Comparative analysis of genome tiling array data reveals many novel primate-specific functional RNAs in human

BACKGROUND: Widespread transcription activities in the human genome were recently observed in high-resolution tiling array experiments, which revealed many novel transcripts that are outside of the boundaries of known protein or RNA genes. Termed as "TARs" (Transcriptionally Active Regions), these novel transcribed regions represent "dark matter" in the genome, and their origin and functionality need to be explained. Many of these transcripts are thought to code for novel proteins or non-protein-coding RNAs. We have applied an integrated bioinformatics approach to investigate the properties of these TARs, including cross-species conservation, and the ability to form stable secondary structures. The goal of this study is to identify a list of potential candidate sequences that are likely to code for functional non-protein-coding RNAs. We are particularly interested in the discovery of those functional RNA candidates that are primate-specific, i.e. those that do not have homologs in the mouse or dog genomes but in rhesus. RESULTS: Using sequence conservation and the probability of forming stable secondary structures, we have identified ~300 possible candidates for primate-specific noncoding RNAs. We are currently in the process of sequencing the orthologous regions of these candidate sequences in several other primate species. We will then be able to apply a "phylogenetic shadowing" approach to analyze the functionality of these ncRNA candidates. CONCLUSION: The existence of potential primate-specific functional transcripts has demonstrated the limitation of previous genome comparison studies, which put too much emphasis on conservation between human and rodents. It also argues for the necessity of sequencing additional primate species to gain a better and more comprehensive understanding of the human genome

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease

The Diploid Genome Sequence of an Individual Human

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Structural Variation in the Human Genome

The study of variation found in DNA is fundamental in human genetic studies. Single nucleotide polymorphisms (SNPs) are simple to document because they can be captured in single DNA sequence reads. Larger structural variation including duplications, insertions, deletions, termed as copy number variation (CNV), inversions and translocations are more challenging to discover. Recent studies using microarray and sequencing technologies have demonstrated the prevalence of structural variation in humans. They can disrupt genic and regulatory sequences, be associated with disease, and fuel evolution. Therefore, it is important to identify and characterize both SNPs and structural variants to fully understand their impact. This thesis presents the analysis of structural variation in the human genome. The primary DNA sample used for my experiments is the DNA of J. Craig Venter, also termed HuRef. It was the first personal human genome sequenced. I combined computational re-analysis of sequence data with microarray-based analysis, and detected 12,178 structural variants covering 40.6 Mb that were not reported in the initial sequencing study. The results indicated that the genomes of two individuals differed 1.3% by CNV, 0.3% by inversion and 0.1% by SNP. Structural variation discovery is dependent on the strategy used. No single approach can readily capture all types of variation, and a combination of strategies is required. I analyzed the formation mechanisms of all HuRef structural variants. The results showed that the relative proportion of mutational processes changed across size range: the majority of small variants (<1kb) were associated with nonhomologous processes and microsatellite events; median size variants (<10kb) were commonly related to minisatellites and retrotransposons; and large variants were associated with nonallelic homologous recombination. Eight new breakpoint-resolved HuRef inversions were genotyped in populations to elucidate these understudied variants. I discovered that the structures of inversion could be complex, could create conjoined genes, and their frequencies could exhibit population differentiation. The data here contributes to our understanding of structural variation in humans. It shows the need to use multiple strategies to identify variants, and it emphasizes the importance to examine the full complement of variation in all biomedical studies.Ph

Comparative Benchmarking of Optical Genome Mapping and Chromosomal Microarray Reveals High Technological Concordance in CNV Identification and Additional Structural Variant Refinement

The recommended practice for individuals suspected of a genetic etiology for disorders including unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and multiple congenital anomalies (MCA) involves a genetic testing workflow including chromosomal microarray (CMA), Fragile-X testing, karyotype analysis, and/or sequencing-based gene panels. Since genomic imbalances are often found to be causative, CMA is recommended as first tier testing for many indications. Optical genome mapping (OGM) is an emerging next generation cytogenomic technique that can detect not only copy number variants (CNVs), triploidy and absence of heterozygosity (AOH) like CMA, but can also define the location of duplications, and detect other structural variants (SVs), including balanced rearrangements and repeat expansions/contractions. This study compares OGM to CMA for clinically reported genomic variants, some of these samples also have structural characterization by fluorescence in situ hybridization (FISH). OGM was performed on IRB approved, de-identified specimens from 55 individuals with genomic abnormalities previously identified by CMA (61 clinically reported abnormalities). SVs identified by OGM were filtered by a control database to remove polymorphic variants and against an established gene list to prioritize clinically relevant findings before comparing with CMA and FISH results. OGM results showed 100% concordance with CMA findings for pathogenic variants and 98% concordant for all pathogenic/likely pathogenic/variants of uncertain significance (VUS), while also providing additional insight into the genomic structure of abnormalities that CMA was unable to provide. OGM demonstrates equivalent performance to CMA for CNV and AOH detection, enhanced by its ability to determine the structure of the genome. This work adds to an increasing body of evidence on the analytical validity and ability to detect clinically relevant abnormalities identified by CMA. Moreover, OGM identifies translocations, structures of duplications and complex CNVs intractable by CMA, yielding additional clinical utility

A Chromosome-Length Assembly of the Hawaiian Monk Seal (Neomonachus schauinslandi): A History of &ldquo;Genetic Purging&rdquo; and Genomic Stability

The Hawaiian monk seal (HMS) is the single extant species of tropical earless seals of the genus Neomonachus. The species survived a severe bottleneck in the late 19th century and experienced subsequent population declines until becoming the subject of a NOAA-led species recovery effort beginning in 1976 when the population was fewer than 1000 animals. Like other recovering species, the Hawaiian monk seal has been reported to have reduced genetic heterogeneity due to the bottleneck and subsequent inbreeding. Here, we report a chromosomal reference assembly for a male animal produced using a variety of methods. The final assembly consisted of 16 autosomes, an X, and portions of the Y chromosomes. We compared variants in this animal to other HMS and to a frequently sequenced human sample, confirming about 12% of the variation seen in man. To confirm that the reference animal was representative of the HMS, we compared his sequence to that of 10 other individuals and noted similarly low variation in all. Variation in the major histocompatibility (MHC) genes was nearly absent compared to the orthologous human loci. Demographic analysis predicts that Hawaiian monk seals have had a long history of small populations preceding the bottleneck, and their current low levels of heterozygosity may indicate specialization to a stable environment. When we compared our reference assembly to that of other species, we observed significant conservation of chromosomal architecture with other pinnipeds, especially other phocids. This reference should be a useful tool for future evolutionary studies as well as the long-term management of this species