Number Of Amplification Cycles Research Articles

Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Descriptive analysis of Cycle Threshold in patients with hematologic malignancies infected with SARS-COV-2

SARS-CoV-2 has caused over 200 million documented infections, more than 4 million deaths, and unprecedented consequences worldwide. The cycle threshold (Ct), the number of amplification cycles required to obtain a product detectable through fluorescence during a quantitative RT-PCR test, is an indirect measurement of viral load. Patients with hematologic malignancies have an increased risk of death by the SARS-CoV-2. We conducted a retrospective, observational, descriptive analysis of the Ct obtained from patients with history of hematologic malignancies who tested positive for SARS-CoV-2 in our hospital, from March 3rd, 2020, to August 17th, 2021. We used the mean Ct at diagnosis. 15 adults, with previous diagnosis of lymphomas, acute leukemias and chronic lymphocytic leukemia, were included. 9 of the 15 patients (60 %) developed pneumonia, 6 of them required supplementary oxygen and 5 mechanical ventilation. 5 patients died between 7-86 days from symptom onset. Ct was lower among the group of patients who died (15.5 cycles; SD= 2.28, CI95%= 9.17-21.86) compared with those who survived (20.2 cycles; SD= 8.87, CI95%= 13.9-26.6). Ct was also lower in the pneumonia group (18.2 cycles; SD= 2.28, CI95%= 12.98-23.51) than in the no-pneumonia group (19.3 cycles; SD= 4.11; CI95%= 8.73-29.9). Ct was lowest in severe forms of CoViD-19. Further studies with larger populations of patients with hematologic malignancies could establish the validity of Ct as a quantitative laboratory determination as a course-prediction and infectivity tool.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer.

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIMTo use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODSThe primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.RESULTSThe best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.CONCLUSIONIn summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.

Quantitative detection of Vibrio parahaemolyticus in aquatic products by duplex droplet digital PCR combined with propidium monoazide

Based on the combination of droplet digital PCR (ddPCR) and propidium monoazide (PMA), a new method called PMA-duplex ddPCR was developed for the quantitative determination of viable Vibrio parahaemolyticus in aquatic products. Specific primers and probes targeting a new gene, vp0488, coding for putative DNA polymerase III beta chain, which was self-screened by this laboratory, and a traditional target gene, thermolabile hemolysin (tlh), were designed and verified. The duplex ddPCR system and conditions and PMA treatment conditions were optimized. The limit of detection (LOD) for PMA-duplex ddPCR was evaluated and applied to detect V. parahaemolyticus in artificially contaminated samples and actual samples. The results indicate that the optimal duplex ddPCR annealing temperature was 60 °C, the optimal number of amplification cycles was 40, and the optimal ratio of primer to probe was 1:0.5. The optimal PMA treatment conditions were as follows: incubation time 5 min, concentration 30 μmol/L, and exposure time 15 min. The LOD of PMA-duplex ddPCR (7.32 × 101 CFU/mL) was lower than that of duplex quantitative PCR (qPCR) (7.32 × 102 CFU/mL). The lowest detectable V. parahaemolyticus in oysters was 8.15 × 101 CFU/g without any pre-enrichment. The method established in this study has the advantages of good specificity, high sensitivity and accuracy, which provides an effective method for the detection of viable Vibrio parahaemolyticus in aquatic foods.

Rapid SARS-CoV-2 diagnosis using disposable strips and a metal-oxide-semiconductor field-effect transistor platform.

