Master Sequence Research Articles

Quasispecies productivity.

The quasispecies theory is a helpful concept in the explanation of RNA virus evolution and behaviour, with a relevant impact on methods used to fight viral diseases. It has undergone some adaptations to integrate new empirical data, especially the non-deterministic nature of mutagenesis, and the variety of behavioural motifs in cooperation, competition, communication, innovation, integration, and exaptation. Also, the consortial structure of quasispecies with complementary roles of memory genomes of minority populations better fits the empirical data than did the original concept of a master sequence and its mutant spectra. The high productivity of quasispecies variants generates unique sequences that never existed before and will never exist again. In the present essay, we underline that such sequences represent really new ontological entities, not just error copies of previous ones. Their primary unique property, the incredible variant production, is suggested here as quasispecies productivity, which replaces the error-replication narrative to better fit into a new relationship between mankind and living nature in the twenty-first century.

Analysis of Junior High School Students' Errors in Solving Mathematical Literacy Problems based on Newman Procedure Viewed from Students’ Learning Styles

This study aimed (1) to determine students' mathematical literacy skills viewed from learning styles; (2) to know and describe students' mistakes in solving mathematical literacy questions based on Newman procedure viewed from students' learning styles; and (3) to know the causes of students' mistakes in solving mathematical literacy questions based on Newman procedures viewed from learning styles. This research was a qualitative research with a case study type and used a descriptive approach. The subjects of this study were 53 students of class VIII, 11 Public Junior High School, Tegal, Indonesia. The data were collected using questionnaires, tests, and interviews, which were then analysed. The results showed that: (1) the students' learning styles were visual, auditory, kinesthetic, and auditory-kinesthetic; (2) visual learning style students have the best mathematical literacy skills because they reach the lowest level 3 and the highest level 4, whereas kinesthetic learning style students made the most mistakes; and (3) the causes of students' errors in solving mathematical literacy questions viewed from their learning styles tend to be the same, i.e.: students do not master patterns and sequence material, and students felt rushed when working on the questions.

Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

In-Host HEV Quasispecies Evolution Shows the Limits of Mutagenic Antiviral Treatments.

Here, we report the in-host hepatitis E virus (HEV) quasispecies evolution in a chronically infected patient who was treated with three different regimens of ribavirin (RBV) for nearly 6 years. Sequential plasma samples were collected at different time points and subjected to RNA extraction and deep sequencing using the MiSeq Illumina platforms. Specifically, we RT-PCR amplified a single amplicon from the core region located in the open-reading frame 2 (ORF2). At the nucleotide level (genotype), our analysis showed an increase in the number of rare haplotypes and a drastic reduction in the frequency of the master (most represented) sequence during the period when the virus was found to be insensitive to RBV treatment. Contrarily, at the amino acid level (phenotype), our study revealed conservation of the amino acids, which is represented by a high prevalence of the master sequence. Our findings suggest that using mutagenic antivirals concomitant with high viral loads can lead to the selection and proliferation of a rich set of synonymous haplotypes that express the same phenotype. This can also lead to the selection and proliferation of conservative substitutions that express fitness-enhanced phenotypes. These results have important clinical implications, as they suggest that using mutagenic agents as a monotherapy treatment regimen in the absence of sufficiently effective viral inhibitors can result in diversification and proliferation of a highly diverse quasispecies resistant to further treatment. Therefore, such approaches should be avoided whenever possible.

Minimizing the sum of completion times on a single machine with health index and flexible maintenance operations

This paper is motivated by the development of Industry 4.0 and the need to better integrate production and maintenance decisions. Our problem considers a single machine on which jobs of different families are scheduled to minimize the sum of completion times. The machine has a health index which decreases when jobs are processed. To restore the machine health, maintenance operations must be scheduled. Moreover, to be scheduled, each job requires the machine to have a minimum health index which depends on the job family. Two cases are studied: (1) The daily case with a single flexible maintenance operation, and (2) The weekly case with two flexible maintenance operations. The second case is shown to be NP-complete. Two Mixed Integer Linear Programming models are presented for each case. The first model uses “classical” positional variables, while the second model improves the first model by using the notion of master sequence. Different valid inequalities are also proposed. Computational experiments show that the second model is much more efficient than the first model when solved with a standard solver, and the impact of the valid inequalities is discussed.

