Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Risk factors for placental malaria, sulfadoxine-pyrimethamine doses, and birth outcomes in a rural to urban prospective cohort study on the Bandiagara Escarpment and Bamako, Mali

Abstract Background Malaria in Mali remains a primary cause of morbidity and mortality, with women at high risk during pregnancy for placental malaria (PM). Risk for PM and its association with birth outcomes was evaluated in a rural to urban longitudinal cohort on the Bandiagara Escarpment and the District of Bamako. Methods Placental samples (N = 317) were collected from 249 mothers who were participants in a prospective cohort study directed by BIS in the years 2011 to 2019. A placental pathologist and research assistant evaluated the samples by histology in blinded fashion to assess PM infection stage and parasite density. Generalized estimating equations (GEE) were used to model the odds of PM infection. Results In a multivariable model, pregnancies in Bamako, beyond secondary education, births in the rainy season (instead of the hot dry season), and births to women who had ≥ 3 doses of sulfadoxine-pyrimethamine (SP) instead of no doses were associated with reduced odds of experiencing PM (active and past infections combined). Births in later years of the study were strongly associated with reduced odds of PM. Maternal age, which was positively associated with offspring year of birth, was significant as a predictor of PM only if offspring year of birth was omitted from the model. Gravidity was positively associated with both maternal age and offspring year of birth such that if either variable was included in the model, then gravidity was no longer significant. However, if maternal age or year of offspring birth were not adjusted for, then the odds of PM were nearly two-fold higher in primigravida compared to multigravida. Birth outcomes improved (+ 285 g birth weight, + 2 cm birth length, + 75 g placental weight) for women who had ≥ 3 doses of SP compared to no doses, but no difference was detected in birth weight or length for women who had 2 instead of ≥ 3 SP doses. However, at 2 instead of ≥ 3 doses placentas were 36 g lighter and the odds of low birth weight ( 10% erythrocytes infected) were significantly associated with decreases in birth weight, birth length, and placental weight, as were chronic PM infections. The women who received no SP during pregnancy (7% of the study total) were younger and lacked primary school education. The women who received ≥ 3 doses of SP came from more affluent families. Conclusions Women who received no doses of SP during pregnancy experienced the most disadvantageous birth outcomes in both Bamako and on the Bandiagara Escarpment. Such women tended to be younger and to have had no primary school education. Targeting such women for antenatal care, which is the setting in which SP is most commonly administered in Mali, will have a more positive impact on public health than focusing on the increment from two to three doses of SP, although that increment is also desirable.http://deepblue.lib.umich.edu/bitstream/2027.42/173706/1/12936_2022_Article_4125.pd

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

<div><p>Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R² = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R² = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R² = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R² = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10⁷-fold range was reduced more than 10,000-fold, from 2.3×10⁹ to 1.4×10⁵ sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.</p></div

Performance of competitive amplicon library preparation with ERCC Reference Materials.

<p><b>a)</b> Measured signal abundance of ERCC targets in samples A, B, C and D. X-axis units are derived from Ambion product literature for the known concentration of ERCC spike-in controls (n = 104). <b>b)</b> Difference plots of data in panel A ordered numerically by ERCC ID. Each ERCC target depicted was measured at least once in all four samples A–D. For purposes of clarity, ERCC-170 is highlighted orange in panels A and B (n = 104). <b>c)</b> Samples C and D represent a 3∶1 and 1∶3 mixture, respectively, of samples A and B. These ratios were used to calculate expected measurements for samples C and D (X-axis). Actual measurements of samples C and D are plotted on the Y-axis (n = 52). <b>d)</b> Coefficient of variation (CV) in measurements of ERCC targets in samples A-D, for those assays with at least two IS dilution points. Red line depicts expected CV based on a Poisson sampling (n = 95). <b>e)</b> ROC curves to detect fold change with corresponding area under the curve (AUC) with 95% confidence intervals. ROC curves are derived from the comparison of differential ratio subpools of ERCC targets in samples: A vs. B, A vs. C, A vs. D, B vs. C, B vs. D and C vs. D. Results for 1.1-fold change represent a range of differential ratio subpools [1.05–1.174] (controls n = 100, tests n = 96); 1.25-fold change [1.175–1.374] (controls n = 163, tests n = 163); 1.5-fold change [1.375–1.74] (controls n = 229, tests n = 227); 2.0-fold change [1.75–2.49] (controls n = 229, tests n = 223); ≥4.0-fold change [2.5–10.0] (controls n = 286, tests n = 290).</p

Schematic depiction of how competitive amplicon library preparation reduces oversampling.

<p><b>a)</b> Depicted are two native targets (NT) within a hypothetical cDNA sample. One NT is in high abundance, 10⁸ copies (“Abundant” NT), while another is in low abundance, 10² copies (“Rare” NT), representing a one million-fold difference in abundance between targets. This hypothetical cDNA sample is combined with a mixture of internal standards (IS) with a fixed relationship of concentrations at 10⁵ copies. <b>b)</b> Depicted is the competitive multiplex-PCR library preparation for panel A. The PCR amplification plots for both the “Abundant” and “Rare” NT are separated for purposes of clarity, but occur in the same reaction. During competitive multiplex-PCR, each NT competes equally with its respective competitive IS for dNTPs, polymerase and a limiting concentration of primers. Because the starting concentration of each target’s primer-pair is the same, each competitive reaction will plateau around the same end-point concentration (∼10⁹ copies). <b>c)</b> The equal competition between each NT and respective IS preserves the proportional relationship between NT in the original sample, allowing for measurement of native target abundance without signal compression (also see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079120#pone.0079120.s012" target="_blank">Animation S1</a></b>). Yet, a 10⁶ fold range of templates is reduced to 10³ after competitive multiplex-PCR library preparation resulting in a 1,000-fold reduction in oversampling/sequencing of the high abundance target.</p

Performance of competitive amplicon library preparation with endogenous cDNA targets.

<p><b>a–d)</b> Absolute signal abundance of cDNA targets in sample A in units of copies per library preparation measured on separate days, at different sites (OU = Ohio University; UTMC = University of Toledo Medical Center), and between different reverse transcription preparations (RT1 and RT2). <b>a)</b> Inter-day effect (n = 88). <b>b</b>) Inter-day and Inter-site effect (n = 81). <b>c)</b> Inter-day and Inter-library effect (n = 92). <b>d)</b> Inter-day, Inter-site and Inter-library effect (n = 80). <b>e–f)</b> Samples C and D represent a 3∶1 and 1∶3 mixture, respectively, of total RNA from samples A and B. These ratios were used to calculate expected measurements for samples C and D (X-axis) from measurements of A and B. Plotted on the Y-axis are actual measurements of samples C (n = 86) and D (n = 90).</p