template mismatches at their 3' end may represent a general issue for dengue virus RT-qPCR detection methods that merits more attention in their development process. Our approach is also available as a public tool, and demonstrates how utilizing genomic data can provide meaningful insights in an applied public health setting such as the detection of viral species in human diagnostics.

A simplified protocol for molecular sexing in the emu (Dromaius novaehollandiae).

Male and female emus are nearly identical both as chicks and as adults. Although morphological differences of the internal genital tract can be used to distinguish the sexes, a high degree of diagnostic skill is required for accurate sexing. DNA-based sexing methods are highly accurate and can be used to diagnose sex without requiring a high degree of technical skill. However, conventional PCR-RFLP is time consuming and costly, requiring the digestion of PCR products. In this study, we simplified the protocol for sexing the emu by using multiplex PCR without restriction enzyme treatment. Multiplex PCR based on a W-specific primer, with the commonly designed primer set on both Z and W chromosomes, amplified both 197-bp and 272-bp bands in the female, and only the 272-bp band in the male. Sexing results obtained in this way were completely concordant with results obtained using the conventional PCR-RFLP method. Thus, we succeeded in simplifying the protocol for sexing the emu, and suggest that our protocol improves production efficiency by facilitating rapid pairing and selection of individuals to establish high-quality pedigrees.

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems.

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex®21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR®. Identifiler® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets.

A Culture-Independent PCR-Based Assay to Detect the Root Rot Pathogen Fusarium solani Species Complex 11 from Soybean Roots and Soil.

Fusarium solani species complex (FSSC) 11 is the primary phylogenetic species of FSSC causing root rot in soybean in the north-central United States. A polymerase chain reaction (PCR)-based assay was developed to identify and differentiate FSSC 11 from the less aggressive FSSC 5 and other Fusarium and Pythium spp. associated with soybean roots. The primer set FSSC11-F and FSSC11-R designed from the RNA polymerase second largest subunit gene yielded the expected amplicon of about 900 bp with DNA from all 22 FSSC 11 isolates tested in PCR. However, it did not produce an amplicon with DNA from 29 isolates of FSSC 5, seven other Fusarium spp., three Pythium spp., and soybean tested in PCR. Furthermore, the primer set successfully detected FSSC 11 from a DNA mixture containing the DNA of FSSC 11, FSSC 5, other Fusarium spp., and soybean. The primer set also detected FSSC 11 from both soil and soybean roots. Additionally, the prevalence of FSSC 11 in soybean roots was determined in five fields in North Dakota by both a culture-independent PCR approach with FSSC11-F and FSSC11-R and a culture-dependent approach. Results from both the culture-dependent and culture-independent approaches with FSSC11-F and FSSC11-R were consistent and revealed the presence of the FSSC 11 in three of five fields sampled.

Genome-Wide Development of MicroRNA-Based SSR Markers in Medicago truncatula with Their Transferability Analysis and Utilization in Related Legume Species.

Microsatellite (simple sequence repeats, SSRs) marker is one of the most widely used markers in marker-assisted breeding. As one type of functional markers, MicroRNA-based SSR (miRNA-SSR) markers have been exploited mainly in animals, but the development and characterization of miRNA-SSR markers in plants are still limited. In the present study, miRNA-SSR markers for Medicago truncatula (M. truncatula) were developed and their cross-species transferability in six leguminous species was evaluated. A total of 169 primer pairs were successfully designed from 130 M. truncatula miRNA genes, the majority of which were mononucleotide repeats (70.41%), followed by dinucleotide repeats (14.20%), compound repeats (11.24%) and trinucleotide repeats (4.14%). Functional classification of SSR-containing miRNA genes showed that all targets could be grouped into three Gene Ontology (GO) categories: 17 in biological process, 11 in molecular function, and 14 in cellular component. The miRNA-SSR markers showed high transferability in other six leguminous species, ranged from 74.56% to 90.53%. Furthermore, 25 Mt-miRNA-SSR markers were used to evaluate polymorphisms in 20 alfalfa accessions, and the polymorphism information content (PIC) values ranged from 0.39 to 0.89 with an average of 0.71, the allele number per marker varied from 3 to 18 with an average of 7.88, indicating a high level of informativeness. The present study is the first time developed and characterized of M. truncatula miRNA-SSRs and demonstrated their utility in transferability, these novel markers will be valuable for genetic diversity analysis, marker-assisted selection and genotyping in leguminous species.

Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods.

