IABS/DCVMN webinar on next generation sequencing.

Next Generation Sequencing (NGS) is a new technology that could overcome some of the limitations of the current viral testing methods for demonstrating the absence of adventitious agents in biologics. This report is for the webinar that was organized by the International Alliance for Biological Standardization (IABS) and the Developing Countries Vaccine Manufacturers Network (DCVMN), held on July 20, 2022, as an introduction to the technical and bioinformatics concepts of NGS and to some of the strengths and limitations of using the technology for those working in vaccine production or development. The current state of scientific knowledge and readiness of NGS to replace or supplement the current viral tests was further discussed in the 3rd Conference on NGS for Adventitious Virus Detection in Biologics for Humans and Animals that was held in Rockville, Maryland, USA, on September 27-28, 2022. The application of NGS to supplement or replace current in vivo and in vitro assays in adventitious virus testing during vaccine production is promising; however, assay performance (sensitivity, specificity, and reproducibility) needs to be demonstrated, which may include laboratory and bioinformatics work. Efforts from regulatory authorities, industry, and researchers are ongoing to facilitate validation and establishment of NGS as a new method for virus detection.

Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals.

Next-generation sequencing (NGS) has been proven to address some of the limitations of the current testing methods for adventitious virus detection in biologics. The International Alliance for Biological Standardization (IABS), the U.S. Food and Drug Administration (FDA), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) co-organized the "3rd Conference on Next-generation Sequencing for Adventitious Virus Detection in Biologics for Humans and Animals", which was held on September 27-28, 2022, in Rockville, Maryland, U.S.A. The meeting gathered international representatives from regulatory and public health authorities and other government agencies, industry, contract research organizations, and academia to present the current status of NGS applications and the progress on NGS standardization and validation for detection of viral adventitious agents in biologics, including human and animal vaccines, gene therapies, and biotherapeutics. Current regulatory expectations were discussed for developing a scientific consensus regarding using NGS for detection of adventitious viruses. Although there are ongoing improvements in the NGS workflow, the development of reference materials for facilitating method qualification and validation support the current use of NGS for adventitious virus detection.

Energy intake misreport: how different methods affect its prevalence and nutrient intake estimates.

BACKGROUND: Although different methods for the evaluation of energy intake (EI) misreport have been described, it is unclear which one is the most appropriate. AIM: To assess the performance of these methods in the prevalence of EI misreports and accuracy of nutrient intake estimates. METHODS: Reports of 3,639 adults from the Portuguese National Food, Nutrition and Physical Activity Survey 2015-2016 were classified using univariate (Willett; interquartile range) and multivariate (Goldberg; predicted total energy expenditure [pTEE], testing different standard deviations [SD]) methods. Self-reported intakes were compared to their respective estimates by urinary excretion in a sub-sample of 80. The effect of the exclusion of misreporters on nutrient estimates was assessed by the differences in linear regression coefficients between plausible and total sample. RESULTS: The highest prevalence of EI misreport was observed using pTEE 1SD (63.9%). Differences in the associations between nutrient self-reported intake and estimated intake using urinary biomarkers were verified with misreporters' exclusion by pTEE 1SD method (ß-protein = 0.209; 95% CI = 0.074-0.529; ß-potassium = 0.276; 95% CI = 0.060-0.560) and Goldberg 2SD (ß-protein = 0.080; 95% CI = 0.025-0.235; ß-potassium = 0.106; 95% CI = -0.048-0.246). CONCLUSIONS: Multivariate methods lead to a higher prevalence of misreports and larger differences in nutrient estimates. The application of the pTEE 1SD and Goldberg 2SD methods resulted in more accurate nutrient estimates.

Impact of iodine supplementation during preconception, pregnancy and lactation on maternal thyroid homeostasis and offspring psychomotor development: protocol of the IodineMinho prospective study.