The SARS-CoV-2 pandemic has had a significant impact worldwide. Currently, the most common detection methods for the virus are polymerase chain reaction (PCR) and lateral flow tests. PCR takes more than an hour to obtain the results and lateral flow tests have difficulty with detecting the virus at low concentrations. In this study, 60 clinical human saliva samples, which included 30 positive and 30 negative samples confirmed with RT-PCR, were screened for COVID-19 using disposable glucose biosensor strips and a reusable printed circuit board. The disposable strips were gold plated and functionalized to immobilize antibodies on the gold film. After functionalization, the strips were connected to the gate electrode of a metal-oxide-semiconductor field-effect transistor on the printed circuit board to amplify the test signals. A synchronous double-pulsed bias voltage was applied to the drain of the transistor and strips. The resulting change in drain waveforms was converted to digital readings. The RT-PCR-confirmed saliva samples were tested again using quantitative PCR (RT-qPCR) to determine cycling threshold (Ct) values. Ct values up to 45 refer to the number of amplification cycles needed to detect the presence of the virus. These PCR results were compared with digital readings from the sensor to better evaluate the sensor technology. The results indicate that the samples with a range of Ct values from 17.8 to 35 can be differentiated, which highlights the increased sensitivity of this sensor technology. This research exhibits the potential of this biosensor technology to be further developed into a cost-effective, point-of-care, and portable rapid detection method for SARS-CoV-2.

Clinical Course of COVID-19 and Cycle Threshold in Patients with Haematological Neoplasms

The SARS-CoV-2 viral load in a respiratory sample can be inversely quantified using the cycle threshold (Ct), defined as the number of amplification cycles required to detect the viral genome in a quantitative PCR assay using reverse transcriptase (RT-qPCR). It may be classified as high (Ct < 25), intermediate (25-30) and low (Ct > 30). We describe the clinical course of 3 patients with haematological neoplasms who contracted COVID-19. None of them had been vaccinated. Firstly, a 22-year-old male with a refractory acute lymphoblastic leukaemia experienced an oligosymptomatic COVID-19 and had a Ct of 23 with an ascending curve. Another male, aged 23, had recently begun treatment for a promyelocytic leukaemia. He had a subacute course with high oxygen requirements. His Ct dropped from 28, when he only experienced fever, to 14.8, during the most critical period and on the edge of ventilatory support. Viral clearance was documented 126 days after the beginning of the symptoms. Finally, a 60-year-old male had received rituximab as maintenance therapy for a follicular lymphoma 3 months before contracting COVID-19. He had a fulminant course and required mechanical ventilation a few days later. We highlight the association between the course of CoViD-19 and the Ct. Viral shedding was longer than in immunocompetent hosts.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

Evaluating tests for diagnosing COVID-19 in the absence of a reliable reference standard: pitfalls and potential solutions

Evaluating tests for diagnosing COVID-19 in the absence of a reliable reference standard: pitfalls and potential solutions

The Truth about SARS-CoV-2 Cycle Threshold Values Is Rarely Pure and Never Simple.

Nucleic acid amplification tests (NAATs) are the reference standard methods for SARS-CoV-2 detection because of their high analytical sensitivity and specificity. Many NAATs, which use reverse-transcription real-time PCR (RT-PCR), are clinically validated, technically validated, and authorized by the U.S. Food & Drug Administration (FDA) to be interpreted qualitatively as "detected" (positive for SARS-CoV-2 RNA) or "not detected" (negative for SARS-CoV-2 RNA). As of this writing there are over 250 SARS-CoV-2 molecular diagnostic tests that have obtained emergency use authorization from the FDA. The primary results generated by RT-PCR are fluorescent light emissions; serial detection of this fluorescence is plotted and the amplification curves visualized. Positive or negative interpretation depends on whether or not the curve exceeds a specified signal threshold. Part of the resulting process includes determination of the number of cycles needed before the fluorescent signal crosses this threshold (Ct value). In general, the more viral RNA in the initial specimen, the fewer the number of amplification cycles required to generate a positive signal; thus, the lower the Ct value, the higher the viral burden in the primary sample. Though all current SARS-CoV-2 NAATs are authorized only for qualitative interpretation, as of 10 December 2020, the FDA explicitly states that the Ct value results may be reported by the clinical laboratory in addition to the qualitative interpretation. Throughout the pandemic, many scientists, physicians, politicians, and public citizens have attempted to emphasize the importance (or unimportance) of certain pandemic-related interventions, mitigation strategies, the disease itself, and testing approaches. Some have advocated that a specific variable is most important in a testing approach and should be maximized to the potential detriment of the others: analytical sensitivity and specificity, cost, turnaround time, sample type, or accessibility of collection. If the truth was obvious, then there would be little debate, but the debate has been important and earnest. As Oscar Wilde wrote, "The truth is rarely pure and never simple." We suggest that this quote describes the current situation on the debate over the relevance of Ct values, and we will explore the clinical utility of quantitative SARS-CoV-2 testing here.

