Mapping and phasing of structural variation in patient genomes using nanopore sequencing

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline—NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications

The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology

Tuberculosis screening among ambulatory people living with HIV: a systematic review and individual participant data meta-analysis.

BackgroundThe WHO-recommended tuberculosis screening and diagnostic algorithm in ambulatory people living with HIV is a four-symptom screen (known as the WHO-recommended four symptom screen [W4SS]) followed by a WHO-recommended molecular rapid diagnostic test (eg Xpert MTB/RIF [hereafter referred to as Xpert]) if W4SS is positive. To inform updated WHO guidelines, we aimed to assess the diagnostic accuracy of alternative screening tests and strategies for tuberculosis in this population.MethodsIn this systematic review and individual participant data meta-analysis, we updated a search of PubMed (MEDLINE), Embase, the Cochrane Library, and conference abstracts for publications from Jan 1, 2011, to March 12, 2018, done in a previous systematic review to include the period up to Aug 2, 2019. We screened the reference lists of identified pieces and contacted experts in the field. We included prospective cross-sectional, observational studies and randomised trials among adult and adolescent (age ≥10 years) ambulatory people living with HIV, irrespective of signs and symptoms of tuberculosis. We extracted study-level data using a standardised data extraction form, and we requested individual participant data from study authors. We aimed to compare the W4SS with alternative screening tests and strategies and the WHO-recommended algorithm (ie, W4SS followed by Xpert) with Xpert for all in terms of diagnostic accuracy (sensitivity and specificity), overall and in key subgroups (eg, by antiretroviral therapy [ART] status). The reference standard was culture. This study is registered with PROSPERO, CRD42020155895.FindingsWe identified 25 studies, and obtained data from 22 studies (including 15 666 participants; 4347 [27·7%] of 15 663 participants with data were on ART). W4SS sensitivity was 82% (95% CI 72-89) and specificity was 42% (29-57). C-reactive protein (≥10 mg/L) had similar sensitivity to (77% [61-88]), but higher specificity (74% [61-83]; n=3571) than, W4SS. Cough (lasting ≥2 weeks), haemoglobin (2), and lymphadenopathy had high specificities (80-90%) but low sensitivities (29-43%). The WHO-recommended algorithm had a sensitivity of 58% (50-66) and a specificity of 99% (98-100); Xpert for all had a sensitivity of 68% (57-76) and a specificity of 99% (98-99). In the one study that assessed both, the sensitivity of sputum Xpert Ultra was higher than sputum Xpert (73% [62-81] vs 57% [47-67]) and specificities were similar (98% [96-98] vs 99% [98-100]). Among outpatients on ART (4309 [99·1%] of 4347 people on ART), W4SS sensitivity was 53% (35-71) and specificity was 71% (51-85). In this population, a parallel strategy (two tests done at the same time) of W4SS with any chest x-ray abnormality had higher sensitivity (89% [70-97]) and lower specificity (33% [17-54]; n=2670) than W4SS alone; at a tuberculosis prevalence of 5%, this strategy would require 379 more rapid diagnostic tests per 1000 people living with HIV than W4SS but detect 18 more tuberculosis cases. Among outpatients not on ART (11 160 [71·8%] of 15 541 outpatients), W4SS sensitivity was 85% (76-91) and specificity was 37% (25-51). C-reactive protein (≥10 mg/L) alone had a similar sensitivity to (83% [79-86]), but higher specificity (67% [60-73]; n=3187) than, W4SS and a sequential strategy (both test positive) of W4SS then C-reactive protein (≥5 mg/L) had a similar sensitivity to (84% [75-90]), but higher specificity than (64% [57-71]; n=3187), W4SS alone; at 10% tuberculosis prevalence, these strategies would require 272 and 244 fewer rapid diagnostic tests per 1000 people living with HIV than W4SS but miss two and one more tuberculosis cases, respectively.InterpretationC-reactive protein reduces the need for further rapid diagnostic tests without compromising sensitivity and has been included in the updated WHO tuberculosis screening guidelines. However, C-reactive protein data were scarce for outpatients on ART, necessitating future research regarding the utility of C-reactive protein in this group. Chest x-ray can be useful in outpatients on ART when combined with W4SS. The WHO-recommended algorithm has suboptimal sensitivity; Xpert for all offers slight sensitivity gains and would have major resource implications.FundingWorld Health Organization

Warm ocean processes and carbon cycling in the Eocene

Sea surface and subsurface temperatures over large parts of the ocean during the Eocene epoch (55.5–33.7?Ma) exceeded modern values by several degrees, which must have affected a number of oceanic processes. Here, we focus on the effect of elevated water column temperatures on the efficiency of the biological pump, particularly in relation to carbon and nutrient cycling. We use stable isotope values from exceptionally well-preserved planktonic foraminiferal calcite from Tanzania and Mexico to reconstruct vertical carbon isotope gradients in the upper water column, exploiting the fact that individual species lived and calcified at different depths. The oxygen isotope ratios of different species' tests are used to estimate the temperature of calcification, which we converted to absolute depths using Eocene temperature profiles generated by general circulation models. This approach, along with potential pitfalls, is illustrated using data from modern core-top assemblages from the same area. Our results indicate that, during the Early and Middle Eocene, carbon isotope gradients were steeper (and larger) through the upper thermocline than in the modern ocean. This is consistent with a shallower average depth of organic matter remineralization and supports previously proposed hypotheses that invoke high metabolic rates in a warm Eocene ocean, leading to more efficient recycling of organic matter and reduced burial rates of organic carbon

A 40-million-year history of atmospheric CO2

The alkenone–pCO2 methodology has been used to reconstruct the partial pressure of ancient atmospheric carbon dioxide (pCO2) for the past 45 million years of Earth's history (Middle Eocene to Pleistocene epochs). The present long-term CO2 record is a composite of data from multiple ocean localities that express a wide range of oceanographic and algal growth conditions that potentially bias CO2 results. In this study, we present a pCO2 record spanning the past 40 million years from a single marine locality, Ocean Drilling Program Site 925 located in the western equatorial Atlantic Ocean. The trends and absolute values of our new CO2 record site are broadly consistent with previously published multi-site alkenone–CO2 results. However, new pCO2 estimates for the Middle Miocene are notably higher than published records, with average pCO2 concentrations in the range of 400–500?ppm. Our results are generally consistent with recent pCO2 estimates based on boron isotope-pH data and stomatal index records, and suggest that CO2 levels were highest during a period of global warmth associated with the Middle Miocene Climatic Optimum (17–14 million years ago, Ma), followed by a decline in CO2 during the Middle Miocene Climate Transition (approx. 14 Ma). Several relationships remain contrary to expectations. For example, benthic foraminiferal ?18O records suggest a period of deglaciation and/or high-latitude warming during the latest Oligocene (27–23?Ma) that, based on our results, occurred concurrently with a long-term decrease in CO2 levels. Additionally, a large positive ?18O excursion near the Oligocene–Miocene boundary (the Mi-1 event, approx. 23?Ma), assumed to represent a period of glacial advance and retreat on Antarctica, is difficult to explain by our CO2 record alone given what is known of Antarctic ice sheet history and the strong hysteresis of the East Antarctic Ice Sheet once it has grown to continental dimensions. We also demonstrate that in the Neogene with low CO2 levels, algal carbon concentrating mechanisms and spontaneous biocarbonate–CO2 conversions are likely to play a more important role in algal carbon fixation, which provides a potential bias to the alkenone–pCO2 method