BACKGROUND: Iodine deficiency is the most common cause of preventable brain harm and cognitive impairment in children. Portuguese women of childbearing age, pregnant women and their progeny were shown to have inadequate iodine intake. Consequently, the Portuguese Health Authorities have recommended a daily supplementation with 150-200 µg iodine in preconception, pregnancy, and lactation. The IodineMinho study intends to evaluate whether (i) this recommendation impacted on the prevalence of iodine deficiency in pregnant women from the Minho region of Portugal, (ii) the time of initiation of iodine supplementation (if any) influences the serum levels of thyroid hormones at several intervals during pregnancy and (iii) there are serum thyroid-hormone parameters in the 1st trimester of pregnancy that predict psychomotor development of the child at 18 months of age. METHODS: Most Portuguese women are followed throughout pregnancy in community Family Health Units, where family physicians may choose to follow the National recommendation or other, concerning iodine sufficiency. This study will recruit women (N = 304) who intend to become pregnant or are already pregnant from 10 representative Units. Physician's approach and prescriptions, sociodemographic, nutrition and clinical information will be obtained at baseline and throughout pregnancy. To evaluate endocrine function, blood and urine samples will be collected at recruitment, once in each trimester of pregnancy, at delivery and 3 months after delivery. Breastmilk samples will be collected for iodine and energy content analysis. Children will be evaluated for psychomotor development at 18 months. Maternal thyroid volume will be evaluated by ultrasound scan at baseline, in the 3rd trimester and at 3 months after delivery. DISCUSSION: Iodine deficiency early during development precludes children from achieving full intellectual capabilities. This protocol describes a study that is innovative and unique in its detailed and comprehensive evaluation of maternal and child endocrine and psychomotor parameters. By evaluating the effectiveness of the iodine supplementation recommendation, it will contribute to the public health systems' efforts to provide excellence in maternal and infant care. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04288531 . Registered 28 February 2020-Retrospectively registered.

Validation of a new software eAT24 used to assess dietary intake in the adult Portuguese population.

OBJECTIVE: The aim of the current study was to evaluate the accuracy of the new software eAT24 used to assess dietary intake in the National Food, Nutrition and Physical Activity Survey (IAN-AF) against urinary biomarkers: N (nitrogen), K (potassium) and Na (sodium). DESIGN: We conducted a cross-sectional study. Two non-consecutive 24-h dietary recalls (24-HDR) were applied, and a 24-h urine sample was collected. We examined differences between estimates from dietary and urine measures, Pearson correlation coefficients were calculated and the Bland-Altman plots were drawn. Multiple linear regression was used to evaluate the factors associated with the difference between estimates. SETTING: Sub-sample from the Portuguese IAN-AF sampling frame. PARTICIPANTS: Ninety-five adults (men and women) aged 18-84 years. RESULTS: The estimated intake calculated using the dietary recall data was lower than that estimated from urinary excretion for the three biomarkers studied (protein 94·3 v. 100·4 g/d, K 3212 v. 3416 mg/d and Na 3489 v. 4003 mg/d). Considering 2 d of recall, the deattenuated correlation coefficients were 0·33, 0·64 and 0·26 for protein, K and Na, respectively. For protein, differences between dietary and urinary estimates varied according to BMI (ß = -1·96, P = 0·017). The energy intake and 24-h urine volume were significantly associated with the difference between estimates for protein (ß = 0·03, P < 0·001 and ß = -0·02, P = 0·002, respectively), K (ß = 0·71, P < 0·001 and ß = -0·42, P = 0·040, respectively) and Na (ß = 1·55, P < 0·001 and ß = -0·81, P = 0·011, respectively). CONCLUSIONS: The new software eAT24 performed well in estimating protein and K intakes, but lesser so in estimating Na intake, using two non-consecutive 24-HDR.

Sequence comparison alignment-free approach based on suffix tree and L-words frequency.

The vast majority of methods available for sequence comparison rely on a first sequence alignment step, which requires a number of assumptions on evolutionary history and is sometimes very difficult or impossible to perform due to the abundance of gaps (insertions/deletions). In such cases, an alternative alignment-free method would prove valuable. Our method starts by a computation of a generalized suffix tree of all sequences, which is completed in linear time. Using this tree, the frequency of all possible words with a preset length L-L-words--in each sequence is rapidly calculated. Based on the L-words frequency profile of each sequence, a pairwise standard Euclidean distance is then computed producing a symmetric genetic distance matrix, which can be used to generate a neighbor joining dendrogram or a multidimensional scaling graph. We present an improvement to word counting alignment-free approaches for sequence comparison, by determining a single optimal word length and combining suffix tree structures to the word counting tasks. Our approach is, thus, a fast and simple application that proved to be efficient and powerful when applied to mitochondrial genomes. The algorithm was implemented in Python language and is freely available on the web.

