To implement parent-oriented discharge planning (Train-to-Home) for preterm infants in neonatal care.Before and after study, investigating the effects of the intervention during two 11-month periods before and after implementation.Four local neonatal units (LNUs) in South West England.Infants without major anomalies born at 27–33 weeks’ gestation admitted to participating units, and their parents.A family-centred discharge package to increase parents’ involvement and understanding of their baby's needs, comprising a train graphic and supporting care pathways to facilitate parents’ understanding of their baby's progress and physiological maturation, combined with improved estimation of the likely discharge date.Perceived Maternal Parenting Self-Efficacy (PMP S-E) scores, infant length of stay (LOS) and healthcare utilisation for 8 weeks following discharge.Parents reported that the Train-to-Home improved understanding of their baby's progress and their preparedness for discharge. Despite a lack of change in PMP S-E scores with the intervention, the number of post-discharge visits to emergency departments (EDs) fell from 31 to 20 (p<0.05), with a significant reduction in associated healthcare costs  after discharge. In both study phases, over 50% of infants went home more than 3 weeks before their estimated date of delivery (EDD), though no reduction in LOS occurred.Despite the lack of measurable effect on the parental self-efficacy scores, the reduction in ED attendances and associated costs supports the potential value of this approach. This is the first study to measure the impact of a neonatal family-centred care intervention on parental self-efficacy or use of emergency department (ED) post-discharge for moderately preterm infants.Health economic data collection was available for most families which facilitated a detailed analysis of the costs of healthcare usage following discharge.The lack of time for implementing the Train-to-Home intervention meant that some staff were not confident in using the family-centred approach to discharge planning.The quasi-experimental study design (before and after) was also a limitation in that the changes in outcome measures were not randomised between units, but there were no significant differences in the infant or maternal demographics between the two study periods.The improvements in survival of preterm infants over the past 20 years mean that more than 90% of infants born at 27 weeks’ or more gestation will survive to go home.2Parents of preterm infants need to learn how to care for them after discharge home, and to prepare themselves and their home environment. Evidence suggests that ex-preterm infants make a disproportionate demand on emergency and ‘out-of-hours’ health services.6A structured approach to discharge planning using care pathways and predictable timings for discharge improves the quality of care before and after discharge and reduces the need for unexpected re-admission after discharge, as well as improving patient satisfaction.10Many parents of preterm infants are routinely informed by neonatal staff that their baby will be discharged home at or around the time the baby was due to be born—that is, the estimated date of delivery (EDD). This continues despite increasing evidence that improvements in neonatal care over recent years have led to shorter stays in hospital and earlier discharge to home.13In an audit of the length of stay (LOS) of preterm infants in local neonatal units 11Building on work from McMaster Children's Hospital, Canada,Neonatal care is an expensive and limited health resource with prematurely born infants occupying the majority of neonatal hospital bed-days.17The specific aims of the study were to investigate whether introducing the parent-centred neonatal discharge package (Train-to-Home) increased parental confidence in caring for their infant (self-efficacy), reduced infants’ length of hospital stay and reduced healthcare resource use after discharge from hospital.Soon after admission to the neonatal unit, an accurate estimate of when the baby is likely to be discharged from hospital is provided, based on the locally derived 50th–75th centiles for LOS for each gestation. The discharge date range is displayed on a laminated train which has five labelled carriages: breathing, feeding, growth, temperature and sleeping . Using aParents of infants born between 27 weeks 0 days and 33 weeks 6 days were recruited in four large LNUs in South West England. Two of the units had associated level 1 units to which some infants were transferred before going home. Parents were recruited during two 11-month periods (phase 1: October 2012–August 2013 and phase 2: October 2013–August 2014) before and after the introduction in the LNUs of the Train-to-Home (with parent pathways). For the research study, infants with major congenital anomalies or with mothers under 16 years were not recruited. Parental assent was sought by the LNU nurses and consent gained by a study researcher.Demographic and clinical information was collected by the researchers for all participating infants. Both parents were asked to complete a validated standardised measure, the Perceived Maternal Parenting Self-Efficacy (PMP S-E)A short healthcare resource use data collection tool was developed for parents to record all healthcare contacts for the baby, from which data were collected at telephone follow-up 4 and 8 weeks after discharge. Qualitative data were collected from parents 8 to 10 weeks following discharge by semi-structured telephone interviews exploring their experiences of the unit and perceptions of the intervention. All data were anonymised before analysis. Focus groups were also held to explore the views of nursing staff, and telephone interviews conducted with senior medical staff from all LNUs. Parent interviews and nursing staff focus groups were audio recorded, transcribed verbatim and analysed using thematic methods facilitated by the qualitative package NVivo.2 tests were used to n−1 degrees of freedom. A test of normality on continuous data was conducted using the Shapiro-Wilk test and observing the Q–Q plots. The Mann-Whitney U test was used for non-parametric data, which were described using medians and IQRs. Resource use data in volume units were combined with price and unit cost information from published sourcesStatistical analysis was performed using IBM SPSS Statistics V.21 and Stata V.13. For proportional data, χA total of 245 families participated in the study, 128 families in phase 1 and 117 families in phase 2 as shown in The length of hospital stay in phase 1  was not significantly different (p=0.32) from phase 2 . Almost 75% of infants were discharged home on or before the initially estimated Train-to-Home discharge dates. More infants in phase 2 were discharged home being mixed fed with breast milk and formula and fewer were exclusively formula fed .Cost analysis of healthcare after discharge is based on 173 infants, 85/128 in phase 1 and 88/117 in phase 2, for whom sufficient information was provided . There were significantly more attendances at emergency departments (EDs) by infants in phase 1 than in phase 2 , with an associated significantly higher estimated cost in phase 1 than phase 2 . There was no difference between phases 1 and 2 in the number of hospital re-admissions, or hospital outpatient appointments attended by the infants after discharge, and no difference in primary care attendances .Results from the qualitative interviews with 37 parents and 24 staff are reported in detail in a separate paper (Ingram in preparation). Parents were overwhelmingly positive about the ‘Train-to-Home’ package and reported feeling better prepared for home in phase 2 than phase 1. Most found the Train-to-Home helpful in showing them visually that their baby was progressing and described feelings of being given hope and feeling in control. Mothers, fathers and siblings enjoyed using it. Medical and nursing staff generally agreed that the intervention materials were helpful in explaining a baby's physiological progress to parents, but some nursing staff had concerns that the estimated discharge dates were too optimistic. The materials were introduced over a 6-week period which was not long enough to embed the materials into each LNU, and without this period of normalisation, some staff were uncertain about using the package. However, staff in one unit were particularly positive about the Train-to-Home intervention and were keen for it to continue.There was a small but significant reduction in out-of-hours ED visits and associated costs after the introduction of the Train-to-Home intervention. This was particularly notable in view of an increase in ED attendances nationally over this period, which coincided with the introduction of the ‘111 out-of-hours’ service, which encouraged more callers to attend ED. There was no significant difference in the changes in PMP S-E scores between the two phases of the study; however, the change was slightly greater and parents reported feeling more confident in phase 2. PMP S-E scores increased between admission and discharge of the babies in both study phases, indicating improved parental self-efficacy.The predicted discharge dates helped parents to prepare for home. The ways that staff engaged with the materials when communicating with them helped them feel more confident, as well as having something visual to show their baby's progress and stage of physiological readiness. The questions in the leaflets encouraged parents to ask appropriate questions in a timely fashion to improve their knowledge and understanding. Monitoring compliance was difficult to measure but staff feedback and attitudes expressed in the interviews indicated that staff engagement was different between the units.Others have shown that a risk factor for increased use of health services is the parents’ perception that their prematurely born infant is vulnerable.6http://www.unicef.org.uk/BabyFriendly) and were therefore encouraging and supporting mothers to go home breast feeding, as reflected by the increased proportion of infants receiving some breast milk at discharge in phase 2. Breastfeeding is more difficult for preterm babies and is often a reason for a longer stay while mothers and babies learn how to breastfeed.There was no significant change in LOS in the LNU, although more than half the infants went home at least 3 weeks before the EDD in both phases. During phase 2, all the LNUs were working towards gaining full WHO/UNICEF Baby Friendly Initiative accreditation  was also a limitation but was felt to be the most appropriate design for implementing a complex intervention in neonatal care. The ‘before and after’ design meant that the intervention and any changes in outcome measures were not randomised between units, but we found no significant differences in the infant or maternal demographics between the two study periods. Our study was also limited to infants of 27–33 weeks’ gestation based in four LNUs. In the future, it would be important to implement the package on a network-wide basis to ensure equity so that infants transferred between units would all be using similar discharge planning packages. It also needs to include the wider range of gestational ages cared for in neonatal units so that staff can use it for all infants.Although our initial primary outcome measure (PMP S-E score) did not show any significant differences between the groups, the improvement in preparedness for discharge home reported by the parents and the measured reduction in ED attendances with associated cost reduction suggest the intervention had significant benefits. This approach to educating and involving parents in the care and needs of preterm babies in hospital has potential value and warrants further study and more widespread adoption.


