Domestication through clandestine cultivation constrained genetic diversity in magic mushrooms relative to naturalized populations.

Fungi that are edible or fermentative were domesticated through selective cultivation of their desired traits. Domestication is often associated with inbreeding or selfing, which may fix traits other than those under selection, and causes an overall decrease in heterozygosity. A hallucinogenic mushroom, Psilocybe cubensis, was domesticated from its niche in livestock dung for production of psilocybin. It has caused accidental poisonings since the 1940s in Australia, which is a population hypothesized to be introduced from an unknown center of origin. We sequenced genomes of 38 isolates from Australia and compared them with 86 genomes of commercially available cultivars to determine (1) whether P. cubensis was introduced to Australia, and (2) how domestication has impacted commercial cultivars. Our analyses of genome-wide SNPs and single-copy orthologs showed that the Australian population is naturalized, having recovered its effective population size after a bottleneck when it was introduced, and it has maintained relatively high genetic diversity based on measures of nucleotide and allelic diversity. In contrast, domesticated cultivars generally have low effective population sizes and hallmarks of selfing and clonal propagation, including low genetic diversity, low heterozygosity, high linkage disequilibrium, and low allelic diversity of mating-compatibility genes. Analyses of kinship show that most cultivars are founded from related populations. Alleles in the psilocybin gene cluster are identical across most cultivars of P. cubensis with low diversity across coding sequence; however, unique allelic diversity in Australia and some cultivars may translate to differences in biosynthesis of psilocybin and its analogs.

Whole genome sequencing of colonies derived from cannabis flowers and the impact of media selection on benchmarking total yeast and mold detection tools.

BACKGROUND: Cannabis products are subjected to microbial testing for human pathogenic fungi and bacteria. These testing requirements often rely on non-specific colony forming unit (CFU/g) specifications without clarity on which medium, selection or growth times are required. We performed whole genome sequencing to assess the specificity of colony forming units (CFU) derived from three different plating media: Potato Dextrose Agar (PDA), PDA with chloramphenicol and Dichloran Rose Bengal with chloramphenicol (DRBC). METHODS: Colonies were isolated from each medium type and their whole genomes sequenced to identify the diversity of microbes present on each medium selection. Fungal Internal Transcribed Spacer (ITS3) and Bacterial 16S RNA(16S) quantitative polymerase chain reactions (qPCR) were performed, to correlate these CFUs with fungi- and bacterial- specific qPCR. RESULTS: Each plating medium displayed a ten-fold difference in CFU counts. PDA with chloramphenicol showed the highest diversity and the highest concordance with whole genome sequencing. According to ITS3 and 16S qPCR confirmed with whole genome sequencing, DRBC under counted yeast and mold while PDA without chloramphenicol over counted CFUs due to bacterial growth without selection. CONCLUSIONS: Colony Forming Unit regulations lack specificity. Each medium produces significant differences in CFU counts. These are further dependent on subjective interpretation, failure to culture most microbes, and poor selection between bacteria and fungi. Given the most human pathogenic microbes found on cannabis are endophytes which culture fails to detect, molecular methods offer a solution to this long-standing quantification problem in the cannabis testing field.

A draft reference assembly of the Psilocybe cubensis genome.

We describe the use of high-fidelity single molecule sequencing to assemble the genome of the psychoactive Psilocybe cubensis mushroom. The genome is 46.6Mb, 46% GC, and in 32 contigs with an N50 of 3.3Mb. The BUSCO completeness scores are 97.6% with 1.2% duplicates. The Psilocybin synthesis cluster exists in a single 3.2Mb contig. The dataset is available from NCBI BioProject with accessions PRJNA687911 and PRJNA700437.

Bayes Lines Tool (BLT): a SQL-script for analyzing diagnostic test results with an application to SARS-CoV-2-testing.

The performance of diagnostic tests crucially depends on the disease prevalence, test sensitivity, and test specificity. However, these quantities are often not well known when tests are performed outside defined routine lab procedures which make the rating of the test results somewhat problematic. A current example is the mass testing taking place within the context of the world-wide SARS-CoV-2 crisis. Here, for the first time in history, laboratory test results have a dramatic impact on political decisions. Therefore, transparent, comprehensible, and reliable data is mandatory. It is in the nature of wet lab tests that their quality and outcome are influenced by multiple factors reducing their performance by handling procedures, underlying test protocols, and analytical reagents. These limitations in sensitivity and specificity have to be taken into account when calculating the real test results. As a resolution method, we have developed a Bayesian calculator, the Bayes Lines Tool (BLT), for analyzing disease prevalence, test sensitivity, test specificity, and, therefore, true positive, false positive, true negative, and false negative numbers from official test outcome reports. The calculator performs a simple SQL (Structured Query Language) query and can easily be implemented on any system supporting SQL. We provide an example of influenza test results from California, USA, as well as two examples of SARS-CoV-2 test results from official government reports from The Netherlands and Germany-Bavaria, to illustrate the possible parameter space of prevalence, sensitivity, and specificity consistent with the observed data. Finally, we discuss this tool's multiple applications, including its putative importance for informing policy decisions.