Holocene and Upper Pleistocene stratigraphy of Xochimilco lake, central Mexico

The lacustrine system developed in the basin of Mexico has been of great interest in studies of various geological and social disciplines. Its sediments preserve the history of the environmental changes that occurred during the late Quaternary. Between 2016 and 2018, a set of lacustrine sediment cores were collected in the Xochimilco sub-basin, located south of the basin of Mexico. From these sedimentary sequences, a master sequence was constructed that represents the upper 66.5 m of the lacustrine deposits. For the establishment of its stratigraphic column, 15 lithotypes were defined from their sedimentary components in which the clastic components made up of silicates dominate, mostly silt and clay in size. The most abundant biogenic fraction is diatoms that frequently form oozes, and ostracods are a common component in most of the sequence, which locally form hashes. Interbedded with the clastic and biogenic lithotypes are volcaniclastic deposits. Eight lithological units were defined based on their dominant lithotypes. The time scale of the sequence is still uncertain for the oldest sediments; however, it is estimated that it covers the last 300000 to 350000 years. From this temporality, environmental oscillations of millennial scales are inferred. Diatom-rich laminated sediments represent the deepest phases in the lake. The deposits with higher biogenic and authigenic calcareous components indicate the phases of higher ionic concentration during which the lake was shallower. The detailed analysis of the stratigraphy and the description of the sedimentary components in Xochimilco allows to establish an initial interpretation of the variations in the depositional environments, and provides the base from which the detailed studies of paleoclimatic indicators will be supported.

The Intrapopulation Genetic Diversity of RNA Virus May Influence the Sensitivity of Chlorine Disinfection.

RNA virus populations are not clonal; rather, they comprise a mutant swarm in which sequences are slightly different from the master sequence. Genetic diversity within a population (intrapopulation genetic diversity) is critical for RNA viruses to survive under environmental stresses. Disinfection has become an important practice in the control of pathogenic viruses; however, the impact of intrapopulation genetic diversity on the sensitivity of disinfection, defined as –log10 (postdisinfected infectious titer/predisinfected titer), has not been elucidated. In this study, we serially passaged populations of rhesus rotavirus. We demonstrated that populations with reduced chlorine sensitivity emerged at random and independently of chlorine exposure. Sequencing analysis revealed that compared with sensitive populations, less-sensitive ones had higher non-synonymous genetic diversity of the outer capsid protein gene, suggesting that changes in the amino acid sequences of the outer capsid protein were the main factors influencing chlorine sensitivity. No common mutations were found among less-sensitive populations, indicating that rather than specific mutations, the diversity of the outer capsid protein itself was associated with the disinfection sensitivity and that the disinfection sensitivity changed stochastically. Simulation results suggest that the disinfection sensitivity of a genetically diverse population is destabilized if cooperative viral clusters including multiple sequences are formed. These results advocate that any prevention measures leading to low intrapopulation genetic diversity are important to prevent the spread and evolution of pathogenic RNA viruses in society.

Biased Pol II fidelity contributes to conservation of functional domains in the Potato spindle tuber viroid genome.

Accurate calculation of mutation rates for viruses and viroids is necessary for evolutionary studies and to evaluate adaptation potential. However, estimation of in vivo mutation rates is complicated by selection, which leads to loss or proliferation of certain mutations. To minimize this concern, lethal mutations, including nonsense and non-synonymous mutations, have been used to determine mutation rates for several viruses and viroids, including Potato spindle tuber viroid (PSTVd). However, this approach has limitations, including focus on a relatively small number of genome sites and the possibility that mutations may not actually be lethal or may be maintained by wild type individuals. To avoid selection bias altogether, we sequenced minus-strand PSTVd dimers from concatemeric replication intermediates. The underlying rationale is that mutations found in only one of the monomers were likely generated de novo during RNA polymerase II (Pol II) transcription of the circular plus-strand RNA genome. This approach yielded an apparent Pol II error rate of ~1/1837 nucleotides per transcription cycle, and an estimated mutation rate of ~1/919 nucleotides for a single replication cycle. Remarkably, de novo mutations were nearly absent from the most conserved, replication-critical regions of the PSTVd genome, suggesting that sequence conservation is a consequence of both essential function and template optimization for greater Pol II fidelity. Such biased fidelity may constitute a novel strategy to ensure population success while allowing abundant sampling of sequence space in other genome regions. Comparison with variants in progeny populations derived from a cloned, wild type PSTVd master sequence revealed that most de novo mutations were lost through selection.

Insights Into Potato Spindle Tuber Viroid Quasi-Species From Infection to Disease.

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the “master” sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid’s accumulation by inducing the RNA silencing of the host’s defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid’s interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

Functional analysis reveals G/U pairs critical for replication and trafficking of an infectious non-coding viroid RNA.