Our goal was to test whether short-term intermittent hypobaric hypoxia (IHH) at a level well tolerated by healthy humans could, in combination with muscle electro-stimulation (ME), mobilize circulating progenitor cells (CPC) and increase their concentration in peripheral circulation.Nine healthy male subjects were subjected, as the active group (HME), to a protocol involving IHH plus ME. IHH exposure consisted of four, three-hour sessions at a barometric pressure of 540 hPa . These sessions took place on four consecutive days. ME was applied in two separate 20-minute periods during each IHH session. Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment, and then 24 h, 48 h, 4 days, 7 days and 14 days after the last day of hypoxic exposure. Four months later a control study was carried out involving seven of the original subjects (CG), who underwent the same protocol of blood samples but without receiving any special stimulus.In comparison with the CG the HME group showed only a non-significant increase in the number of CPC CD34+ cells on the fourth day after the combined IHH and ME treatment.CPC levels oscillated across the study period and provide no firm evidence to support an increased CPC count after IHH plus ME, although it is not possible to know if this slight increase observed is physiologically relevant. Further studies are required to understand CPC dynamics and the physiology and physiopathology of the hypoxic stimulus. Human circulating progenitor cells (CPC) have generally been defined as circulating cells that express a variety of cell surface markers similar to those expressed by vascular endothelial cells, that adhere to endothelium at sites of hypoxia/ischemia, and which participate in new vessel formation, contributing to the maintenance of endothelial function and organ perfusion through mechanisms that range from endothelial repair to neovasculogenesis-3. SeverHypoxia enhances proliferation and tissue formation derived from mesenchymal stem cells in human bone marrow. FunctioResearch also suggests that exercise may influence the mobilization of CPC, and probably tissue homing as well, both in healthy people-23 and iA recent study by our group suggested that a short protocol of exposure to IHH in a hypobaric chamber with simultaneous ME is able to increase the concentration of CPC in peripheral blood in humans. This prThe present research sought to reproduce our previous study with a larger sample size, more post-stimulus blood samples and the inclusion of a control group, the aim being to determine whether short-term IHH at a level well tolerated by healthy humans could, in combination with ME, increase CPC.Nine healthy males were subjected to a protocol involving IHH plus ME, hypoxic muscle electro-stimulation as the active group (HME). Their median age was 29 years [interquartile range (IQR) 9]. Six of them performed regular athletic exercise (three fewer than 3 times/week and three more than 3 times/week) and one was a smoker. Median height was 176 cm (IQR 11.5), mean weight was 80 kg (IQR 14) and median BMI was 25 (IQR 3).IHH exposure consisted of four, three-hour sessions at a barometric pressure of 540 hPa . These sessions were conducted on four consecutive days in the hypobaric chamber of the University of Barcelona at temperature between 24.1 and 26.7°C and relative humidity between 32 and 39%. During the IHH sessions ME was applied using the Compex Vitality® vascular and capillarization program, with electrodes being fixed to the quadriceps and abdominal muscles. ME was Blood samples were obtained from an antecubital vein on three consecutive days immediately before the experiment, and then 24 h, 48 h, 4 days, 7 days and 14 days after the last day of hypoxic exposure.A control study was conducted four months later with seven of these same participants (CG). On this occasion they did not receive any special stimulus, neither IHH nor ME was used, but blood samples were obtained according to the same schedule and on the same days of the week.All blood samples were obtained in the morning and following the same extraction methodology, as detailed below. Peripheral blood samples were collected by puncture of an antecubital vein and deposited into tubes treated with 0.34 M di-potassium ethylenediaminetetraacetic acid anticoagulant. All samples were stored at room temperature and processed within six hours of arrival at the laboratory. Samples were incubated for cytometric absolute count with anti-human fluorescein isothiocyanate (FITC)-conjugated CD45 monoclonal antibody (BD Bioscience) and anti-human phycoerythrin (PE)-conjugated anti-CD34 (BD Bioscience) for 15 min at room temperature. Red blood cells were lysed with 1 ml of quick lysis solution  for 15 min at room temperature. Samples were incubated under dark conditions and analysed immediately. To ensure accuracy, reverse pipetting was used to dispense the volumes.A single-platform protocol with Perfect-Count microspheres  was used according to manufacturer’s instructions. The Perfect-Count microsphere system contains two different fluorospheres in a known proportion (A and B beads), thereby assuring the accuracy of the assay by verifying the proportion of both types of beads. Known volumes (50 μL) of Perfect-Count microspheres were added to the same known volume (50 μL) of stained blood using a lyse-no-wash technique, with the beads being counted along with cells. Cell viability was measured by staining the samples with the vital dye 7-aminoactinomycin D (7-AAD), as proposed by ISHAGE guidelines. SamplesAll aspects of the study were approved by our Bellvitge University Hospital’s Research Ethics Committee, and informed consent was obtained from all participants.Data are expressed as mean, median, standard deviation and interquartile range, as appropriate. Continuous variables were compared between groups by means of analysis of variance (ANOVA) or the Mann-Whitney U test, as appropriate. The Wilcoxon signed-rank test and the Friedman test for repeated measures were also used. All tests were performed using SPSS v.13. Statistical significance was set at p < .05.The initial values of CPC CD34+/μL ranged from 1.54 to 5.41 in the control group (CG) and from 1.83 to 6.88 in the active group (HME). Compared with the CG the HME group showed a very slight increase in the number of CPC on the fourth day after IHH and ME, but this difference was not significant. Figure 2 and heart rate (HR) were measured five times per session per participant with a pulse oximeter (180 measures). Mean SaO2 was 75.6% (SD 5.6) and mean HR was 89 beats per min (SD 15).During hypobaric hypoxic periods at a simulated 5000 m altitude, SaO-1 (range: 0.5–2.1) to a median level of 6.65 cells · μL-1 (range: 3.7-10.7), this increase being highly significant[This study found no a significant increase in the number of CPC after IHH plus ME, as compared with a control group. This result contrasts with the findings of our previous study, in which IHH plus ME seemed able to increase the concentration of CPC in four healthy men aged around 50 years of age. In this previous experiment the CPC count increased from a median value of 0.95 cells · μLnificant. As CPC The opposite results have been found in some studies using different protocols. Specifically, hypoxia decreased CPC count in two normobaric hypoxia studies involving a total of 20 subjects,33, and 2max[Hypoxia with exercise increased CPC count in three studies,29,38 an2max. Wang et2max.The small samples used in these different studies make it difficult to draw any firm conclusions, since there are many factors that might influence the results. It is worth noting, however, that whereas many types of standard physical exercise can be difficult to implement in the context of hypoxic procedures or when osteoarticular conditions are present, ME is easier to apply and has been shown to be efficient in reproducing training effects,28. ThatInter-individual CPC variability,43 and tAge has been related with less capacity to produce neurogenesis and withIntermittent hypoxia (IH) has been shown to be physiologically beneficial in various groups of healthy and ill people,46,47. IThe present study does have certain limitations, notably the small sample size and the variability of the techniques used to detect CPC in blood. It is lTaking these limitations into account, the results of the present pilot study fail to confirm the significant increase in CPC after IHH plus ME previously reported by our group. It is not possible to know whether the slight increase observed is physiologically relevant, or whether there was also increase at other points after exercise. These results are controversial, and further studies including a larger number of subjects and more exhaustive blood sampling times and technique are required to clarify the possible effect of IHH and ME on the release of progenitor cells. This is particularly important with regard to their possible physiological or pathophysiological significance.The authors declare that they have no conflict of interest to disclose.All six authors have contributed to the study in accordance with the international consensus on authorship, and they have approved the final draft submitted. LC contributed to the design and implementation of the study, to data collection and analysis, and to manuscript writing. CJ contributed to the conception and design of the study, to its implementation, to data collection and analysis, and to manuscript writing. JB contributed to implementation of the study, performed the measurements of circulating progenitor cells, and contributed to data analysis and manuscript writing. GV contributed to the conception and design of the study, to its implementation, to data collection and analysis, and to manuscript writing. AR contributed to the design and implementation of the study and to manuscript writing. JV made a significant contribution to the conception and design of the study, as well as to data analysis and manuscript writing. All authors read and approved the final manuscript.