A two-step PCR assembly for construction of gene variants across large mutational distances.

Construction of empirical fitness landscapes has transformed our understanding of genotype–phenotype relationships across genes. However, most empirical fitness landscapes have been constrained to the local genotype neighbourhood of a gene primarily due to our limited ability to systematically construct genotypes that differ by a large number of mutations. Although a few methods have been proposed in the literature, these techniques are complex owing to several steps of construction or contain a large number of amplification cycles that increase chances of non-specific mutations. A few other described methods require amplification of the whole vector, thereby increasing the chances of vector backbone mutations that can have unintended consequences for study of fitness landscapes. Thus, this has substantially constrained us from traversing large mutational distances in the genotype network, thereby limiting our understanding of the interactions between multiple mutations and the role these interactions play in evolution of novel phenotypes. In the current work, we present a simple but powerful approach that allows us to systematically and accurately construct gene variants at large mutational distances. Our approach relies on building-up small fragments containing targeted mutations in the first step followed by assembly of these fragments into the complete gene fragment by polymerase chain reaction (PCR). We demonstrate the utility of our approach by constructing variants that differ by up to 11 mutations in a model gene. Our work thus provides an accurate method for construction of multi-mutant variants of genes and therefore will transform the studies of empirical fitness landscapes by enabling exploration of genotypes that are far away from a starting genotype.

Coamplification of Mycobacterium leprae genome sections by real-time PCR: Detection of the pathogen and the possibility of a semi-quantitative assessment of the bacterial load

Aim. To develop a method for coamplification of single-copy genes and repetitive elements of the Mycobacterium leprae genome in the analysis of clinical material from leprosy patients with an assessment of the clinical significance of the study results. Materials and methods . Skin scarification and biopsy samples from patient R. with a diagnosis of “A30.5 Leprosy. Multibacillary form. Lepromatous type. Active stage” were used as empirical material for the study. A search for M. leprae DNA in the clinical material was performed by the method of real-time PCR (RT-PCR) using primers and hydrolysis probes for the single-copy species-specific genes rpoB (encodes the β -subunit of bacterial RNA polymerase), sodA (encodes the superoxide dismutase enzyme) and mntH (encodes the manganese transport protein), as well as for RLEP — the non-coding repetitive element of the genome . Results. Using various RT-PCR assays, consistent results were obtained concerning the presence or absence of M. leprae DNA in the studied clinical samples. The high sensitivity of PCR was confirmed for the detection of the repetitive element RLEP compared to the single-copy genes rpoB , sodA and mntH , which consists in reducing the number of amplification cycles (Ct) needed for exceeding the threshold fluorescence value of hydrolysis probes and leading to the maximum intensity of the fluorescence signal. When constructing standard graphs for calibrating the accumulation of a fluorescent signal for simultaneously analyzed portions of the M. leprae genome in dilutions from 1 to 1,000, significant differences in the results of co-amplification were noted depending on the quantitative presence of the DNA being detected. Conclusion. Coamplification of M. leprae genome sections with varying degrees of copy number variation by the RTPCR method provides for effective detection of the M. leprae DNA in clinical material and forms a basis for a quantitative assessment of the bacterial load in skin scarification and biopsy samples.

Genome-wide SNP typing of ancient DNA: Determination of hair and eye color of Bronze Age humans from their skeletal remains.