The global epidemiology of chikungunya from 1999 to 2020: A systematic literature review to inform the development and introduction of vaccines.

Chikungunya fever is an acute febrile illness that is often associated with severe polyarthralgia in humans. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus. Since its reemergence in 2004, the virus has spread throughout the tropical world and several subtropical areas affecting millions of people to become a global public health issue. Given the significant disease burden, there is a need for medical countermeasures and several vaccine candidates are in clinical development. To characterize the global epidemiology of chikungunya and inform vaccine development, we undertook a systematic literature review in MEDLINE and additional public domain sources published up to June 13, 2020 and assessed epidemiological trends from 1999 to 2020. Observational studies addressing CHIKV epidemiology were included and studies not reporting primary data were excluded. Only descriptive analyses were conducted. Of 3,883 relevant sources identified, 371 were eligible for inclusion. 46% of the included studies were published after 2016. Ninety-seven outbreak reports from 45 countries and 50 seroprevalence studies from 31 countries were retrieved, including from Africa, Asia, Oceania, the Americas, and Europe. Several countries reported multiple outbreaks, but these were sporadic and unpredictable. Substantial gaps in epidemiological knowledge were identified, specifically granular data on disease incidence and age-specific infection rates. The retrieved studies revealed a diversity of methodologies and study designs, reflecting a lack of standardized procedures used to characterize this disease. Nevertheless, available epidemiological data emphasized the challenges to conduct vaccine efficacy trials due to disease unpredictability. A better understanding of chikungunya disease dynamics with appropriate granularity and better insights into the duration of long-term population immunity is critical to assist in the planning and success of vaccine development efforts pre and post licensure.

Iodine supplementation: compliance and association with adverse obstetric and neonatal outcomes.

Objectives: Over 1.9 billion people worldwide are living in areas estimated to be iodine insufficient. Strategies for iodine supplementation include campaigns targeting vulnerable groups, such as women in pre-conception, pregnancy and lactation. Portuguese women of childbearing age and pregnant women were shown to be mildly-to-moderately iodine deficient. As a response, in 2013, the National Health Authority (NHA) issued a recommendation that all women considering pregnancy, pregnant or breastfeeding, take a daily supplement of 150-200 µg iodine. This study explored how the iodine supplementation recommendation has been fulfilled among pregnant and lactating women in Portugal, and whether the reported iodine supplements intake impacted on adverse obstetric and neonatal outcomes. Design and methods: Observational retrospective study on pregnant women who delivered or had a fetal loss in the Braga Hospital and had their pregnancies followed in Family Health Units. Results: The use of iodine supplements increased from 25% before the recommendation to 81% after the recommendation. This was mostly due to an increase in the use of supplements containing iodine only. Iodine supplementation was protective for the number of adverse obstetric outcomes (odds ratio (OR) = 0.791, P = 0.018) and for neonatal morbidities (OR = 0.528, P = 0.024) after controlling for relevant confounding variables. Conclusion: The recommendation seems to have succeeded in implementing iodine supplementation during pregnancy. National prospective studies are now needed to evaluate the impact of iodine supplementation on maternal thyroid homeostasis and offspring psychomotor development and on whether the time of the beginning of iodine supplementation (how early during preconception or pregnancy) is relevant to consider.

Deoxynivalenol exposure assessment through a modelling approach of food intake and biomonitoring data - A contribution to the risk assessment of an enteropathogenic mycotoxin.