Replacement of sugar-sweetened by non-nutritively sweetened beverages or water may reduce excess weight gain in children. However, it is unclear whether children like non-nutritively sweetened beverages as much as sugar-sweetened beverages. We examined whether children could taste a difference between non-nutritively sweetened beverages and matching sugar-sweetened beverages, and which of the two types of beverage they liked best.89 children aged 5 to 12 tasted seven non-nutritively sweetened beverages and matching sugar-sweetened beverages, for a total of 14 beverages. We used Triangle tests to check their ability to discriminate between the matched versions, and a 5-point scale to measure how much the children liked each individual beverage.Overall, 24% of children appeared to be genuinely capable of distinguishing between non-nutritively sweetened and sugar-sweetened beverages. The mean ± SD score for how much the children liked the non-nutritively sweetened beverages was 3.39±0.7 and that for the sugar-sweetened beverages 3.39±0.6 (P = 0.9) on a scale running from 1 (disgusting) to 5 (delicious). The children preferred some beverages to others irrespective of whether they were sugar-sweetened or non-nutritively sweetened (P = 0.000). Children who correctly identified which of three drinks contained the same sweetener and which one was different also showed no preference for either type.We found that about one in four children were able to discriminate between non-nutritively sweetened and sugar-sweetened beverages but children liked both varieties equally. Non-nutritively sweetened beverages may therefore be an acceptable alternative to sugar-sweetened beverages although water remains the healthiest beverage for children. The increased prevalence of obesity in children is a major health problem However, little is known about the ability of children to discriminate between NNB and SSB and their liking of NNB. The taste of sugar is difficult to mimic with non-nutritive sweeteners. Most non-nutritive sweeteners are perceived as bitter and as having non-sweet aftertastes Also, little is known about the liking of NNB in children, and results in adults are inconsistent. One study found similar ratings of pleasantness for aspartame and sucrose We examined whether children could taste a difference between NNB and matching SSB, and which of the two varieties they liked best.Written informed consent was provided by a parent or guardian who had obtained assent from the child. The Medical Ethical Committee of VU University Medical Centre Amsterdam approved the study protocol. The investigation has been conducted according to the principles expressed in the Declaration of Helsinki.The study was done at an elementary school in the town of Purmerend near Amsterdam. In preparation for this study we recorded what drinks the children brought from home to drink during the breaks in three school classes: one with 22 children aged 4 to 6, one with 25 children aged 6 to 8, and one with 27 children aged 9 to 11. We found that 92% of the beverages consumed were sugar-sweetened (SS), and 8% were water, milk or non-nutritively sweetened (NNS). The low number of beverages sweetened with non-nutritive sweeteners agrees with findings in Dutch children in general We used seven matched pairs of NNB and SSB, for a total of 14 beverages . We obtaWe performed a single-blind sensory study in November 2008 during school hours. We administered the test in the staff room, and tested the children individually. The test lasted approximately 15 minutes per child. A total of 45 children first performed a Pleasantness test and then a Triangle test; 44 children performed these tests in reverse order. We offered 15 mL of each beverage at room temperature in a transparent 25 mL medication cup. They tasted the beverage, and then swallowed the liquid. If a child felt that one sip was not enough, she or he was allowed to take another sip. We did not ask the children to finish the whole cup. Children could drink water between beverages if they so desired.In a Triangle test two samples are the same and one is different. The child is requested to pick out the ‘different’ sample. This test has been used previously in children This test has been validated for biscuits for children in this age group For the Triangle test, all children received the beverages in the same randomly determined order obs is the proportion correct (X/n), X is the actual number of correct statements, n is the total number of statements, p is the chance probability of 1/3, and q = 1–p = 2/3 For all data underlying the findings reported in this manuscript, see Tables S5 and S6 in Correct answers in the Triangle test were given by children who genuinely recognized the aberrant sample, but also by children who picked out the aberrant sample by chance. We calculated the number of genuinely correct answers G as follows. Let F be the number of false answers, X is the actual number of correct statements, and n the total number. X equals n – F . The numWe analyzed the Pleasantness test with a Wilcoxon signed rank test because the data were ordinal. We analysed the results within each of the 7 drinks, within groups of drinks that contained the same blend of sweeteners, and for all NNB versus all SSB. Data were available for 89 children, except for Spa apple/cherry (N = 86) and Spa forest fruits (N = 88) where the test leader had offered the wrong samples to some children. We used both the one-way ANOVA and Kruskal-Wallis tests to analyze whether children preferred some beverages to others, irrespective of whether they were SS or NNS, including corrections for multiple testing.For both the Triangle test and Pleasantness test, we also performed analyses for younger and older children separately. Younger children were defined as the children below the median group age of 9.3 years and older children above the median. We used the Statistical Package for the Social Sciences (SPSS) version 17.0.The mean age of the 89 participants was 9.2±2.0 years (±SD). 51 (57%) were girls. The school was located in an area with a z score for socio-economic status of 0.51 The percentage of children who correctly pointed out the ‘different’ sample ranged from 37% to 61% between the seven beverages . When reFor each of the seven pairs separately, children liked the SS and the NNS beverages similarly, The children preferred some beverages to others irrespective of whether they were SS or NNS . For instance, we found that children liked the SS Spa forest fruits significantly better than both the SS and NNS variety of non-commercial mango, non-commercial peach, Roosvicee forest fruit, Roosvicee peach drink, see Table S4a and Table S4b in We found that about one in four children was genuinely able to discriminate between SSB and NNB when they were asked to identify the odd sample out of three. Evidently the blends of non-nutritive sweeteners used in our beverages tasted somewhat different from sugar, but the difference was slight Interestingly, the beverages were liked equally, even by the children who were able to discriminate between SSB and NNB. This result is in line with a previous three-week study in adults with beverages We calculated that only some 24% of children was genuinely competent to distinguish between non-nutritive sweeteners and sugar. We would not expect a higher proportion in other populations, because at the school where our study was done the large majority of children habitually consumed SSB and very few drank NNB during school breaks See 2.2  section.Our seven NNB still contained small amounts of sugar. Small amounts of sugar are sometimes added to NNB to improve the taste. However, our data do not support such an effect. The three NNB with 0.1% of sugar were liked as much as the four others that contained 1.7% to 3.5% sugar. Apparently, residual sugar content is not a major determinant of the taste of this type of NNB in children.Our study has several strengths. To our best knowledge, this was the first sensory study of non-nutritive sweeteners in children. We included 89 subjects while other studies had only 16 to 31 participants Both the one-way ANOVA and Kruskal-Wallis analyses showed that children preferred some beverages to others irrespective of whether they were SS or NNS. On one hand the Kruskal-Wallis test is more reliable here because it takes into account the ordinal structure of the data and the lack of normal distributions in our outcome measures. However, a disadvantage of this test is that it uses the median instead of the mean and is therefore not able to pick up more subtle differences in liking between drinks. We therefore present the outcomes of both analyses, see Table S4a and S4b in Our participants were healthy Dutch children. Future studies should be carried out to investigate whether our findings hold for other ethnic groups, obese children, habitual consumers of NNB, and different age groups.In conclusion, although one in four children were able to discriminate between NNB and SSB, they liked both types equally. Recent large randomized controlled trials have shown that replacement of SSB by NNB reduces weight gain in children and adolescents S1 FileSupplementary Appendix.Table S1, Ability of children to discriminate between non-nutritively sweetened and sugar-sweetened beverages by all participants, older and younger children. Table S2, Rating of pleasantness of non-nutritively sweetened and sugar-sweetened beverages by all participants, older and younger children. Table S3, Frequencies of scores on the five point scale for each drink on the Pleasantness test by 89 children. Table S4, A)The extent to which the 89 children rated one beverage better than the other irrespective of the sweetener used analyzed with one-way ANOVA analyses. B) The extent to which the 89 children rated one beverage better than the other irrespective of the sweetener used analyzed with Kruskal-Wallis tests. Table S5, The full dataset of both the Triangle and Pleasantness test by 89 children. Table S6, The full dataset of both the Triangle and Pleasantness test by 89 children.(DOCX)Click here for additional data file.