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson–Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.

Lateral Extrapolation of Coastal Catenary Sequences Using the Biophysical Cross-shore Classification System (BCCS) to Create Shore-parallel Situational Zonation Mapping Units

Finkl, C.W. and Makowski, C., 2020. Lateral extrapolation of coastal catenary sequences using the Biophysical Cross-shore Classification System (BCCS) to create shore-parallel situational zonation mapping units. Journal of Coastal Research, 36(3), 457–471. Coconut Creek (Florida), ISSN 0749-0208.The Biophysical Cross-shore Classification System (BCCS), which uses transects to assess shore-normal ecological and geomorphological successions from offshore to onshore within a coastal belt (Finkl and Makowski, 2020a), also provides a basis for extrapolating cross-shore catenas into shore-parallel units. This paper shows that three-dimensional transects can be parameterized in terms of alongshore breadth as well as cross-shore width and depth below or elevation above sea level. The codification of cross-shore environments and habitats in the framework of the BCCS provides an interpretative basis for determining the lateral extent of transect units (archetypes and sub archetypes) by lateral extrapolation to polygonal mapping units. The method discussed here is akin to geological cores or cross-sections that are used to laterally extrapolate units within vertical sequences. Repetitive successions of archetypes, based on cross-shore ecological interpretation of satellite imagery, results in a common master sequence referred to as a Dominant Catenary Sequence (DCS). The DCS is composed of generic archetypes, such as Barrier, Beach, Beach Ridge, Cliff, Coral Reef, Delta, Dune, Flat, Ice, Lagoon, Mountain, Rock, Till (Glacial Material), Upland, and Wetland. The more detailed Coastal Ecological Sequence (CES) of a coastal belt, which is defined by a discrete codification sequence built up from the DCS, is formulated by cognitive geovisual analytics to link the dominant catena with a numbered shore-parallel shape distinction and subscripted sub archetypes to refine the sequential composite archetypes in a DCS. Once the DCS- or CES-labeled transect has been plotted on a satellite image, the identified shore-normal units can be extrapolated into shore-parallel polygons by traditional (i.e. visual, cognitive) image interpretive and mapping techniques to show the spatial extent of classified archetypes and sub archetypes.

ELM Dendrochronology

Elm is generally considered to be unsuitable for dendrochronology, usually having too few rings, or having abrupt growth-rate changes that do not result from the external weather conditions. Samples rarely match each other in the same structure. A further difficulty is that even where it is known that the sequence is complete (the bark is still present), it is often not possible to distinguish sapwood rings by their appearance, even under a microscope. This is a significant problem as elm has been an important structural component of many British vernacular buildings over many centuries, but whereas dendrochronological dating of oak has transformed our understanding of thousands of buildings, by 2015 only four instances of dating elm had been made, two of those involving a single timber. When elm has been encountered, it has generally been dismissed from further dendrochronological study as a result of these known issues, but no systematic study has been undertaken to see whether these prejudices are justified. In order to get some evidence-based information about how elm might behave dendrochronologically, Historic England initiated a study: Developing the dendrochronology of elm in historic buildings, Project 7350, funded through its Heritage Protection Commissions. This resulted in over 70 buildings being looked at, with several being sampled, and the results are discussed here. At some sites an elm site master sequence could be derived, but potential matches with local oak chronologies were generally not strong enough to be considered dated. In one instance good matches with local oak sites were found, but subsequent radiocarbon analysis found these matches to be erroneous. At another site, five trees gave an 89-year ring sequence, but no acceptable matches were found with oak chronologies. Radiocarbon dating and oxygen isotope dating both gave the same dating results at this site, however, showing that these two methods appear to give the best hope of dating elm in the future.

A basic model of mutations

We study a basic model for mutations. We derive exact formulae for the mean time needed to discover the master sequence, the mean returning time to the initial state, or to any Hamming class. These last two formulae are the same than the formulae obtained by Mark Kac for the Ehrenfest model.