Deoxynivalenol (DON), an enteropathogenic mycotoxin produced by Fusarium species, is usually associated with adverse health outcomes such as gastrointestinal diseases and immunotoxicity. To estimate DON exposure of the Portuguese population at national level, a modelling approach, based on data from 94 Portuguese volunteers, was developed considering the inputs of the food consumption data generated within the National Food and Physical Activity Survey and the human biomonitoring data used to assess the exposure to DON. Ten models of association between DON urinary biomarkers and food items (pasta, cookies, biscuits, sweets, bread, rusks, nuts, oilseeds, beer, meat, milk) were established. Applying the most adequate model to the consumption data (n = 5811) of the general population, the exposure estimates of the Probable Daily Intake revealed that a fraction (0.1%) of the Portuguese population might exceed the Tolerable Daily Intake defined for DON. The analysis stratified by age revealed children (3.2%) and adolescents (6.0%) are more likely to exceed the Tolerable Daily Intake for DON. Although the unavoidable uncertainties, these results are important contributions to understand the exposure to this mycotoxin in Portugal, to assess the associated risk and the potential public health consequences.

Calystegines are Potential Urine Biomarkers for Dietary Exposure to Potato Products.

SCOPE: Metabolites derived from specific foods present in urine samples can provide objective biomarkers of food intake (BFIs). This study investigated the possibility that calystegines (a class of iminosugars) may provide BIFs for potato (Solanum tuberosum L.) product exposure. METHODS AND RESULTS: Calystegine content is examined in published data covering a wide range of potato cultivars. Rapid methods are developed for the quantification of calystegines in cooked potato products and human urine using triple quadrupole mass spectrometry. The potential of calystegines as BFIs for potato consumption is assessed in a controlled food intervention study in the United Kingdom and validated in an epidemiological study in Portugal. Calystegine concentrations are reproducibly above the quantification limit in first morning void urines the day after potato consumption, showing a good dose-response relationship, particularly for calystegine A3 . The design of the controlled intervention mimicks exposure to a typical UK diet and showed that neither differences in preparation/cooking method or influence of other foods in the diet has significant impact on biomarker performance. Calystegine biomarkers also perform well in the independent validation study. CONCLUSION: It is concluded that calystegines have many of the characteristics needed to be considered as specific BFIs for potato product intake.

Challenges Associated With the Design and Deployment of Food Intake Urine Biomarker Technology for Assessment of Habitual Diet in Free-Living Individuals and Populations-A Perspective.

Improvement of diet at the population level is a cornerstone of national and international strategies for reducing chronic disease burden. A critical challenge in generating robust data on habitual dietary intake is accurate exposure assessment. Self-reporting instruments (e.g., food frequency questionnaires, dietary recall) are subject to reporting bias and serving size perceptions, while weighed dietary assessments are unfeasible in large-scale studies. However, secondary metabolites derived from individual foods/food groups and present in urine provide an opportunity to develop potential biomarkers of food intake (BFIs). Habitual dietary intake assessment in population surveys using biomarkers presents several challenges, including the need to develop affordable biofluid collection methods, acceptable to participants that allow collection of informative samples. Monitoring diet comprehensively using biomarkers requires analytical methods to quantify the structurally diverse mixture of target biomarkers, at a range of concentrations within urine. The present article provides a perspective on the challenges associated with the development of urine biomarker technology for monitoring diet exposure in free-living individuals with a view to its future deployment in "real world" situations. An observational study (n = 95), as part of a national survey on eating habits, provided an opportunity to explore biomarker measurement in a free-living population. In a second food intervention study (n = 15), individuals consumed a wide range of foods as a series of menus designed specifically to achieve exposure reflecting a diversity of foods commonly consumed in the UK, emulating normal eating patterns. First Morning Void urines were shown to be suitable samples for biomarker measurement. Triple quadrupole mass spectrometry, coupled with liquid chromatography, was used to assess simultaneously the behavior of a panel of 54 potential BFIs. This panel of chemically diverse biomarkers, reporting intake of a wide range of commonly-consumed foods, can be extended successfully as new biomarker leads are discovered. Towards validation, we demonstrate excellent discrimination of eating patterns and quantitative relationships between biomarker concentrations in urine and the intake of several foods. In conclusion, we believe that the integration of information from BFI technology and dietary self-reporting tools will expedite research on the complex interactions between dietary choices and health.