In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA “superbarcodes” for testing hypotheses regarding global patterns of diversity. This is expected to reduce overall assembly success, exhibited as a tendency toward generating complete mitogenomes for some species and fragmented sequences for others. Sequence contiguity for fragmented species may be improved through the use of different assemblers and by combining their output , but theThe huge amount of primary data generated by the metagenomic approach, and the low expected proportion of mitochondrial data . Genomic DNA was extracted destructively from each whole specimen individually and DNA extracts of all specimens were pooled using an equal volume per sample to simulate a bulk extraction from unsorted samples. Two such pools were made for Illumina TruSeq library preparation (average insert sizes approximately 480 and 850 bp). Illumina MiSeq sequencing  yielded 7.9 and 8.1 Gb of raw data for the small and large-insert libraries, respectively. Following removal of sequencing adapters, the proportion of paired reads retained for analysis was 62.96% and 70.78%. After this initial step, the data were further filtered with BLASTn against a database of 245 identified coleopteran mitogenomes , hereafter “MitoDB” , at E ≤ 1e-5, to retain only “mitochondrial-like” reads for analysis, for a total of 9.91% and 11.24% in the short- and long-insert libraries, respectively. A conservative estimate of the proportion of true mitochondrial reads was obtained by requiring long BLASTn alignments  for both reads of each pair to the MitoDB. This indicated that approximately 1.12% (480 bp) and 1.43% (850 bp) were sufficiently similar to the MitoDB sequences to be confidently classed as mitochondrial  not identifiable at family-level . Note that the number of mitochondrial contigs reported is conservative as the threshold used to distinguish true mitochondrial sequences was a ≥1 kb BLASTn alignment to MitoDB . This effectively discards mitochondrial contigs less than 1 kb in addition to the non-mitochondrial sequences.De novo assembly of mitochondrial-like reads was undertaken with Celera Assembler (CA)  and IDBAsupplementary fig. S1, Supplementary Material online). To explore the effect of the quality-control step, an additional assembly was obtained from IDBA-UD following more stringent quality-control . This reduced the number of reads used in the assembly by 13.9% but decreased assembly contiguity only slightly with a 9% reduction in contigs greater than 15 kb and a complementary increase in contigs of 5–10 kb .In order to maximize the number of species recovered and the contiguity of their representative sequence, the two assemblies were combined in Geneious  by reasssupplementary table S3, Supplementary Material online; see example contig in supplementary fig. S2, Supplementary Material online). Maximizing coverage values would suggest the SMALT software  with default parameters as the optimum choice; however, this program otherwise performs poorly . In contrast, using SMALT with a percentage identity threshold of 98% gives a higher quality result at a cost of reduced coverage. BWA-backtrack . Average coverage values between contigs are highly variable, ranging from 1.6× to 313.1× with a mean of 27.7× . This reflects, at least in part, the high variability in input DNA per species; under equimolar pooling for 209 species we would have expected an average coverage of 44× for this amount of data. Plotting average coverage estimates against contig length for the two assemblies and the non-redundant contigs shows that the merging of CA and IDBA-UD increases contiguity of many high coverage partial sequences but not all, indicating that this process could be further optimized .Mapping the quality-controlled reads to the final set of non-redundant contigs with widely used software packages gave inconsistent results. In all cases, some bases were not covered by the reads . In total, 70.32% of the assembled nucleotides were contained within contigs covering at least 11 protein-coding loci, whereas contigs containing three or fewer loci represented just 15.65%.Putative protein-coding gene (PCG) sequences were identified following tRNA-based fragmentation of the contigs and the mapping of these fragments to a mitogenome reference sequence . The PCGs were aligned with transAlign  is illustrated in figure 1b. This average coverage varies between 18× and 25× in the 13 PCGs, with an overall average of 22.2×.The least frequently recovered gene was cox1-5′) were matched to 275 barcode sequences obtained by polymerase chain reaction (PCR) on the individual DNA extracts (see below), successfully linking 124 contigs to morphospecies at an identity threshold of 98% with megablast. Of these, 96 were contigs with 10 or more genes (10+) while 28 were shorter contigs containing between one and eight PCGs (but not nad4l). Of the remaining 10+ contigs (28 of 124), 25 contained the barcode region but could not be linked to specimens due to the incompleteness of our barcode database (see below), whereas three contigs did not contain cox1.157 contigs containing the barcode region  and 240  mitogenomes, respectively. Four analyses were run: 1) 10+ contigs with Mito34, 2) 10+ contigs with Mito240, 3) 10+ contigs alone, and 4) nad4l-centered contigs .Phylogenetic analysis was conducted 1) on the 124 contigs of 10+ genes and 2) on a set of All trees obtained with PhyloBayes  on the acox1 barcodes. We first assessed the taxonomic assignment against Mito34 . Given the limited sampling of many families by this reduced reference set only 30 contigs were assigned to family , the failure to make a taxon assignment to family level was due to limited representations of small families .The reliability of phylogenetic assignment relative to the fully identified Mito240 sequences was tested using the 96 10+ contigs that could be linked to specimens and family-level identification through the o family , but theo family , 73 conto family , as the The representation of superfamilies by the contigs in a) was generally similar to the full tree obtained with Mito240. Archostemata was sister to the Caraboidea (suborder Adephaga) and they combined were sister to the Polyphaga. Within Polyphaga, the Scirtoidea was sister to all remaining Polyphaga. Further branches were occupied by a paraphyletic Elateriformia , a monophyletic, albeit poorly represented Staphylinoidea, and a monophyletic Cucujiformia, itself composed of the Cleroidea + Erotylid series (formerly considered part of Cucujoidea), Tenebrionoidea, the remaining lineages of Cucujoidea and Chrysomeloidea + Curculionoidea. The latter two superfamilies consisting of the highly species-rich weevils, leaf beetles and longhorns usually grouped as “Phytophaga,” were paraphyletic with respect to each other and this clade also included the Cucujoidea. These problems were alleviated to some extent when the shorter contigs of the nad4l-centred matrix were included (b), as the Chrysomeloidea were monophyletic, but a subset of Curculionoidea (Anthribidae) remained in a distant position from the main clade. Generally, both trees were similar overall and after pruning the short sequences the symmetric difference  , representing 275 unique haplotypes. When combined with the 157 cox1-5′ sequences from the assembled mitochondrial contigs, the resulting 326 unique haplotypes were grouped into 232 species-level entities using the GMYC  in case a species-level identification was possible. Only one sequence returned a hit, at 98.75% similarity. This was a circular mitogenome  that had no matching PCR barcode but was assigned to Chrysomeloidea in the tree with Mito34 and Chrysomelidae in the tree with Mito240. The BOLD match was Liroetiella antennata, a species of Chrysomelidae  described from Danum Valley   .nt database using discontiguous-megablast searches. Based on these analyses, out of 96 identified 10+ contigs with these genes available (93 for cob), a total of 32, 77, 54 and 42 contigs were correctly assigned to family based on the cox1-5′ (barcode), cox1-3′, cox2, and cob loci, respectively, and 82 contigs using any of these genes. The 3′ portion of cox1 was therefore by far the best performing marker in this analysis, attaining a success rate of 80.2% as compared with 75% for the phylogeny with the Mito240. Taxonomic assignment with MEGAN  to be included in the phylogenetic analysis, limiting the community phylogeny to 175 species. Conventional DNA barcoding gave a similar result, recovering sequences for 161 morphospecies and 197 GMYC groups. Given the low rate of barcoding success, possibly due to low DNA quality obtainable from canopy-trapped specimens that were left in situ for up to 24 h, this is clearly an underestimate of the true diversity of the sample. Incorporating the barcode sequences from the contigs we recovered 232 GMYC groups, indicating that the species included in the community phylogeny represent approximately 75% of the total diversity of the sample.Samples from highly biodiverse sites can be accumulated rapidly, but the effort for identification and phylogenetic assignment of the recovered specimens frequently is prohibitive. This has resulted in calls for procedures that can avoid the conventional taxonomic process by large-scale DNA sequencing . This stsupplementary information S2, Supplementary Material online). Here we used the contigs assembled from the lower stringency settings to maximize the length of the assembled sequences, at the risk of incorporating a small number of erroneous bases, as the impact on the phylogeny would be negligible and mostly lead to slightly increased terminal branch lengths. Filtering the assembled contigs to retain only mitochondrial sequences is complicated by the lack of close reference sequences for the species in the sample. Here we require a BLAST alignment length of 1 kb to a MitoDB sequence, effectively removing long non-mitochondrial contigs but at the cost of mitochondrial contigs less than 1 kb. Thus, we underestimate the number of assembled mitochondrial contigs but the phylogenetic analysis is unaffected due to the 2 kb (with nad4l) criterion for inclusion . In general we find that assembly quality is difficult to assess, either from general assembly statistics or by mapping reads to the contigs. We suggest that the length distribution of mitochondrial contigs is the most appropriate metric for comparing the suitability of different assemblers for mitochondrial metagenomics, particularly as statistics obtained from mapping are highly dependent on the software and parameters used. The variable mapping quality encountered herein  may have been exacerbated by variable intra- and interspecific sequence divergences in the sample, as read mapping softwares are not optimized to find the best alignment solution for reads derived from the same portion of the genome from multiple species simultaneously. In addition the insect mitogenome is AT-rich, especially in the (repetitive) control region, which may present an additional challenge to both mapping and assembly software . Ultimately the phylogenetic trees resulting from the assembled contigs, both with and without the MitoDB sequences, are consistent with existing hypotheses and where we are able to link contigs with a morphological identification their placement is correct. Thus, major errors in assembly are unlikely.The methodology represents a balance between economical low coverage sequencing of complex samples and the errors and omissions in the assembled contigs that might arise as a result. Greater efficiency of metagenomic sequencing to obtain mitogenome sequences will be key for the wider utility of this methodology, particularly for large-scale community ecology. The estimated proportion of truly mitochondrial reads for both libraries (1.1–1.4%) was more than 2-fold higher than the 0.5% reported from studies on the Illumina HiSeq and shorter insert sizes of the TruSeq libraries of 200 or 250 bp . Althougc–esupplementary fig. S3, Supplementary Material online). IDBA-UD produces the larger contig set, but both assemblers contribute unique