The Biophysical Cross-shore Classification System (BCCS): Defining Coastal Ecological Sequences with Catena Codification to Classify Cross-shore Successions Based on Interpretation of Satellite Imagery

Finkl, C.W. and Makowski, C., 2020. The Biophysical Cross-shore Classification System (BCCS): Defining coastal ecological sequences with catena codification to classify cross-shore successions based on interpretation of satellite imagery. Journal of Coastal Research, 36(1), 1–29. Coconut Creek (Florida), ISSN 0749-0208.Coastal classification is a complicated endeavor due to the complexity of coasts and the application of special purpose characterizations for a wide range of tasks. The conundrum of coastal classification in general is also partly related to variable definitions and uses of common terms such as coast, coastline, shore, shoreline, and seashore. This research effort was not aimed at replacing extant systems but rather investigating the possibility of using the new Biophysical Cross-shore Classification System (BCCS) to define or classify cross-shore ecological successions in coastal belts based visual analytics and cognitive interpretation of satellite imagery. Approximately 200 coastal images from equatorial to polar regions showed that specific types of ecological successions were repetitive and could be organized by dominant characteristics. Certain ecological characterizations were so prominent and common that they became identified as Archetypes, which included Barrier, Beach, Beach Ridge, Cliff, Coral Reef, Delta, Dune, Flat, Ice, Lagoon, Mountain, Rock, Till (Glacial Material), Upland, and Wetland. When several archetypes are sequentially linked together based on the cross-shore ecological interpretation of the imagery, a generalized or idealized common master sequence is created and deemed the Dominant Catenary Sequence (DCS; e.g., Beach-Dune-Wetland). The more detailed Coastal Ecological Sequence (CES) of a coastal belt, which is presented as a codification sequence, can be formulated by cognitively linking the Dominant Catenary Sequence with a numbered shore-parallel shape distinction and subscripted sub archetypes that further refine the archetypes present in the DCS. Overall, the BCCS was found to be an effective method for the classification of cross-shore ecological successions in coastal belts. Descriptive headers, extended captions, and Coastal Ecological Sequences are provided for randomly selected satellite images, with some examples shown in this paper.

Stages, periods, epochs, and phases in Paracas and Nasca chronology: another look at John Rowe’s Ica valley master sequence

Chronology on the south coast of Peru is adrift. Researchers have a choice of using developmental stages or historical periods. Increasingly, fuzzy thinking has led to the publication of chronologies employing both stages and periods in the same chart. Authors seem unaware that stages and periods are fundamentally different ways of organizing the past, underpinned by different sets of assumptions which ask different questions. The current work is specifically concerned with the relative chronologies for the south coast Paracas and Nasca cultures, but it also reviews the fundamental principles of stages and periods, examines the workings of John Rowe’s Master Sequence, and provides clear definitions for terminology. In conclusion, an updated chronology for the south coast Early Horizon and Early Intermediate Period is introduced.

Long-term context-dependent genetic adaptation of the viral genetic cloud.

RNA viruses generate a cloud of genetic variants within each host. This cloud contains high-frequency genotypes, and many rare variants. The dynamics of these variants is crucial to understand viral evolution and their effect on their host. We use an experimental evolution system to show that the genetic cloud surrounding the Coxsackie virus master sequence slowly, but steadily, evolves over hundreds of generations. This movement is determined by strong context-dependent mutations, where the frequency and type of mutations are affected by neighboring positions, even in silent mutations. This context-dependent mutation pattern serves as a spearhead for the viral population's movement within the adaptive landscape and affects which new dominant variants will emerge. The non-local mutation patterns affect the mutated dinucleotide distribution, and eventually lead to a non-uniform dinucleotide distribution in the main viral sequence. We tested these results on other RNA viruses with similar conclusions. Supplementary data are available at Bioinformatics online.

Lithostratigraphy and physical properties of lacustrine sediments of the last ca. 150 kyr from Chalco basin, central México

The recognition of past climatic fluctuations in sedimentary sequences in central Mexico is relevant for understanding the forcing mechanisms and responses of climatic system in the northern American tropic. Moreover, in this active volcanic setting the sedimentary record preserves the history of past volcanic activity. Climatic and environmental variability has been documented for the last tenths of thousands of years from the upper lacustrine sediments in Chalco basin. A series of cores drilled down to 122 m depth in this basin offer a long, continuous and high resolution record of past climatic changes of the last ca. 150 kyr in this region. Here we present the detailed lithostratigraphy and some physical properties (magnetic susceptibility and density) of the master sequence. Sedimentary components and their abundance were identified and quantified in smear slides and direct core observations. Age model is based on 13 14C and one 230Th/U dates. Based on their facies association seven lithostratigraphic units were defined, which reflect the main stages of lake Chalco evolution. These phases closely match the marine isotopic stages. The data reveal that at the end of MIS6 Chalco was a relatively deep and stratified freshwater lake. During MIS5 the depositional environment fluctuated between low lake stands to marshy and marginal playa settings with sporadic flooding events, and severe arid periods resulted in aerial exposure of lake sediments. Low lake stands persisted during MIS4 and MIS3, with minor fluctuations towards slightly deeper phases. The Last Glacial Maximum (LGM) and the deglacial period (21-13 kyr) are characterized by intense volcanism. The early and mid-Holocene high calcareous content and alkaline-subsaline lake suggest dry conditions. The fluctuations of lake levels inferred provide the basis for future paleoclimatic works.