A genome-wide high-throughput single nucleotide polymorphism (SNP) typing method was tested with respect of the applicability to ancient and degraded DNA. The results were compared to mini-sequencing data achieved through single base extension (SBE) typing. The SNPs chosen for the study allow to determine the hair colors and eye colors of humans. The DNA samples were extracted from the skeletal remains of 59 human individuals dating back to the Late Bronze Age. The 3,000 years old bones had been discovered in the Lichtenstein Cave in Lower Saxony, Germany. The simultaneous typing of 24 SNPs for each of the ancient DNA samples was carried out using the 192.24 Dynamic Array™ by Fluidigm®. Thirty-eight of the ancient samples (=64%) revealed full and reproducible SNP genotypes allowing hair and eye color phenotyping. In 10 samples (=17%) at least half of the SNPs were unambiguously determined, in 11 samples (=19%) the SNP typing failed. For 23 of the 59 individuals, a comparison of the SNP typing results with genotypes from an earlier performed SBE typing approach was possible. The comparison confirmed the full concordance of the results for 90% of the SNP typings. In the remaining 10% allelic dropouts were identified. The high genotyping success rate could be achieved by introducing modifications to the preamplification protocol mainly by increasing the DNA input and the amplification cycle number. The occurrence of allelic dropouts indicates that a further increase of DNA input to the preamplification step is desirable.

Evaluating and Correcting Inherent Bias of microRNA Expression in Illumina Sequencing Analysis.

microRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3′-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3′-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3′-adaptor ratios displayed a steady value of 0.3 +/− 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum.

\xb5LAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5\u2009minutes

We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.

Field Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Platform for the Detection of Schistosoma japonicum Infection in Oncomelania hupensis Snails.

Schistosoma infection in snails can be monitored by microscopy or indirectly by sentinel mice. As both these approaches can miss infections, more sensitive tests are needed, particularly in low-level transmission settings. In this study, loop-mediated isothermal amplification (LAMP) technique, designed to detect a specific 28S ribosomal Schistosoma japonicum (Sj28S) gene with high sensitivity, was compared to microscopy using snail samples from 51 areas endemic for schistosomiasis in five Chinese provinces. In addition, the results were compared with those from polymerase chain reaction (PCR) by adding DNA sequencing as a reference. The testing of pooled snail samples with the LAMP assay showed that a dilution factor of 1/50, i.e., one infected snail plus 49 non-infected ones, would still result in a positive reaction after the recommended number of amplification cycles. Testing a total of 232 pooled samples, emanating from 4006 snail specimens, showed a rate of infection of 6.5%, while traditional microscopy found only 0.4% positive samples in the same materials. Parallel PCR analysis confirmed the diagnostic accuracy of the LAMP assay, with DNA sequencing even giving LAMP a slight lead. Microscopy and the LAMP test were carried out at local schistosomiasis-control stations, demonstrating that the potential of the latter assay to serve as a point-of-care (POC) test with results available within 60–90 min, while the more complicated PCR test had to be carried out at the National Institute of Parasitic Diseases (NIPD) in Shanghai, China. In conclusion, LAMP was found to be clearly superior to microscopy and as good as, or better than, PCR. As it can be used under field conditions and requires less time than other techniques, LAMP testing would improve and accelerate schistosomiasis control.

726. Viral Genomic Load, Cytokine Profiles and Life-Threatening Respiratory Syncytial Virus Infection in Previously Healthy Infants

BackgroundData are controversial regarding the role of viral load and the host immune response in the severity of respiratory syncytial virus (RSV) infection. The objective of this study was t. o analyze the relationship between viral load (VL) and host cytokine responses with RSV life-threatening disease (LTD).MethodsProspective cohort study including previously healthy infants <12 months, hospitalized with a first RSV infection in 2017. Viral titers were assessed by qPCR and cytokine levels measured in nasopharyngeal aspirates obtained on admission. All patients with LTD were admitted to the intensive care unit.ResultsFifty-one patients, median age 3 months (IQR 2–4), 29(56.9%) male. Eight developed LTD (1,569 LTD cases/10,000 RSV-hospitalizations/year [95% CI 702–2.859]). Antibiotic prescription was significantly higher (42.9 vs. 87.5%, P < 0.001) and length of hospitalization significantly prolonged (5.2 ± 1.9 vs. 16.1 ± 12.7 days, P < 0.001) in infants with LTD. No differences were seen in the number of amplification cycles needed for a positive qPCR test (CT) nor in the viral titers of patients with LTD compared with those with better outcome (P = 0.71), Figure 1. VL was not a predictor of LTD (AUC = 0.53); however, no LTD was seen with ≤159,200 copies/mL. CT/VL did not correlate with other outcomes (Figure 2). IFN-y levels (Th1 response) were significantly lower in infants with LTD (P = 0.034). We detected no differences in TNF-α (pro-inflammatory), IL-9, IL-13 (Th2), IL-10 or IL-17 (regulatory) levels from mildly/severely or extremely severe ill patients; lower IL-5 levels were seen in LTD (Figure 3). VL correlated with TNF-α (P < 0.001) and IL-10 (P < 0.001) levels. After logistic regression analysis, socioeconomic, pregnancy and infant variables showed no association with bad outcomes; only frequent consumption of fruits and vegetables during pregnancy conferred protection (aOR 0.03; P < 0.001).ConclusionRSV titers did not correlate with LTD. Lower levels of IFN-γ were associated with increased disease severity.These findings could provide additional data for future RSV preventive and therapeutic strategies. Disclosures All authors: No reported disclosures.

