Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments. Cycle sequencing conditions are presented to sequence DNA ...samples isolated from a number of microbial genomes including 750-kb Ureaplasma urealyticum, 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli. Average read lengths of >700 bp from unique primer annealing sites are often sufficient to fill final gaps in microbial genome sequencing projects without additional manipulations of template DNA. The technique can also be applied to sequence-targeted regions, thereby bypassing tedious subcloning steps.
In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining ...clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.
Display omitted
•Clinical LOD is a useful benchmark to assess cfDNA-based test performance•cTAF accounts for cfDNA cancer signal variation across cancer types and stages•cfDNA methylation was the most promising genomic feature for cancer signal detection•The results informed the development of a cfDNA-based multi-cancer early detection test
Jamshidi et al. compare several approaches for circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) tests. A whole-genome methylation-based approach has the best performance among those evaluated. In addition, they define a metric—clinical limit of detection (LOD)—based on tumor fraction to enable future comparison of cfDNA-based tests.
Myeloproliferative neoplasms (MPNs) are blood cancers that are characterized by the excessive production of mature myeloid cells and arise from the acquisition of somatic driver mutations in ...haematopoietic stem cells (HSCs). Epidemiological studies indicate a substantial heritable component of MPNs that is among the highest known for cancers
. However, only a limited number of genetic risk loci have been identified, and the underlying biological mechanisms that lead to the acquisition of MPNs remain unclear. Here, by conducting a large-scale genome-wide association study (3,797 cases and 1,152,977 controls), we identify 17 MPN risk loci (P < 5.0 × 10
), 7 of which have not been previously reported. We find that there is a shared genetic architecture between MPN risk and several haematopoietic traits from distinct lineages; that there is an enrichment for MPN risk variants within accessible chromatin of HSCs; and that increased MPN risk is associated with longer telomere length in leukocytes and other clonal haematopoietic states-collectively suggesting that MPN risk is associated with the function and self-renewal of HSCs. We use gene mapping to identify modulators of HSC biology linked to MPN risk, and show through targeted variant-to-function assays that CHEK2 and GFI1B have roles in altering the function of HSCs to confer disease risk. Overall, our results reveal a previously unappreciated mechanism for inherited MPN risk through the modulation of HSC function.
We report a whole-genome shotgun assembly (called WGSA) of the human genome generated at Celera in 2001. The Celera-generated shotgun data set consisted of 27 million sequencing reads organized in ...pairs by virtue of end-sequencing 2-kbp, 10-kbp, and 50-kbp inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 5.3 times, and the inserts from which pairs of reads were obtained covered the genome 39 times. With the nearly complete human DNA sequence National Center for Biotechnology Information (NCBI) Build 34 now available, it is possible to directly assess the quality, accuracy, and completeness of WGSA and of the first reconstructions of the human genome reported in two landmark papers in February 2001 Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., et al. (2001) Science 291, 1304-1351; International Human Genome Sequencing Consortium (2001) Nature 409, 860-921. The analysis of WGSA shows 97% order and orientation agreement with NCBI Build 34, where most of the 3% of sequence out of order is due to scaffold placement problems as opposed to assembly errors within the scaffolds themselves. In addition, WGSA fills some of the remaining gaps in NCBI Build 34. The early genome sequences all covered about the same amount of the genome, but they did so in different ways. The Celera results provide more order and orientation, and the consortium sequence provides better coverage of exact and nearly exact repeats.
In clinical trials, a placebo response refers to improvement in disease symptoms arising from the psychological effect of receiving a treatment rather than the actual treatment under investigation. ...Previous research has reported genomic variation associated with the likelihood of observing a placebo response, but these studies have been limited in scope and have not been validated. Here, we analyzed whole-genome sequencing data from 784 patients undergoing placebo treatment in Phase III Asthma or Rheumatoid Arthritis trials to assess the impact of previously reported variation on patient outcomes in the placebo arms and to identify novel variants associated with the placebo response. Contrary to expectations based on previous reports, we did not observe any statistically significant associations between genomic variants and placebo treatment outcome. Our findings suggest that the biological origin of the placebo response is complex and likely to be variable between disease areas.
The extent and patterns of linkage disequilibrium (LD) determine the feasibility of association studies to map genes that underlie complex traits. Here we present a comparison of the patterns of LD ...across four major human populations (African-American, Caucasian, Chinese, and Japanese) with a high-resolution single-nucleotide polymorphism (SNP) map covering almost the entire length of chromosomes 6, 21, and 22. We constructed metric LD maps formulated such that the units measure the extent of useful LD for association mapping. LD reaches almost twice as far in chromosome 6 as in chromosomes 21 or 22, in agreement with their differences in recombination rates. By all measures used, out-of-Africa populations showed over a third more LD than African-Americans, highlighting the role of the population's demography in shaping the patterns of LD. Despite those differences, the long-range contour of the LD maps is remarkably similar across the four populations, presumably reflecting common localization of recombination hot spots. Our results have practical implications for the rational design and selection of SNPs for disease association studies.
Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of Aplysia. Each of the two peptides is able to induced egg-laying behavior in recipient animals. ...In vitro recordings from the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 μ M. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration, and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Ley-Ser-Lys-Glu-Asp-Gly -Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated Mrof A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. It is estimated that each atrial gland contains at least 150 μ g of peptide A and 50 μ g of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.
We have identified a rare coding mutation, T835M (rs137875858), in the Netrin receptor
UNC5C
that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset ...Alzheimer’s disease (LOAD), and was associated with disease across four large case/control cohorts (OR = 2.15, Pmeta= 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and
in vitro
studies demonstrate that this mutation leads to increased cell death in several cell types, including neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to multiple neurodegenerative stimuli, including β-Amyloid (Aβ). Based on these data and the enriched hippocampal expression of
UNC5C
in the adult nervous system, we propose one possible mechanism in which T835M
UNC5C
contributes to the risk of Alzheimer’s disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer’s brain.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Gene expression profiling using an AFLP-based technique generates a large number of gene fragments that require identification by sequencing. The DNA fragments vary in length from about 50-500 bp. ...Ion-pair reversed-phase HPLC can be used to purify selected double-stranded DNA fragments that represent differentially expressed genes. The gene fragments are sequenced directly after vacuum drying of the collected HPLC fractions.
The transforming protein of a primate sarcoma virus and a plateletderived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of ...extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.