Cloning of CHO Cells, Productivity and Genetic Stability—A Discussion

While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 1014 cells that have divided for weeks. Regulatory interest in “Proof of Clonality” is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term “stable” will always be used in a context of relative stability—considering time frames of weeks to months at best.) “CHO master sequence” genomes (containing the desired DNAs of interest). Stable cell populations are to be selected for and maintained for the various phases of manufacturing under specific culture conditions reducing trends for the selection of diverse subpopulations. Such conditions are based on insights gained from population genetics, evolutionary landscape fitness principles, and a 40-year old model for evolution of error prone replicating systems—the Quasi-Species concept.

Distribution of the quasispecies for a Galton–Watson process on the sharp peak landscape

Abstract We study a classical multitype Galton–Watson process with mutation and selection. The individuals are sequences of fixed length over a finite alphabet. On the sharp peak fitness landscape together with independent mutations per locus, we show that, as the length of the sequences goes to ∞ and the mutation probability goes to 0, the asymptotic relative frequency of the sequences differing on k digits from the master sequence approaches (σe-a - 1)(ak/k!)∑i≥ 1ik/σi, where σ is the selective advantage of the master sequence and a is the product of the length of the chains with the mutation probability. The probability distribution Q(σ, a) on the nonnegative integers given by the above equation is the quasispecies distribution with parameters σ and a.

Appropriate Genotyping of Hepatitis B Virus in Iran.

Dear Editor We read, with great interest, the article by Karimi et al. (1) published in your journal recently, regarding the characterization of hepatitis B virus (HBV) genotypes in a group of patients with chronic hepatitis B infection. They found a prevalence of 73.9% and 26.1% for genotypes D and C, respectively, in Iranian patients. Bearing this context in mind, some points deserve to be considered. The patients were HBsAg-positive and a conventional polymerase chain reaction (PCR) methodology was used to detect DNA-positive samples. Subsequently, the authors applied genotype-specific real-time PCR to identify the genotypes. Only a fraction of the HBsAg-positive patients (19.82%) tested positive by conventional and real-time PCR. The use of standard PCR as a protocol for detecting DNA-positive samples was the major concern. Basically, all HBsAg-positive individuals should be tested positive by PCR, regardless of the type of PCR applied. The first weakness of the study was the sensitivity of PCR. In the field of clinical virology, nucleic acid amplification technology (NAT) is considered superior to conventional serology; therefore, samples testing positive by serological tests but negative by NAT is indicative of an error in the type of NAT used. Based on the article, it seems that the authors used multiplex real-time PCR to identify HBV genotypes; however, there is no data regarding the optimization of the method. Moreover, there is no information on the sequence of primers and probes used or any references that support the reliability of the kit. In such situations, real-time PCR can provide erroneous results, especially in the identification of minor variants. Therefore, the second weakness of the study was that the real-time PCR results were not confirmed by a specific approach such as sequencing. Previous and recent investigations carried out by the Iranian Hepatitis Network on the distribution of HBV among patients across different clinics have indicated that the main circulating genotype in Iran is D (2, 3). Some reports on the presence of genotypes apart from D in Iran have been published; however, they suffer from the same weaknesses as those mentioned above with respect to the methodology used. Previous surveys used fragment-based conventional PCR (4) and restriction fragment length polymorphism (RFLP) (5, 6). Although the sensitivity of RFLP is highly appreciated, RFLP and primer-specific methods are not very helpful in identifying minor variants in a quasispecies. Essentially, when a dominant variant exists in a population, the minor species might be ignored owing to their low proportion compared to that of the master sequence. Thus, sequencing is the gold standard method for the identification of genotypes. Owing to its adequate sensitivity, it is highly suitable for such purposes. Moreover, even the sensitivity of sequencing is not optimal for identifying all the possible variants. Therefore, the best option for accurate genotyping might be subjecting the amplicons to cloning plus sequencing. The Iranian hepatitis network suggests that all national labs and research centers use a standard (not necessarily unique) methodology for HBV genotypic characterization. A sensitive real-time PCR method could be used as an appropriate screening technique. Thus, regardless of the NAT type, we propose subjecting the amplified cloned extracted material to direct sequencing.