sequences to the alignments indicating that the inclusion of both is beneficial (a). Additionally, the number of unique sequences produced with either assembler varied between genes so there is no clear basis to recommend one over the other. Additional study is needed of the behavior of these assemblers and the effect of parameter choice in the presence of variable inter- and intra-specific sequence divergence, particularly with respect to variation in rates of evolution between genes. Here we observe that the most conserved locus, cox1, was recovered the least frequently and two of the more variable loci, nad4l and nad6, were recovered the most frequently. This may indicate that closely related species have been combined to a single contig in the cox1 region, or that intraspecific diversity has been oversplit into different contigs in the nad4l–nad6 region. This variability in gene recovery does not appear to be related to the average coverage obtained for these alignments (b), which varies only between 18 and 25×, suggesting that this slight enrichment of nad4l and nad6 results from the assembly, rather than any sequencing bias toward this region.Our results indicate that combining the contigs from multiple assemblies can be beneficial to increase sequence contiguity. This is most evident from the increase in contigs greater than 15 kb and the number and proportion of those that were circularized , as wellneficial a. Additiignments b, which b) recovered most of the expected nodes defining major lineages at the superfamily level and a tree topology resembling that of earlier studies based on extensive sampling guided by taxonomic criteria  relationships may be unexpected, but past problems with these markers were encountered mainly at interorder or interphylum levels and in studies with sparse taxon sampling limited by conventional sequencing technology, which presumably exacerbated the effects of great heterogeneity in molecular rates and nucleotide composition . HeterogA phylogenetic approach to the description of complex (undescribed) canopy communities goes beyond existing analyses that mainly group species into guilds or functional types whose relative proportions have been used to characterize forest ecosystems , or simpcox1 barcode, yet in all but one instance no close matches were available in BOLD. In the face of the current incompleteness in barcode coverage at the species level, secure family-level identifications are crucial for describing the taxonomic composition of the sample and understanding the evolutionary relationships. We tested the possibility of assigning contigs to family using the BLAST top hit, MEGAN, and the phylogeny . Thus, even the best-performing locus gives a misleading description and the accuracy of any single identification is unknown. Given the high relative abundance of cox1-3′ sequences for beetles compared with other loci, additional tests would be needed to determine how these findings apply to other insects in mitochondrial metagenomic analyses.A key step in the evolutionary analysis of biodiversity is the appropriate assignment of a species to a clade or higher-level taxonomic entity. Ideally all contigs would be linked to a species-level identification through the hylogeny . The succox1-3′ BLAST top-hit and the phylogeny were similar herein, the phylogeny included just 240 species whereas a recent assessment of GenBank  were made where possible. Specimens were then assigned to morphospecies within each lowest taxonomic level. All observable features were taken into account when sorting to morphospecies, including body shape and proportions, total length, surface sculpturing, patterning, and coloration. A conservative approach to morphospecies assignment was taken in the case of ambiguously imaged specimens, erring on the side of lumping superficially similar specimens into a single group.cox1. Primers CO1F2 and CO1R2 . Sequencing was conducted on an ABI3700 sequencer. Sequences were assembled and edited in Geneious R6.1 (Biomatters Ltd.). A matrix of 648 bp was generated from these sequences, and barcodes were used as “baits” to link the assembled mitogenomes to particular specimens or morphospecies.DNA was extracted from each whole specimen individually following the BioSprint 96 protocol for DNA purification from tissues (Qiagen) and eluted to a volume of 200 µl. The DNA extracts were used for PCR amplification and Sanger sequencing of the 5′ portion (barcode region) of nd CO1R2  were usend CO1R2  , were used for phylogenetic reconstruction with RAxML  in R v3.1.1 . All unique haplotypes were searched against the BOLD database  to obtain a species-level identification where possible.For a barcode-based estimate of total species richness, MAFFT v7  and uniqth RAxML  .supplementary table S4, Supplementary Material online. A quality assessment of raw FASTQ files for each library was made using FastQC v0.10.1  prior to the removal of adapter sequences with Trimmomatic v0.30 (ILLUMINACLIP:2:30:10) . MitoDB includes a set of 245 partial and complete mitogenomes covering all major lineages of Coleoptera, including all four established suborders, all 18 superfamilies within Polyphaga and a total 97 families . Putative mitochondrial reads were extracted with a Perl script , allowing for a BLAST hit from either one or both reads per pair.A list of all programs and settings used to assemble mitochondrial genome contigs is included in 2:30:10) . To simp2:30:10)   and converted to FASTA. Assemblies with IDBA-UD used a similarity threshold of 98% and minimum and maximum k values of 80 and 230 bp, respectively. The same settings were used for a second assembly with IDBA-UD using more stringent quality controls to check for the effect of this step on assembly contiguity .Assembly of all putative mitochondrial reads from the two libraries was conducted with CA v7.0  and IDBAE value 1e-5; maximum target sequences 1). All contigs with a BLAST alignment length of ≥1 kb  were extracted with cdbfasta and annotated for tRNA sequences using COVE v2.4.4 . Annotated contigs over 15 kb were manually checked in Geneious for identical or near-identical overlapping terminal regions and were circularized where possible. Combining multiple assemblies correctly to maximize sequence contiguity while minimizing error requires some flexibility to allow genuine SNPs while discarding assembly errors. Here this is achieved by iterative reassembly and manual curation in Geneious over several steps, as the automated application of thresholds at the whole-contig level will in some cases introduce errors into the consensus sequence for a species, while in other cases con-specific sequences will not be identified as such and will inflate contig-based estimates of species diversity. Initially, the circular contigs from each assembly were assembled at 99% identity to make a non-redundant set against which the linear contigs were then filtered (99% identity) to remove linear sequences from one assembly which were circularized in the other. The remaining linear contigs were then de novo assembled in two steps, merging the most similar contigs first (99% identity), and later incorporating slightly more divergent sequences where appropriate (98% identity). In each step, the assembled contigs were checked manually and edited to resolve any discrepancies between the two assemblies and generate a final set of high-confidence, non-redundant contigs. The observed discrepancies fell mainly into two categories: First, SNPs were observed in several cases, presumably arising from genuine intraspecific variation; second, discrepancies were sometimes observed at the ends of contigs. In general the longer contig was taken to be the correct sequence, otherwise the IDBA-UD contig was preferentially selected due to the application of user-defined quality control parameters and a requirement for 98% identity during assembly. Where indels were observed in one contig relative to the other, the open-reading frame prediction tool in Geneious was used to determine which contig was most likely to be correct, given the expected sizes and arrangements of the PCGs.Following assembly, the contigs were filtered against MitoDB with BLASTn , and SMALT , with the latter additionally being used to map reads at 98% identity (−y 0.98). This gave the most plausible result but was highly conservative, thus read mapping could not be used to aid contig verification in the reassembly step. Mapping statistics were obtained with Qualimap . These fragments were then mapped with low stringency against a single reference mitochondrial genome  in Geneious to sort the fragments into genes . These fragments were extracted gene-by-gene and all fragments covering at least 50% of the length of the corresponding gene in the reference mitogenome were retained.Inter-tRNA regions of all non-redundant contigs ≥1 kb were extracted using FeatureExtract 1.2   followinPCGs were aligned using transAlign v1.2 . Each sequence per alignment was then scored as present in CA, IDBA-UD, or both assemblies. The mean coverage of the sequences in each alignment was also calculated based on megablast searches against a database of the quality-controlled reads . For each alignment, the estimated coverage is calculated as the total number of contributing bases divided by the total number of aligned nucleotides . For PCGs longer than 400 bp , the number of contributing bases is calculated as the sum of the lengths of the BLAST alignment for each read with an alignment length of at least 200 bp at 98% identity. For the three shortest genes, the number of contributing bases is calculated as the sum of the lengths of the BLAST alignment for each read with an alignment identity of 98% and alignment length of ≥50% of the length of the gene in the Tribolium castaneum reference genome (GenBank: NC_003081) .To assess the impact of combining two assemblies on the rate of recovery of unique gene sequences, the curated sequences from each alignment were searched against a database of inter-tRNA fragments obtained from FeatureExtract for each of the two original assemblies  and concatenated to form a supermatrix of 3,730 amino acids. We constructed two supermatrices, one requiring the presence of nad4l (the most highly represented gene in the data set) and minimum contig length of 2 kb, and another requiring a minimum of ten PCGs per contig (10+ contigs).The alignments were translated into protein sequences in Mesquite v2.75  to assess the impact of low reference taxon sampling on phylogeny-based taxonomic assignment.To incorporate the most informative novel sequences from this community into the growing mitochondrial phylogeny for beetles, we combined the 10+ contigs with the 245 complete and nearly complete mitogenomes in the MitoDB. The alignments described above were realigned with the equivalent data for the MitoDB, curated, translated and concatenated as previously, for a new data matrix of 3,695 amino acids under a criterion of a minimum representation of ten genes . A subset of this supermatrix formed a fourth data set, including the 10+ contigs and a reduced MitoDB set of 34 complete mitogenomes  using megablast  to link the contigs with a morphological identification where possible. A 98% identity threshold was used to allow identification of contigs representing multi-haplotype species even when the assembled haplotype was not represented in the bait database due to PCR or Sanger sequencing failure.All contig-derived cox1-5′, cox1-3′, cox2 and cob fragments in the NCBI nt database, using discontiguous-megablast . The degree of congruence between the top hit in each case and the morphological identification was assessed. For comparison, MEGAN5 . These BLAST-based identifications were compared against the assignations suggested by the phylogenetic placement of these contigs in the trees incorporating Mito34 and Mito240. In this case, identifications were to the lowest rank permitted by the tree topology and required monophyly of the contig with the reference sequences.All morphospecies-linked mitogenome sequences from the 10+ contigs were tested for hits of the , MEGAN5  .