DPCR - the digital polymerase chain reaction.

Quantitative real-time PCR (qPCR) is based on the assumption that the amplification of DNA target molecules is exponential. DNA targets may be quantified by comparing the number of amplification cycles required to achieve a predetermined signal threshold to that obtained for a calibrant. However, many factors complicate this calculation, creating uncertainties and inaccuracies. Digital PCR (dPCR) is a modification of the qPCR method that can be employed to quantify precisely defined nucleic acid targets. The technique is based on the concept of limiting dilutions, which involves the partitioning of a PCR reaction into multiple sub-reactions such that each sub-reaction either contains none or one or more DNA targets. Following thermal cycling, reactions are classified as either positive (target detected) or negative (no target detected), hence providing the basis for a digital output format. By determining the proportion of empty partitions, Poissonian statistics can be applied and the initial number of target molecules present can be estimated.

Microsatellite Length Scoring by Single Molecule Real Time Sequencing - Effects of Sequence Structure and PCR Regime.

Microsatellites are DNA sequences consisting of repeated, short (1–6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.

Preferential amplification of repetitive DNA during whole genome sequencing library creation from historic samples

Repetitive microsatellite DNA forms a universal component of eukaryote genomes and specific biochemical properties of such repeat regions may influence the outcome of laboratory protocols. The Atlantic cod (Gadus morhua) genome contains an order of magnitude more dinucleotide repeats than the majority of vertebrates, with over eight percent of its genome that can be classified as either AC or AG dinucleotide repeat. We find that the abundance of these repeats can be inflated in ancient DNA (aDNA) whole genome sequencing (WGS) data generated from this species, in particular in samples with a lower fragment length. This inflation is suppressed by a reduced number of amplification cycles and by the inclusion of manufactured dinucleotide repeat oligonucleotides during amplification. These data indicate that a biased amplification reaction leads to artificially high levels of AC and AG repeats. This process appears to be particularly efficient in Atlantic cod –likely due to its high genomic content of repeats with relatively simple sequence complexity. While the extend of such bias in other studies is unclear, we nonetheless urge caution when quantifying repeat content in aDNA WGS data, given that amplification bias can be difficult to detect if this process affects more complex repeat structures than dinucleotide repeats.

Selection of reference genes in Saccharopolyspora spinosa for real-time PCR

Reverse transcription quantitative PCR(RT-qPCR)combined with the published genome information of Saccharopolyspora spinosa can allow sophisticated studies about S. spinosa, including studying the regulation of spinosyn biosynthesis, finding new target genes for engineering, and discovering and exploiting other macrolide secondary metabolites. Studies have demonstrated that appropriate internal control is needed to normalize target genes at transcription levels. However, many studies have shown that no single reference gene is universal for all strains under all experimental conditions. Thus, eight candidate reference genes of three different S. spinosa strains in two different cultures were studied to find suitable reference gene(s). The number of amplification cycles of these candidate genes was calculated by BestKeeper, NormFinder and geNorm. The results indicated that the most suitable reference genes for normalization during the fermentation of S. spinosa were 16S rRNA and rbL13.