The performance of detection methods (culture methods and polymerase chain reaction assay) and plating media used in the same type of samples were determined as well as the specificity of PCR primers to detected Salmonella spp. contamination in layer hen farms. Also, the association of farm characteristics with Salmonella presence was evaluated. Environmental samples (feces, feed, drinking water, air, boot-swabs) and eggs were taken from 40 layer hen houses. Salmonella spp. was most detected in boot-swabs taken around the houses (30% and 35% by isolation and PCR, respectively) follow by fecal samples (15.2% and 13.6% by isolation and PCR, respectively). Eggs, drinking water, and air samples were negative for Salmonella detection. Salmonella Schwarzengrund and S. Enteritidis were the most isolated serotypes. For plating media, relative specificity was 1, and the relative sensitivity was greater for EF-18 agar than XLDT agar in feed and fecal samples. However, relative sensitivity was greater in XLDT agar than EF-18 agar for boot-swab samples. Agreement was between fair to good depending on the sample, and it was good between isolation and PCR (feces and boot-swabs), without agreement for feed samples. Salmonella spp. PCR was positive for all strains, while S. Typhimurium PCR was negative. Salmonella Enteritidis PCR used was not specific. Based in the multiple logistic regression analyses, categorization by counties was significant for Salmonella spp. presence (P-value = 0.010). This study shows the importance of considering different types of samples, plating media and detection methods during a Salmonella spp. monitoring study. In addition, it is important to incorporate the sampling of floors around the layer hen houses to learn if biosecurity measures should be strengthened to minimize the entry and spread of Salmonella in the houses. Also, the performance of some PCR methods and S. Enteritidis PCR should be improved, and biosecurity measures in hen farms must be reinforced in the region of more concentrated layer hen houses to reduce the probability of Salmonella spp. presence.

The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance.

The measurement of functional gene abundance in diverse microbial communities often employs quantitative PCR (qPCR) with highly degenerate oligonucleotide primers. While degenerate PCR primers have been demonstrated to cause template-specific bias in PCR applications, the effect of such bias on qPCR has been less well explored. We used a set of diverse, full-length nifH gene standards to test the performance of several universal nifH primer sets in qPCR. We found significant template-specific bias in all but the PolF/PolR primer set. Template-specific bias caused more than 1000-fold mis-estimation of nifH gene copy number for three of the primer sets and one primer set resulted in more than 10,000-fold mis-estimation. Furthermore, such template-specific bias will cause qPCR estimates to vary in response to beta-diversity, thereby causing mis-estimation of changes in gene copy number. A reduction in bias was achieved by increasing the primer concentration. We conclude that degenerate primers should be evaluated across a range of templates, annealing temperatures, and primer concentrations to evaluate the potential for template-specific bias prior to their use in qPCR.

Direct Visualization of RNA-DNA Primer Removal from Okazaki Fragments Provides Support for Flap Cleavage and Exonucleolytic Pathways in Eukaryotic Cells.

During DNA replication in eukaryotic cells, short single-stranded DNA segments known as Okazaki fragments are first synthesized on the lagging strand. The Okazaki fragments originate from ∼35-nucleotide-long RNA-DNA primers. After Okazaki fragment synthesis, these primers must be removed to allow fragment joining into a continuous lagging strand. To date, the models of enzymatic machinery that removes the RNA-DNA primers have come almost exclusively from biochemical reconstitution studies and some genetic interaction assays, and there is little direct evidence to confirm these models. One obstacle to elucidating Okazaki fragment processing has been the lack of methods that can directly examine primer removal in vivo In this study, we developed an electron microscopy assay that can visualize nucleotide flap structures on DNA replication forks in fission yeast (Schizosaccharomyces pombe). With this assay, we first demonstrated the generation of flap structures during Okazaki fragment processing in vivo The mean and median lengths of the flaps in wild-type cells were ∼51 and ∼41 nucleotides, respectively. We also used yeast mutants to investigate the impact of deleting key DNA replication nucleases on these flap structures. Our results provided direct in vivo evidence for a previously proposed flap cleavage pathway and the critical function of Dna2 and Fen1 in cleaving these flaps. In addition, we found evidence for another previously proposed exonucleolytic pathway involving RNA-DNA primer digestion by exonucleases RNase H2 and Exo1. Taken together, our observations suggest a dual mechanism for Okazaki fragment maturation in lagging strand synthesis and establish a new strategy for interrogation of this fascinating process.

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities.

UNLABELLED: The use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3' end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest. IMPORTANCE: The quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

Bulk development and stringent selection of microsatellite markers in the western flower thrips Frankliniella occidentalis.

Recent improvements in next-generation sequencing technologies have enabled investigation of microsatellites on a genome-wide scale. Faced with a huge amount of candidates, the use of appropriate marker selection criteria is crucial. Here, we used the western flower thrips Frankliniella occidentalis for an empirical microsatellite survey and validation; 132,251 candidate microsatellites were identified, 92,102 of which were perfect. Dinucleotides were the most abundant category, while (AG)n was the most abundant motif. Sixty primer pairs were designed and validated in two natural populations, of which 30 loci were polymorphic, stable, and repeatable, but not all in Hardy-Weinberg equilibrium (HWE) and linkage equilibrium. Four marker panels were constructed to understand effect of marker selection on population genetic analyses: (i) only accept loci with single nucleotide insertions (SNI); (ii) only accept the most polymorphic loci (MP); (iii) only accept loci that did not deviate from HWE, did not show SNIs, and had unambiguous peaks (SS) and (iv) all developed markers (ALL). Although the MP panel resulted in microsatellites of highest genetic diversity followed by the SNI, the SS performed best in individual assignment. Our study proposes stringent criteria for selection of microsatellites from a large-scale number of genomic candidates for population genetic studies.

Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains.

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.