The 1000 Genomes Project provides a unique source of whole genome sequencing data for studies of human population genetics and human diseases. The last release of this project includes more than 2,500 sequenced individuals from 26 populations. Although relationships among individuals have been investigated in some of the populations, inbreeding has never been studied. In this article, we estimated the genomic inbreeding coefficient of each individual and found an unexpected high level of inbreeding in 1000 Genomes data: nearly a quarter of the individuals were inbred and around 4% of them had inbreeding coefficients similar or greater than the ones expected for first-cousin offspring. Inbred individuals were found in each of the 26 populations, with some populations showing proportions of inbred individuals above 50%. We also detected 227 previously unreported pairs of close relatives (up to and including first-cousins). Thus, we propose subsets of unrelated and outbred individuals, for use by the scientific community. In addition, because admixed populations are present in the 1000 Genomes Project, we performed simulations to study the robustness of inbreeding coefficient estimates in the presence of admixture. We found that our multi-point approach (FSuite) was quite robust to admixture, unlike single-point methods (PLINK). The 1000 Genomes Project (TGP)134In June 2014, the third and final phase of this project consisted in more than 2,500 individuals from 26 populations , i.e. to have ancestry from different populations. In this case, the previous assumption is violated, which could bias FSuite f estimates. Indeed, it has been shown that single-point methods to estimate kinship and inbreeding coefficients are biased in presence of admixture in the population12f estimates on admixed individuals by simulation.The goal of this article is to describe the inbreeding patterns in the 26 populations of the final phase of this panel by using the genotype data obtained from the sequencing. We applied our FSuite pipelinef of an individual can thus be defined as the proportion of the genome that is HBD. Different approaches have been developed to estimate this coefficient from the genotype data of an individual without known genealogy, and can be classified in 2 main categories. First are single-point methods that rely only on the allele frequencies at each marker. Second are multi-point methods that use both allele frequencies and the fact that HBD genotypes come in blocks.The genome of an inbred individual is characterized by large regions homozygous-by-descent (HBD), i.e. regions where this individual has inherited two haplotypes identical-by-descent from a common ancestor. The genomic inbreeding coefficient f estimations. Here, we ran FSuite on 100 submaps by selecting several random markers in each genomic regions delimited by recombination hotspots. This strategy optimizes the f estimation accuracy and the detection of inbred individualsOur pipeline FSuite is a multi-point method, which requires the markers to be in minimal linkage disequilibrium (LD). FSuite therefore creates multiple random sparse marker maps (submaps) to minimize LD of the data while providing robust f estimates by simulating 100 replicates of a sample of 300 admixed individuals with different levels of inbreeding and different levels of European and African ancestry. FSuite multi-point estimates were first compared to the single-point estimates implemented in PLINKWe investigated the accuracy of FSuite f value (Δf) against the true genomic proportion of European ancestry (ADMCEU) of the individual, for first-cousin offspring (1C), second-cousin offspring (2C), and offspring of unrelated parents  (see f estimation (f root mean square errors (RMSEs) whatever the set of allele frequencies , especially when ADMCEU is far from 0.5. However, it did not significantly improve the accuracy of FSuite  had a low-quality estimate (Q-score ≤ 50). Among the 2,497 remaining individuals, 595 individuals were inferred as inbred, which represents nearly a quarter of the panel . These if equals to 0.035). This is in accordance with the population history of the Finns: a small number of founders and very little immigrationMost (501) of the 595 inbred individuals were inferred as 2C offspring and can be considered as descending from remote inbreeding. On the opposite, 94 individuals can be considered as descending from recent inbreeding, i.e. being offspring of first-cousin or closest relationships. These individuals belonged to 14 populations, including all SAS and AMR populations, but only one AFR population (6 from GWD) and one EUR population (one from GBR). Three-quarters of the inbred individuals came from 4 populations: ITU (10), PJL (22), STU (32) and CLM (8). Finally, note that GIH and PUR populations, that had a high proportion of inbred individuals , had only one individual who exhibited recent inbreeding. Of interest, the most inbred European population is the Finnish with one third of individuals detected as remote inbreeding  of the 1000 Genome Project, we found that nearly a quarter of the individuals in this panel were inbred and that around 4% of them had inbreeding coefficients similar or greater than the ones expected for first-cousin offspring. This level of inbreeding was unexpectedly high, and is much higher than the 4% of inbred individuals that we detected on HapMap IIIFinally, we also detected pairs of close relatives (up to first-cousin) not reported from pedigree information. We thus propose two subsets of unrelated and outbred individuals. The first one, labeled TGP 2457, removes relatedness of first and second degree, similarly to what has been performed previously for HGDP-CEPH panelf and a, defining f as the individual inbreeding coefficient and 1/(a(1 − f)) as the expected length of HBD segments (here cM), that are estimated by maximum likelihood. FSuite estimates f as the median value of the estimates obtained on the different submaps after removing the ones with a > 1, and thus calculates a quality Q-score of each estimate as the number of submaps that have not been removed. FSuite also uses FEstim likelihood computed on the different submaps to estimate whether an individual is inbred or not by a likelihood ratio test testing if f is significantly different from 0.001  and if he/she was inferred as AV, 2 × 1C, 1C or 2C.To improve the accuracy of inbreeding detection, we created 100 submaps by selecting several random markers in each genomic regions delimited by HapMap II recombination hotspots24et al.In order to investigate the accuracy of FSuite estimates in admixed populations, we simulated samples in which individuals had different proportions of European and African ancestry. For this purpose, we used the same simulation process as in Gazal Here, we used 232 and 226 haplotypes of CEU and YRI, respectively, with 987,221 SNPs coming from the Illumina Human 1M chip or the Affymetrix v6.0 chip. To simulate an individual genome, we first assigned randomly to each pedigree founder a haplotype origin (CEU or YRI). For example, a 1C offspring will have randomly between 0 and 8 pedigree founders with a CEU origin. Then, according to this haplotype origin, HapMap reference haplotypes were randomly drawn without replacement for each chromosome and were assigned to pedigree founders to construct the genotype data of the individual.ftrue) was calculated by dividing the genome length in cM that is HBD by the total genome length. The genome length was obtained by adding the genetic distance between the first and the last marker on each autosome. The CEU admixture component (ADMCEU) of an individual was calculated as the proportion of genome in cM with CEU reference haplotypes.Pedigree founders were used to detect the true HBD segments of an individual, and the haplotypes of CEU and YRI origin. The true inbreeding coefficient of an individual  and YRI (estimated on the 226 ones), to investigate inbreeding estimation of an admixed individual in a homogeneous sample. Then, to investigate the impact of inappropriate allele frequencies, we used 340 HapMap III haplotypes coming from JPT and CHB populations (JPT/CHB). Finally, we estimated allele frequencies as it is done by default, i.e. on the 300 individuals of the sample.f in each replicate through the single-point method implemented in PLINK version 1.90b2b. Then, to take into account the admixture of the individuals, we also ran single-point method implemented in REAP version 1.2 on each simulated sample. Ancestry proportion of each individual and allele frequencies of each of the ancestral populations were estimated by running software Admixture version 1.23 on each sample; LD within each sample was removed as advised in the Admixture documentation (PLINK pruning option–indep-pairwise 50 10 0.1), and the number of ancestral populations was set to K = 2. All PLINK and REAP negative estimates were set to 0. The performances of the different estimators were compared on the 5 different types of offspring (1-4C and OUT) by randomly drawing one individual of each type from each replicate with ftrue > 0. Accuracy of the different estimators was assessed by measuring the difference between the estimated f and their corresponding ftrue (Δf), and by computing its root mean square error (RMSE) as described previsoulyFSuite was thus run on each replicate with these 4 different sets of allele frequencies. To compare the benefit of using multi-point methods, such as the one implemented in FSuite, to single-point ones, we first estimated ADMCEU) and YRI admixture components, respectively.Finally, to investigate the benefits of using individual allele frequencies with FSuite, we computed for each individual their theoretical individual allele frequencies, obtained by weighting CEU allele frequencies and the YRI allele frequencies by their true CEU (−5 in at least one population) were excluded. After these different filters, 3,033,793 SNPs were retained. The genetic positions of these SNPs were extrapolated from HapMap II genetic map.The final release of the Phase III consists in sequencing data of 2,535 individuals representing 26 populations divided into 5 super populations. We downloaded via ftp the vcf file of the final variant set (release v5 20130502), from which 31 related individuals have been removed based on pedigree information and on the results of the genetic analysis that has been performed to uncover unknown relationships of the phase I of this projectWe then ran RELPAIR version 2.0.1 on every pair of individuals from each population in order to remove unknown first or second-degree relationships to estimate population allele frequencies. The submap approach used in FSuite was extended to RELPAIR. We created 100 submaps using recombination hotspots having recombination intensity >13 cM/Mb, this threshold allowing selecting less than the 9,999 SNPs allowed by RELPAIR  while minimizing LD. For each pair, the relationship inferred the largest number of times on the 100 submaps was reported. Note that none of the detected relationships was previously reported by the phase I of this project.How to cite this article: Gazal, S. et al. High level of inbreeding in final phase of 1000 Genomes Project. Sci. Rep.5, 17453; doi: 10.1038/srep17453 (2015).


