Framework for quality assessment of whole genome cancer sequences.

Bringing together cancer genomes from different projects increases power and allows the investigation of pan-cancer, molecular mechanisms. However, working with whole genomes sequenced over several years in different sequencing centres requires a framework to compare the quality of these sequences. We used the Pan-Cancer Analysis of Whole Genomes cohort as a test case to construct such a framework. This cohort contains whole cancer genomes of 2832 donors from 18 sequencing centres. We developed a non-redundant set of five quality control (QC) measurements to establish a star rating system. These QC measures reflect known differences in sequencing protocol and provide a guide to downstream analyses and allow for exclusion of samples of poor quality. We have found that this is an effective framework of quality measures. The implementation of the framework is available at: https://dockstore.org/containers/quay.io/jwerner_dkfz/pancanqc:1.2.2 .

Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information.

BACKGROUND: Long DNA reads produced by single-molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short-read DNA fragments. For de novo assembly, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the favorite options. However, PacBio's SMRT sequencing is expensive for a full human genome assembly and costs more than $40,000 US for 30× coverage as of 2019. ONT PromethION sequencing, on the other hand, is 1/12 the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio's SMRT sequencing in relation to the quality. FINDINGS: We performed whole-genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64× coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mb and a total genome length of 2.8 Gb. It was comparable to a KOREF assembly constructed using PacBio at 62× coverage (188 Gb, 2,695 contigs, and N50s of 17.9 Mb). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64× coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mb. CONCLUSION: The pore-based PromethION approach provided a high-quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and was more cost-effective than PacBio at comparable quality measurements.

Cost Effectiveness of Karyotyping, Chromosomal Microarray Analysis, and Targeted Next-Generation Sequencing of Patients with Unexplained Global Developmental Delay or Intellectual Disability.

BACKGROUND: Genetic diagnosis of unexplained global developmental delay and intellectual disability (GDD/ID) often ends the diagnostic odyssey and can lead to changes in clinical management. OBJECTIVE: The objective of this study was to investigate the cost effectiveness of testing scenarios involving several methods used to diagnose GDD/ID: karyotyping, chromosomal microarray analysis (CMA), and targeted next-generation sequencing (NGS). METHODS: We used decision-tree models to estimate the number of genetic diagnoses, the cost from a payers' perspective in the USA, and the incremental cost per additional genetic diagnosis. Model parameters were taken from peer-reviewed literature and governmental fee schedules. RESULTS: CMA testing results in more genetic diagnoses at an incremental cost of US $2692 per additional diagnosis compared with karyotyping, which has an average cost per diagnosis of US $11,033. Performing both tests sequentially results in the same number of diagnoses, but the total cost is less when CMA testing is done first and karyotyping second. Furthermore, when CMA testing yields a variant of unknown significance, additional genetic diagnoses can be obtained at an incremental cost of US $4220 by CMA testing of both parents, and when parents are not available or the patient had a normal CMA result, targeted NGS of the patient can add diagnoses at a further incremental cost of US $12,295. CONCLUSION: These results provide a cost effectiveness rationale for the use of CMA as the first-tier test for the genetic diagnosis of unexplained GDD/ID and further indicate that testing of both parents may be cost effective when a variant of unknown significance is detected in the patient.

Trisomy 12 assessment by conventional fluorescence in-situ hybridization (FISH), FISH in suspension (FISH-IS) and laser scanning cytometry (LSC) in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has an extremely heterogeneous clinical course, and prognostication is based on common genetic abnormalities which are detected by standard cytogenetic methods. However, current methods are restricted by the low number of cells able to be analyzed, resulting in the potential to miss clinically relevant sub-clonal populations of cells. A novel high throughput methodology called fluorescence in situ hybridization in suspension (FISH-IS) incorporates a flow cytometry-based imaging approach with automated analysis of thousands of cells. Here we have demonstrated that the FISH-IS technique is applicable to aneuploidy detection in CLL samples for a range of chromosomes using appropriate centromere probes. This method is able to accurately differentiate between monosomy, disomy and trisomy with a sensitivity of 1% in CLL. An analysis comparing conventional FISH, FISH-IS and laser scanning cytometry (LSC) is presented.

Building the SeqChromMM Markov property atlas of the human genome by analyzing the 200-bp units of the 15 different chromatin regions of ENCODE.

We analyzed the publicly available ChromHMM BED files of the ENCODE project and tested the Markov properties of the different chromatin states in the human genome. Nucleotide frequency profiles of regional chromatin segmentations were analyzed, and Markov chains were built to detect Markov properties in the chromatin states of different ChromHMM regions. By estimating the transition probabilities of 200-base pair nucleotide sequences of the human genome, we constructed a nucleotide-sequence-based Markovian chromatin map called SeqChromMM.