Food Consumption Data as a Tool to Estimate Exposure to Mycoestrogens.

Zearalenone and alternariol are mycotoxins produced by Fusarium and Alternaria species, respectively, that present estrogenic activity and consequently are classified as endocrine disruptors. To estimate the exposure of the Portuguese population to these two mycotoxins at a national level, a modelling approach, based on data from 94 Portuguese volunteers, was developed considering as inputs: i) the food consumption data generated within the National Food and Physical Activity Survey; and ii) the human biomonitoring data used to assess the exposure to the referred mycotoxins. Six models of association between mycoestrogens urinary levels (zearalenone, total zearalenone and alternariol) and food items (meat, cheese, and fresh-cheese, breakfast cereals, sweets) were established. Applying the obtained models to the consumption data (n = 5811) of the general population, the median estimates of the probable daily intake revealed that a fraction of the Portuguese population might exceed the tolerable daily intake defined for zearalenone. A reference intake value for alternariol is still lacking, thus the characterization of risk due to the exposure to this mycotoxin was not possible to perform. Although the unavoidable uncertainties, these results are important contributions to understand the exposure to endocrine disruptors in Portugal and the potential Public Health consequences.

Post-last glacial maximum expansion from Iberia to North Africa revealed by fine characterization of mtDNA H haplogroup in Tunisia.

The first large-scale fine characterization of Tunisian H lineages clarifies that the post-Last glacial maximum expansion originating in Iberia not only led to the resettlement of Europe but also of North Africa. We found that 46% of 81 Tunisian H lineages subscreened for 1,580 bp in mtDNA coding region were affiliated with H1 and H3 subhaplogroups, which are known to have originated in Iberia. Although no signs of local expansion were detected, which would allow a clear dating of their introduction, the younger and less diverse Tunisian H1 and H3 lineages indicate Iberia as the radiating centre. Major contributions from historical migrations to this Iberian genetic imprint in Tunisia were ruled out by the mtDNA gene pool similarity between Berber/Arab/cosmopolitan samples and some "Andalusian" communities, settled by the descendents of the "Moors" who once lived in Iberia for 10 centuries (between 8th and 17th centuries), before being expelled to Tunisia.

Definition of Immunological Nonresponse to Antiretroviral Therapy: A Systematic Review.

BACKGROUND: Terms and criteria to classify people living with HIV on antiretroviral therapy who fail to achieve satisfactory CD4 T-cell counts are heterogeneous, and need revision and summarization. METHODS: We performed a systematic review of PubMed original research articles containing a set of predefined terms, published in English between January 2009 and September 2018. The search retrieved initially 1360 studies, of which 103 were eligible. The representative terminology and criteria were extracted and analyzed. RESULTS: Twenty-two terms and 73 criteria to define the condition were identified. The most frequent term was "immunological nonresponders" and the most frequent criterion was "CD4 T-cell count <350 cells/µL after ≥24 months of virologic suppression." Most criteria use CD4+ T-cell counts as a surrogate, either as an absolute value or as a change after a defined period of time [corrected]. Distinct values and time points were used. Only 9 of the 73 criteria were used by more than one independent research team. Herein we propose 2 criteria that could help to reach a consensus. CONCLUSIONS: The high disparity in terms and criteria here reported precludes data aggregation and progression of the knowledge on this condition, because it renders impossible to compare data from different studies. This review will foster the discussion of terms and criteria to achieve a consensual definition.

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise.

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.

Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale.

The impact of phylogeographic information on mtDNA forensics has been limited to the quality control of published sequences and databases. In this work we use the information already available on Eurasian mtDNA phylogeography to guide the choice of coding-region SNPs for haplogroup H. This sub-typing is particularly important in forensics since, even when sequencing both HVRI and HVRII, the discriminating power is low in some Eurasian populations. We show that a small set (eight) of coding-region SNPs resolves a substantial proportion of the identical haplotypes, as defined by control-region variation alone. Moreover, this SNP set, while substantially increasing the discriminating efficiency in most Eurasian populations by roughly equal amounts, discloses population-specific profiles.