Obstetric hemorrhage is the leading cause of maternal mortality, particularly in low resource settings where delays in obtaining definitive care contribute to high rates of death. The non-pneumatic anti-shock garment (NASG) first-aid device has been demonstrated to be highly cost-effective when applied at the referral hospital (RH) level. In this analysis we evaluate the incremental cost-effectiveness of early NASG application at the Primary Health Center (PHC) compared to later application at the RH in Zambia and Zimbabwe.We obtained data on health outcomes and costs from a cluster-randomized clinical trial (CRCT) and participating study hospitals. We translated health outcomes into disability-adjusted life years (DALYs) using standard methods. Econometric regressions estimated the contribution of earlier PHC NASG application to DALYs and costs, varying geographic covariates  to yield regression models best fit to the data. We calculated cost-effectiveness as the ratio of added costs to averted DALYs for earlier PHC NASG application compared to later RH NASG application.Overall, the cost-effectiveness of early application of the NASG at the primary health care level compared to waiting until arrival at the referral hospital was $21.78 per DALY averted . By country, the results were very similar in Zambia, though not statistically significant in Zimbabwe. Sensitivity analysis suggests that results are robust to a per-protocol outcome analysis and are sensitive to the cost of blood transfusions.Early NASG application at the PHC for women in hypovolemic shock has the potential to be cost-effective across many clinical settings. The NASG is designed to reverse shock and decrease further bleeding for women with obstetric hemorrhage; therefore, women who have received the NASG earlier may be better able to survive delays in reaching definitive care at the RH and recover more quickly from shock, all at a cost that is highly acceptable. Ninety-nine percent of the 800 maternal deaths that occur each day are in developing countries . While gObstetric hemorrhage continues to be the leading cause of maternal mortality and morbidity worldwideC . UterineThe non-pneumatic anti-shock garment (NASG) has been studied as a means to stabilize women with hypovolemic shock secondary to obstetric hemorrhage -7. The NSeveral policy initiatives, such as expanding access to emergency obstetric care, have been established over the past few decades to improve maternal health, yet the evidence base on how to implement these policies and strategies at the health system level remains weak . ScalablThe NASG is considered a cost-effective intervention for referral hospitals (RH), based on quasi-experimental studies in Egypt and Nigeria . For womIn 2012, the World Health Organization (WHO) recommended the NASG be used as a temporizing measure for women with PPH until definitive care, blood transfusions, and/or surgery can be obtained. Our aim was to evaluate the cost-effectiveness from the payer’s perspective of early NASG intervention using evidence from a cluster-randomized controlled trial of early NASG application at the primary health care (PHC) level prior to transport compared to later NASG application at the referral hospital (RH) level . This stThe clinical data for this study was approved by the institutional review boards (IRBs) affiliated with the following institutions: University of California, San Francisco; University of Zambia, Lusaka; University of Zimbabwe-UCSF Collaborative Programme on Health Research; and the Department of Reproductive Health and Research of the World Health Organization. The cost data collected for this study did not involve human subjects, and was thus exempt from IRB oversight.This cost-effectiveness analysis builds on a previously reported clinical trial, summarized here. The clinical trial data belongs to UCSF and is freely available with a UCSF data sharing agreement. The cluster-randomized controlled trial of 38 PHCs in Zimbabwe and Zambia enrolled patients from 2009 to 2012. Eligible PHCs were peri-urban with at least 500 annual deliveries that referred obstetric hemorrhage (OH) cases (≥500 mL blood loss) to one of five study regional hospitals. Participants were admitted at the PHC and were consenting women with any obstetric hemorrhage etiology and hypovolemic shock. Women with antepartum hemorrhage with a viable fetus were excluded. PHCs were randomized to either the early application or later application group using a covariate-constrained procedure to ensure balance across intervention arms on number of deliveries, number of deliveries per midwife, distance to referral hospital (RH), and proportion of OH cases expected .Women who presented at the PHC at < 24 weeks gestation  were enrolled in the outpatient department, while women at ≥ 24 weeks were enrolled in the maternity department. Midwives at the PHC were trained to treat PPH with uterotonics and IV fluids, repair first- and second-degree perineal lacerations, and refer any patient with estimated blood loss > 500 mL to the RH. PHCs were not equipped to provide blood transfusions, surgery, or manual vacuum aspiration (MVA). Each PHC had access to a shared ambulance system to transfer patients to the RH.All eligible women had a perineal pad applied at study entry in the PHC to measure blood loss. Women in the early application arm received the NASG  at the PHC and women in the later application arm received it at the RH per treatment protocol. All women were referred to the RH and were transported by ambulance, private vehicle, or taxi. Oxygen, IV fluids, uterotonics or uterine massage for uterine atony, suturing of lacerations, manual removal of placenta or retained tissues, MVA, surgery, and blood transfusions were available as needed at the RH. More detailed information regarding the design of the CRCT is provided elsewhere [We use a per-protocol analysis . CharactDisability-adjusted life years (DALYs) without age-weighting were used to quantify the burden of disease as a discounted sum of the number of years of life lost (YL) from early death and years lost due to disability (YLD) . The timThere was no evidence of statistically significant differences between earlier and later NASG application across mortality and morbidity outcomes. The odds of death in the early application group were 64% lower (OR 0.36 (95% CI: 0.08 – 1.53) than the later application group Table . There wWe estimated costs using micro-costing methods. Resource use was estimated from clinical trial records. Unit costs were collected from pharmacies, blood banks, and hospital administrators in local currencies and converted into international dollars . Costs oThe material cost of the NASG per use was estimated as $1.04, based on an approximate price of $75 and an estimated life of 72 uses per garment . The cost of cleaning the NASG included bleach, bucket for immersion, personal protection equipment, and personnel, and was estimated at $0.50 per use. The total estimate for cost of NASG was $1.54 per use.Training costs included transportation, facilities, materials, and personnel costs. The model assumes training has a 10-year life, which is conservative given that training is not designed to require a refresher. Estimates of training cost per patient were based on actual costs collected from one facility where two hundred participants attended a stand-alone (NASG only) training. The base-case estimate for the cost of training per NASG use was $1.62.Costs of uterotonics, oxytocin per ampoule (10 IU) and misoprostol per dose, were collected from hospital pharmacies and hospital administrators in one facility in Zambia and one facility in Zimbabwe. Cost of blood transfusions was based on cost per unit of blood in each country during the study period. The cost of one unit of blood and uterotonics in Zimbabwe were significantly higher than in Zambia. In Zimbabwe blood cost $135 for the first unit and $90 per each additional unit, compared to $42 per unit in Zambia. Blood was not always available during the study period.Emergency hysterectomy (EH) costs for complications due to intractable uterine atony and complications of abortion were collected. Costs include personnel, equipment, anesthesia, and operating room costs. Emergency hysterectomies were conservatively estimated to require 6 personnel over 60 to 90 minutes. No other surgeries were included in this analysis, as etiologies differed, and some etiologies require surgery .Eic is the probability of a disability-adjusted life year of individual i in cluster c and Cic is the costs of individual i in cluster c; Jc is an intervention indicator for cluster c where j = 1 for early application group and j = 0 for later application group; Zimc is an indicator for Zimbabwe  for each cluster c; and uic is the error term.We estimated a series of models using Stata 13.0 . We anticipated that variances would differ across clusters due to variations in adherence to treatment protocol; we include random effects to allow individual-level differences to vary across clusters . The genEic, the probability of a disability-adjusted life year of individual i in cluster c, we specified 4 random intercept models. In Model 1, the model was specified as above but excluded the country indicator. In Model 2, we added the country indicator. In Model 3, we estimated an interaction term between country indicator Zimc and early application indicator Jc to understand country-specific effects of early application. In Model 4, we omitted the country indicator and instead used 4 referral hospital indicators, RHc, where the Lusaka hospital was the reference. We