A genome-wide assessment of variations of primary colorectal cancer maintained in metastases.

Colorectal cancer (CRC) is a highly heterogeneous disease that is the third leading cause of cancer-related deaths worldwide. This study presents a genome-wide assessment of variations in primary colorectal cancer maintained in metastases, even in distant metastases. The purpose of this study was to determine whether intratumor heterogeneity is related to disease progression and metastasis in CRC. The results showed that 882 single nucleotide polymorphism (SNP) associated genes and 473 copy number variant (CNV) associated genes specific to metastasis were found. In addition, 57 SNPs mapped to miRNAs showed significant differences between primary tumors and metastases. Functional annotation of metastasis-specific genes suggested that adhesion and immune regulation may be essential in the development of tumors. Moreover, the locus rs12881063 in the fourteenth chromosome was found to have a high rate of the G/C type in metastases. The rate of the G/C type in nearby lymph node metastases was 66.7%, while the rate of the G/C type in distance lymph node metastases was 83.3%. These results indicate that rs12881063 may be the basis for clinical diagnosis of CRC metastasis.

[Result survey analysis of prenatal chromosome karyotyping in an external quality assessment program].

OBJECTIVE: To analyze the results of prenatal karyotype of the external quality assessment program in 2013 in order to provide references and recommendations for improving the capability and performances of karyotype analysis of prenatal screening laboratories. METHODS: Five lots of quality control cell photos were sent to 500 laboratories. The participants were asked to decide whether the photos have demonstrated any abnormal karyotype and determine the abnormal type. The results should be submitted before the deadline and compared with the standard results to evaluate the performances of the laboratory. RESULTS: One hundred forty three laboratories have returned their karyotype results for the survey. The standard answers were 7,XX,+18, 46,X,i(X)(q10), 46,XY,i(21)(q10) or 46,XY,+21,der(21;21)(q10;q10), 46,XY and 47,XY,+21 in sequential order, which were used to estimate the score of each participant. The pass rates for five lots were 97.9%, 97.2%, 95.8%, 100.0% and 97.9%, respectively. The total pass rate was 97.7%. The error rates were 2.1%, 2.8%, 4.2%, 0 and 2.1%, respectively. The total error rate was 2.3%. CONCLUSION: Some laboratories did not correctly identify the abnormal karyotypes, while some could not determine the right type of karyotype. The external quality assessment program of prenatal diagnosis of karyotype analysis should be conducted annually in order to improve the capability and performances of karyotype analysis of prenatal screening laboratories.

A Bayesian hierarchical method to account for random effects in cytogenetic dosimetry based on calibration curves.

The dicentric chromosome assay (DCA) is one of the most sensitive and reliable methods of inferring doses of radiation exposure in patients. In DCA, one calibration curve is prepared in advance by in vitro irradiation to blood samples from one or sometimes multiple healthy donors in considering possible inter-individual variability. Although the standard method has been demonstrated to be quite accurate for actual dose estimates, it cannot account for random effects, which come from such as the blood donor used to prepare the calibration curve, the radiation-exposed patient, and the examiners. To date, it is unknown how these random effects impact on the standard method of dose estimation. We propose a novel Bayesian hierarchical method that incorporates random effects into the dose estimation. To demonstrate dose estimation by the proposed method and to assess the impact of inter-individual variability in samples from multiple donors on the estimation, peripheral blood samples from 13 occupationally non-exposed, non-smoking, healthy individuals were collected and irradiated with gamma rays. The results clearly showed significant inter-individual variability and the standard method using a sample from a single donor gave anti-conservative confidence interval of the irradiated dose. In contrast, the Bayesian credible interval for irradiated dose calculated by the proposed method using samples from multiple donors properly covered the actual doses. Although the classical confidence interval of calibration curve with accounting inter-individual variability in samples from multiple donors was roughly coincident with the Bayesian credible interval, the proposed method has better reasoning and potential for extensions.

Unraveling recombination rate evolution using ancestral recombination maps.

Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it.

Assessment of common variability and expression quantitative trait loci for genome-wide associations for progressive supranuclear palsy.

