Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the ...findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.
Copy number variants (CNVs) play a significant role in human heredity and disease. However, sensitive and specific characterization of germline CNVs from NGS data has remained challenging, ...particularly for hybridization-capture data in which read counts are the primary source of copy number information. We describe two algorithmic adaptations that improve CNV detection accuracy in a Hidden Markov Model (HMM) context. First, we present a method for computing target- and copy number-specific emission distributions. Second, we demonstrate that the Pointwise Maximum a posteriori (PMAP) HMM decoding procedure yields improved sensitivity for small CNV calls compared to the more common Viterbi HMM decoder. We develop a prototype implementation, called Cobalt, and compare it to other CNV detection tools using sets of simulated and previously detected CNVs with sizes spanning a single exon to a full chromosome. In both the simulation and previously detected CNV studies Cobalt shows similar sensitivity but significantly fewer false positive detections compared to other callers. Overall sensitivity is 80-90% for deletion CNVs spanning 1-4 targets and 90-100% for larger deletion events, while sensitivity is somewhat lower for small duplication CNVs.
Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing ...functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.
We report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 ...using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389nm, and a photo-induced metastable state with a maximum at 497nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~50min at 293K and pH7.5 as measured by UV–Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. The neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.
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•Dathail will be useful in imaging and biosensors, particularly pcFRET, high-resolution microscopy, and live cell tracking.•Dathail is a reversibly switching chromoprotein with two spectrally distinct chromogenic states with low quantum yields.•X-ray crystallography shows changes in conformation and coordination of the chromophore during Dathail's photoswitching.•Crystallography suggests photo-induced proton transfer from the chromophore with water-mediated proton relay to bulk solvent.•The first time that neutron crystallography has been used for a protein in the GFP family provides surprising new insights.
To the Editor: There is a great diversity of RAG1- and RAG2-dependent primary immunodeficiencies ranging from severe combined immunodeficiency (SCID) to various late-onset combined ...immunodeficiencies, as complete lack of RAG activity leads to absence of mature B and T cells (T-B- SCID), whereas hypomorphic RAG mutations allow limited generation of T and or B cells, resulting in various clinical phenotypes distinct from SCID.1,2 Currently, there are no peripheral blood-based functional assays for the evaluation of RAG activity. ...the pathogenicity of RAG variants is determined using prediction programs and in vitro V(D)J recombination assays.3 Critical to this evaluation is the population frequency of the variants. The number of individuals who are homozygous or compound heterozygous for decreased-function RAG variants is about 9.68 x 10-7 or approximately 1 in 1,000,000. Because this estimation is substantially lower than the frequency of RAG1/2 mutations observed by newborn screening (1 in 336,000),E4 we conclude that the ExAC database currently underestimates the frequency of RAG1/2 deficiency diseases, and the number of individuals who are homozygous or compound heterozygous for decreased-function RAG variants is estimated to be between 1 in 181,000 and 1 in 336,000.
Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 0.8). We have investigated ...the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low ΦF, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in some His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123T69V (2.0 Å) and eCGP123H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.
Eukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, ...and associate with each other in some of their biologically important contexts. Here we report crystal structures of the Spn1 core alone and in complex with the binding determinant of Spt6. Mutating interface residues greatly diminishes binding in vitro and causes strong phenotypes in vivo, including a defect in maintaining repressive chromatin. Overexpression of Spn1 partially suppresses the defects caused by an spt6 mutation affecting the Spn1 interface, indicating that the Spn1-Spt6 interaction is important for managing chromatin. Spt6 binds nucleosomes directly in vitro, and this interaction is blocked by Spn1, providing further mechanistic insight into the function of the interaction. These data thereby reveal the structural and biochemical bases of molecular interactions that function in the maintenance of chromatin structure.
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► Spn1 core region binds directly to a segment of Spt6 ► Crystal structures and biochemical analysis reveal the basis for this interaction ► Spn1-Spt6 binding is important in vivo for maintaining repressive chromatin ► Spt6 binds nucleosomes directly and this interaction is inhibited by Spn1
Microorganisms that degrade biomass produce diverse assortments of carbohydrate‐active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many ...genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome‐wide screen using phage display to identify cellulose‐binding protein domains from the biomass‐degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right‐handed parallel β‐helix protein. Despite very low sequence identity to known β‐helix or carbohydrate‐active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid‐sugar binding β‐helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.
We report a novel variant in
associated with IKAROS haploinsufficiency in a patient with familial immune thrombocytopenia (ITP). IKAROS, encoded by the
gene, is a hematopoietic zinc-finger ...transcription factor that can directly bind to DNA. We show that the identified
variant (p.His195Arg) alters a completely conserved histidine residue required for the folding of the third zinc-finger of IKAROS protein, leading to a loss of characteristic immunofluorescence nuclear staining pattern. In our case, genetic testing was essential for the diagnosis of IKAROS haploinsufficiency, of which known presentations include infections, aberrant hematopoiesis, leukemia, and age-related decrease in humoral immunity. Our family study underscores that, after infections, ITP is the second most common clinical manifestation of IKAROS haploinsufficiency.
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