re-specified Model 4 for Zambia only (Model 4a) to improve statistical power as 13 of the 15 deaths occurred in Zambia. Models 5-8a were specified identical to Models 1-4a substituting cost of individual i in cluster c for outcome. We used a likelihood ratio test to compare model fit for Models 1–4 and 5–8.In order to determine δ1/β1.We compared the effectiveness and costs between the earlier and later NASG application groups to calculate the incremental cost-effectiveness ratio (ICER) . The ICEWe conducted a sensitivity analysis by simulating probabilistic clinical resource costs and mortalities to provide insight to their contribution to the ICER. We varied the unit cost of blood from $20 to $200 while keeping the blood transfusion rate  and all other variables constant to reflect the probable range of costs of blood within sub-Saharan Africa. We also assessed how varying the relative odds ratio of death given timing of NASG application would impact the ICER, and whether the results from our primary models were consistent when stratifying by severe shock at study entry, defined by mean arterial pressure <60 mm Hg.The model that fit the data best Model 4) showed that women in the early application group had 0.712 fewer DALYs than those in the later application group , indicating that a small number of women had very high costs. The median cost was $19.62 (IQR $13.62- $25.62) in Zimbabwe and $8.82 ($8.02-92.02) in Zambia. The unadjusted mean cost per woman was similar across both countries at $54.56 for the early application group and $52.61 for the later application group  individuals received transfusions. In Zambia, roughly half of the women received blood transfusions and a slightly higher  proportion of women in the early application group received blood transfusion. Across both countries and intervention arms, three out of four women received an uterotonic; more than 97% of those receiving an uterotonic received oxytocin. In Zambia, costs of uterotonics are marginal. In Zimbabwe, costs per uterotonic dose were 4–15 times higher, although rates and costs did not differ meaningfully across intervention groups.Early application of the NASG cost $15.51 more than later application based on estimates from the best-fit model (Model 7). In our first cost model, treatment costs for the early application group were $1.96 higher on average than for the later application group, but this difference was not statistically significant (Table RHc) using the Lusaka hospital as reference and this improved the fit of the model compared to the simple model (Model 5). Compared to the Lusaka hospital, the Harare1, Copperbelt1, and Copperbelt2 hospitals had significantly lower treatment costs ; however, there was no significant difference in cost by timing of application within this model. In Model 8a (restricted to Zambia), the mean treatment cost for the early application group was significantly higher than the later application group, at $76.49 vs. $60.45 respectively. Consistent with Model 8, Model 8a shows significantly lower treatment costs for the Copperbelt1 and Copperbelt2 hospitals compared to the Lusaka hospital.The above estimates may be biased if the availability and use of resources varied significantly across referral hospital. In Model 8, we substituted the country indicator with 4 referral hospital indicators  compared to late application based on the results from our best-fit models. In Zambia, early NASG application costs $22.00 for each DALY averted compared to late application ($16.04/0.729 DALYs). We do not have enough evidence to determine a statistically meaningful result for Zimbabwe.First, to assess for potential non-adherence to treatment protocol that might affect a per-protocol analysis, we ran the same models using an intent-to-treat (ITT) analysis. There were no significant differences in effects between the approaches; the estimates of the per-protocol analysis were attenuated slightly by the smaller sample size, as expected. The low attrition rate and negligible degree of bias suggest that our per-protocol results are what we would expect in a clinical practice setting.We reran Models 1–8, stratified by shock status. Women who entered the study with mean arterial blood pressure of less than 60 are considered to be in severe shock. There were no differences in estimates in our cost models. However, the DALYs averted rose from 0.712 to 0.844 in the best-fitting effectiveness model. This suggests that, in this sample, early application of the NASG before transport appeared to have greater benefit for women who did not enter in severe shock.The ICER of applying the NASG early compared to later was sensitive to the unit cost of blood transfusions and ranged from $9.22-87.85 . In Zambia, early NASG application cost a nearly identical $22.00 for each DALY averted compared to late application ($16.04/0.729 DALYs). This ratio is considered “very cost-effective” by the WHO definition of annual GDP per capita [Mean treatment costs were patterned differently by country, where the early application group was associated with somewhat lower but not statistically different costs in Zimbabwe ($17.16), but significantly higher costs ($15.51) in Zambia in our descriptive analyses, and consistent with the random-effects Model 7.The difference in costs across the two countries is largely driven by the rate and cost of blood transfusion Figure . This isprevent worse shock during transport to definitive treatment. Importantly, this finding is inconsistent with previous results from multiple studies, which found greater NASG benefits for women in severe shock when applied at the RH level only [We found that mild shock (MAP > = 60) on presentation was associated with more DALYs averted per women who received the NASG earlier. This may be due to the considerable benefit of early application of the NASG to vel only . TherefoResults from the previous cost-effectiveness analysis on data from the referral hospital level, which compared NASG application to no application, determined the intervention to be cost saving (Egypt) or extremely cost-effective (Nigeria at $3.13 per DALY averted) . Our couPolicy makers with limited resources may need to make decisions about which levels of the health care system to place the NASG. Should NASG be only at the RH? At both the RH and the PHC? Or should it be used at even lower levels of the health care continuum? Should NASGs be on ambulances or other transport?Given the strength of the evidence for cost-effectiveness at the referral hospital level, the decision to implement the NASG within the RH level is straightforward. In deciding whether to incur the additional costs of adding the NASG at the PHC level, policy makers may want to consider several factors: 1) the proportion of women who first seek care at the PHC level; 2) the proportion of maternal deaths from hemorrhage which occur at the PHC, during transit, or within the first 2 hours after admission to the RH; 3) the clinical capacity for resuscitation at the PHC level; 4) average transport times from the PHC to the RH; and 5) the length of delays in admission and receipt of quality definitive care including blood transfusions and surgery once at the RH. Greater benefits will be obtained from situating the NASG at the PHC level in contexts where the first contact with care is at the PHC level, or where receipt of definitive care is delayed by transportation, less optimal resuscitation or other time delays faced at the PHC or RH. Data on situating the NASG within emergency transport are not available.There are several limitations to this study. The major limitation was low power due to a smaller than expected number of deaths in the CRCT. Also, the study was not designed to explicitly study the availability and provision of blood transfusion; thus, we had less reliable information about the availability of blood transfusions. Given that the earlier and later application arms occurred simultaneously and blood transfusions do not appear to be clustered within any set of patients referred from a PHC, we expect non-differential bias across intervention arms.The analysis did not include the economic benefits and costs from the patient’s perspective. It is likely that early application of the NASG would have health and economic benefits for women and their families.The costs for using the NASG will fluctuate, but may continue to decrease, as competitive sales pricing has resulted in prices as low as $57.50.  Incorporating training into ongoing Emergency Obstetric Newborn Care, Life Saving Skills, and pre-service training will eventually eliminate a separate price for stand-alone in-service NASG training. The differential costs for blood, medications, and surgery will be highly context specific.Applying the NASG earlier at the Primary Health Care level for women in hypovolemic shock secondary to obstetric hemorrhage instead of waiting until the woman has been transferred to the Referral Hospital has the potential to be a cost-effective decision across many clinical settings. Our evidence from Zambia strongly supports this conclusion. Evidence from Zimbabwe is suggestive but not statistically significant. As the NASG is designed to reverse shock and decrease further bleeding for women in obstetric hemorrhage, patients who have received the NASG earlier may be better able to survive delays in reaching definitive care at the RH and recover more quickly from shock, all at a cost that is highly acceptable. Policymakers and administrators need to consider a variety of factors to determine which levels of their health system will cost-effectively save the most lives.