Progressive supranuclear palsy is a rare parkinsonian disorder with characteristic neurofibrillary pathology consisting of hyperphosphorylated tau protein. Common variation defining the microtubule associated protein tau gene (MAPT) H1 haplotype strongly contributes to disease risk. A recent genome-wide association study (GWAS) revealed 3 novel risk loci on chromosomes 1, 2, and 3 that primarily implicate STX6, EIF2AK3, and MOBP, respectively. Genetic associations, however, rarely lead to direct identification of the relevant functional allele. More often, they are in linkage disequilibrium with the causative polymorphism(s) that could be a coding change or affect gene expression regulatory motifs. To identify any such changes, we sequenced all coding exons of those genes directly implicated by the associations in progressive supranuclear palsy cases and analyzed regional gene expression data from control brains to identify expression quantitative trait loci within 1 Mb of the risk loci. Although we did not find any coding variants underlying the associations, GWAS-associated single-nucleotide polymorphisms at these loci are in complete linkage disequilibrium with haplotypes that completely overlap with the respective genes. Although implication of EIF2AK3 and MOBP could not be fully assessed, we show that the GWAS single-nucleotide polymorphism rs1411478 (STX6) is a strong expression quantitative trait locus with significantly lower expression of STX6 in white matter in carriers of the risk allele.

Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos.

Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defect common in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current study aimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development. However, at the blastocyst stage aneuploidies were found to be significantly less common among embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable from euploid embryos. The sex ratio was seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the opposite direction was noted for the most rapidly developing blastocysts. In summary, this study confirms that, at the cleavage stage, chromosome abnormalities have little if any effect on morphological scores assigned using traditional criteria. At the blastocyst stage some forms of aneuploidy begin to affect microscopic appearance, but in most instances the impact is subtle. In the case of the most clinically relevant aneuploidies (those capable of forming a pregnancy) there was no detectable effect on morphology at any preimplantation stage.

Bloom syndrome radials are predominantly non-homologous and are suppressed by phosphorylated BLM.

Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.

Assessment of biodosimetry methods for a mass-casualty radiological incident: medical response and management considerations.

Following a mass-casualty nuclear disaster, effective medical triage has the potential to save tens of thousands of lives. In order to best use the available scarce resources, there is an urgent need for biodosimetry tools to determine an individual's radiation dose. Initial triage for radiation exposure will include location during the incident, symptoms, and physical examination. Stepwise triage will include point of care assessment of less than or greater than 2 Gy, followed by secondary assessment, possibly with high throughput screening, to further define an individual's dose. Given the multisystem nature of radiation injury, it is unlikely that any single biodosimetry assay can be used as a standalone tool to meet the surge in capacity with the timeliness and accuracy needed. As part of the national preparedness and planning for a nuclear or radiological incident, the authors reviewed the primary literature to determine the capabilities and limitations of a number of biodosimetry assays currently available or under development for use in the initial and secondary triage of patients. Understanding the requirements from a response standpoint and the capability and logistics for the various assays will help inform future biodosimetry technology development and acquisition. Factors considered include: type of sample required, dose detection limit, time interval when the assay is feasible biologically, time for sample preparation and analysis, ease of use, logistical requirements, potential throughput, point-of-care capability, and the ability to support patient diagnosis and treatment within a therapeutically relevant time point.

Methods for the assessment of telomere status.

Most methods for examining telomere functionality have relied on measurements of telomeric DNA by hybridization or quantitative PCR. While these techniques yield measures of telomeric DNA length, they generate whole-population results. However, telomeric DNA lengths on different chromatids even in the same cell are usually heterogeneous. Also, these measurements do not reveal whether a particular telomere contains the critical minimum DNA length to be functional. Therefore, in order to gain a more complete knowledge of cellular health, an alternative method that reveals the functional status of each individual telomere is needed. Based on the fact that a dysfunctional telomere induces a DNA damage response, we developed a novel technique which combines a DNA damage marker with fluorescence in situ hybridization (FISH) of telomeric DNA on metaphase chromosomes to assess the functional status of individual telomeres. This technique reveals not only whether the telomeric DNA in each chromatid is significantly shortened, but also whether the telomere has induced a DNA damage response, i.e., has become dysfunctional. We describe here in detail the protocols for simultaneous assessment of telomere length and functionality.

Assessment of anti-recombination and double-strand break-induced gene conversion in human cells by a chromosomal reporter.