Y-chromosomal STR haplotypes in three ethnic groups and one cosmopolitan population from Tunisia.

The 11 Y-chromosomal short tandem repeats (STRs) included in the Promega Corporation PowerPlex Y System (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438 and DYS439) were typed in three ethnic groups ("Andalusians", Berber and Arab) and one cosmopolitan population (Tunis) from Tunisia, summing up 247 individuals, and 139 different haplotypes. Focusing the analysis on the seven Y-STRs of the YHRD Minimal Haplotype Core (DYS385 excepted), "Andalusians" showed no differences from the Cosmopolitan and the Arab samples previously published (our Arab sample presented an extremely low haplotype diversity), but were different from the Berbers. The Berbers from Tunisia were not different from those from Morocco.

Portuguese mitochondrial DNA genetic diversity-An update and a phylogenetic revision.

In recent years a large amount of mitochondrial population data for forensic purposes has been produced. Current efforts are focused at increasing the number of studied populations while generating updated genetic information of forensic quality. However, complete mitochondrial control region sequences are still scarce for most populations and even more so for complete mitochondrial genomes. In the case of Portugal, previous population genetics studies have already revealed the general portrait of HVS-I and HVS-II mitochondrial diversity, becoming now important to update and expand the mitochondrial region analysed. Accordingly, a total of 292 complete control region sequences from continental Portugal were obtained, under a stringent experimental design to ensure the quality of data through double sequencing of each target region. Furthermore, H-specific coding region SNPs were examined to detail haplogroup classification and complete mitogenomes were obtained for all sequences belonging to haplogroups U4 and U5. In general, a typical Western European haplogroup composition was found in mainland Portugal, associated to high level of mitochondrial genetic diversity. Within the country, no signs of substructure were detected. The typing of extra coding region SNPs has provided the refinement or confirmation of the previous classification obtained with EMMA tool in 96% of the cases. Finally, it was also possible to enlarge haplogroup U phylogeny with 28 new U4 and U5 mitogenomes.

Admixture and genetic diversity distribution patterns of non-recombining lineages of Native American ancestry in Colombian populations.

Genetic diversity of present American populations results from very complex demographic events involving different types and degrees of admixture. Through the analysis of lineage markers such as mtDNA and Y chromosome it is possible to recover the original Native American haplotypes, which remained identical since the admixture events due to the absence of recombination. However, the decrease in the effective population sizes and the consequent genetic drift effects suffered by these populations during the European colonization resulted in the loss or under-representation of a substantial fraction of the Native American lineages. In this study, we aim to clarify how the diversity and distribution of uniparental lineages vary with the different demographic characteristics (size, degree of isolation) and the different levels of admixture of extant Native groups in Colombia. We present new data resulting from the analyses of mtDNA whole control region, Y chromosome SNP haplogroups and STR haplotypes, and autosomal ancestry informative insertion-deletion polymorphisms in Colombian individuals from different ethnic and linguistic groups. The results demonstrate that populations presenting a high proportion of non-Native American ancestry have preserved nevertheless a substantial diversity of Native American lineages, for both mtDNA and Y chromosome. We suggest that, by maintaining the effective population sizes high, admixture allowed for a decrease in the effects of genetic drift due to Native population size reduction and thus resulting in an effective preservation of the Native American non-recombining lineages.

mtDNAoffice: a software to assign human mtDNA macro haplogroups through automated analysis of the protein coding region.

We describe a fast, automated process to determine distances between mtDNA sequences allowing their subsequent clustering and haplogroup assignment that may increase the speed of data analysis and avoid human errors. In order to avoid complexities/ambiguities resulting from recurrence and insertion/deletion phenomena and thus improving evolutionary signal-to-noise ratio, protein coding sequences were compared using a vectorial representation method, and the corresponding genetic distance matrix was used for the construction of a neighbor-joining/UPGMA tree or an MDS graphic, which generally agrees with the consensus mtDNA phylogeny. mtDNAoffice software, detailed instructions and example files are freely available on the web at http://www.portugene.com/SupMat/setupmtDNAoffice.rar.