Gene conversion is one of the frequent end results of homologous recombination, and it often underlies the inactivation of tumor suppressor genes in cancer cells. Here, we have developed an integrated assay system that allows simultaneous examination of double-strand break (DSB)-induced gene conversion events at the site of a DSB (proximal region) and at a surrounding region ~1 kb away from the break (distal region). Utilizing this assay system, we find that gene conversion events at the proximal and distal regions are relatively independent of one another. The results also indicate that synthesis-dependent strand annealing (SDSA) plays a major role in DSB-induced gene conversion. In addition, our current study has demonstrated that hMLH1 plays an essential role in anti-recombination and gene conversion. Specifically, the anti-recombination activity of hMLH1 is partially dependent on its interaction with hMRE11. Our data suggests that the role of hMLH1 and hMRE11 in the process of gene conversion is complex, and these proteins play different roles in DSB-induced proximal and distal gene conversions. In particular, the involvement of hMLH1 and hMRE11 in the distal gene conversion requires both hMSH2 and heteroduplex formation.

Inferring coancestry in population samples in the presence of linkage disequilibrium.

In both pedigree linkage studies and in population-based association studies there has been much interest in the use of modern dense genetic marker data to infer segments of gene identity by descent (ibd) among individuals not known to be related, to increase power and resolution in localizing genes affecting complex traits. In this article, we present a hidden Markov model (HMM) for ibd among a set of chromosomes and describe methods and software for inference of ibd among the four chromosomes of pairs of individuals, using either phased (haplotypic) or unphased (genotypic) data. The model allows for missing data and typing error, but does not model linkage disequilibrium (LD), because fitting an accurate LD model requires large samples from well-studied populations. However, LD remains a major confounding factor, since LD is itself a reflection of coancestry at the population level. To study the impact of LD, we have developed a novel simulation approach to generate realistic dense marker data for the same set of markers but at varying levels of LD. Using this approach, we present results of a study of the impact of LD on the sensitivity and specificity of our HMM model in estimating segments of ibd among sets of four chromosomes and between genotype pairs. We show that, despite not incorporating LD, our model has been quite successful in detecting segments as small as 10(6) bp (1 Mpb); we present also comparisons with fastIBD which uses an LD model in estimating ibd.

A model of the cell nucleus for DNA damage calculations.

AIMS: Development of a computer model of genomic deoxyribonucleic acid (DNA) in the human cell nucleus for DNA damage and repair calculations. The model comprises the human genomic DNA, chromosomal domains, and loops attached to factories. MATERIAL AND METHODS: A model of canonical B-DNA was used to build the nucleosomes and the 30-nanometer solenoidal chromatin. In turn the chromatin was used to form the loops of factories in chromosome domains. The entire human genome was placed in a spherical nucleus of 10 micrometers diameter. To test the new target model, tracks of protons and alpha-particles were generated using Monte Carlo track structure codes PITS99 (Positive Ion Track Structure) and KURBUC. Damage sites induced in the genome were located and classified according to type and complexity. RESULTS: The three-dimensional structure of the genome starting with a canonical B-DNA model, nucleosomes, and chromatin loops in chromosomal domains are presented. The model was used to obtain frequencies of DNA damage induced by protons and alpha-particles by direct energy deposition, including single- and double-strand breaks, base damage, and clustered lesions. CONCLUSIONS: This three-dimensional model of the genome is the first such model using the full human genome for the next generation of more comprehensive modelling of DNA damage and repair. The model combines simple geometrical structures at the level of domains and factories with potentially full detail at the level of atoms in particular genes, allowing damage patterns in the latter to be simulated.

Genome scan of age-at-onset in the NIMH Alzheimer disease sample uncovers multiple loci, along with evidence of both genetic and sample heterogeneity.

Alzheimer's disease (AD) is a common neurodegenerative disorder of late life with a complex genetic basis. Although several genes are known to play a role in rare early onset AD, only the APOE gene is known to have a high contribution to risk of the common late-onset form of the disease (LOAD, onset >60 years). APOE genotypes vary in their AD risk as well as age-at-onset distributions, and it is likely that other loci will similarly affect AD age-at-onset. Here we present the first analysis of age-at-onset in the NIMH LOAD sample that allows for both a multilocus trait model and genetic heterogeneity among the contributing sites, while at the same time accommodating age censoring, effects of known genetic covariates, and full pedigree and marker information. The results provide evidence for genomic regions not previously implicated in this data set, including regions on chromosomes 7q, 15, and 19p. They also affirm evidence for loci on chromosomes 1q, 6p, 9q, 11, and, of course, the APOE locus on 19q, all of which have been reported previously in the same sample. The analyses failed to find evidence for linkage to chromosome 10 with inclusion of unaffected subjects and extended pedigrees. Several regions implicated in these analyses in the NIMH sample have been previously reported in genome scans of other AD samples. These results, therefore, provide independent confirmation of AD loci in family-based samples on chromosomes 1q, 7q, 19p, and suggest that further efforts towards identifying the underlying causal loci are warranted.