Genomic characterization of the complete terpene synthase gene family from Cannabis sativa.

Terpenes are responsible for most or all of the odor and flavor properties of Cannabis sativa, and may also impact effects users experience either directly or indirectly. We report the diversity of terpene profiles across samples bound for the Washington dispensary market. The remarkable degree of variation in terpene profiles ultimately results from action of a family of terpene synthase genes, only some of which have been described. Using a recently available genome assembly we describe 55 terpene synthases with genomic context, and tissue specific expression. The family is quite diverse from a protein similarity perspective, and subsets of the family are expressed in all tissues in the plant, including a set of root specific monoterpene synthases that could well have agronomic importance. Ultimately understanding and breeding for specific terpene profiles will require a good understanding of the gene family that underlies it. We intend for this work to serve as a foundation for that.

Rare genetic variants in the endocannabinoid system genes CNR1 and DAGLA are associated with neurological phenotypes in humans.

Rare genetic variants in the core endocannabinoid system genes CNR1, CNR2, DAGLA, MGLL and FAAH were identified in molecular testing data from 6,032 patients with a broad spectrum of neurological disorders. The variants were evaluated for association with phenotypes similar to those observed in the orthologous gene knockouts in mice. Heterozygous rare coding variants in CNR1, which encodes the type 1 cannabinoid receptor (CB1), were found to be significantly associated with pain sensitivity (especially migraine), sleep and memory disorders-alone or in combination with anxiety-compared to a set of controls without such CNR1 variants. Similarly, heterozygous rare variants in DAGLA, which encodes diacylglycerol lipase alpha, were found to be significantly associated with seizures and neurodevelopmental disorders, including autism and abnormalities of brain morphology, compared to controls. Rare variants in MGLL, FAAH and CNR2 were not associated with any neurological phenotypes in the patients tested. Diacylglycerol lipase alpha synthesizes the endocannabinoid 2-AG in the brain, which interacts with CB1 receptors. The phenotypes associated with rare CNR1 variants are reminiscent of those implicated in the theory of clinical endocannabinoid deficiency syndrome. The severe phenotypes associated with rare DAGLA variants underscore the critical role of rapid 2-AG synthesis and the endocannabinoid system in regulating neurological function and development. Mapping of the variants to the 3D structure of the type 1 cannabinoid receptor, or primary structure of diacylglycerol lipase alpha, reveals clustering of variants in certain structural regions and is consistent with impacts to function.

A study of pedestrian compliance with traffic signals for exclusive and concurrent phasing.

This paper describes a comparison of pedestrian compliance at traffic signals with two types of pedestrian phasing: concurrent, where both pedestrians and vehicular traffic are directed to move in the same directions at the same time, and exclusive, where pedestrians are directed to move during their own dedicated phase while all vehicular traffic is stopped. Exclusive phasing is usually perceived to be safer, especially by senior and disabled advocacy groups, although these safety benefits depend upon pedestrians waiting for the walk signal. This paper investigates whether or not there are differences between pedestrian compliance at signals with exclusive pedestrian phasing and those with concurrent phasing and whether these differences continue to exist when compliance at exclusive phasing signals is evaluated as if they had concurrent phasing. Pedestrian behavior was observed at 42 signalized intersections in central Connecticut with both concurrent and exclusive pedestrian phasing. Binary regression models were estimated to predict pedestrian compliance as a function of the pedestrian phasing type and other intersection characteristics, such as vehicular and pedestrian volume, crossing distance and speed limit. We found that pedestrian compliance is significantly higher at intersections with concurrent pedestrian phasing than at those with exclusive pedestrian phasing, but this difference is not significant when compliance at exclusive phase intersections is evaluated as if it had concurrent phasing. This suggests that pedestrians treat exclusive phase intersections as though they have concurrent phasing, rendering the safety benefits of exclusive pedestrian phasing elusive. No differences were observed for senior or non-senior pedestrians.

Metagenomic analysis of medicinal Cannabis samples; pathogenic bacteria, toxigenic fungi, and beneficial microbes grow in culture-based yeast and mold tests.

Background: The presence of bacteria and fungi in medicinal or recreational Cannabis poses a potential threat to consumers if those microbes include pathogenic or toxigenic species. This study evaluated two widely used culture-based platforms for total yeast and mold (TYM) testing marketed by 3M Corporation and Biomérieux, in comparison with a quantitative PCR (qPCR) approach marketed by Medicinal Genomics Corporation. Methods: A set of 15 medicinal Cannabis samples were analyzed using 3M and Biomérieux culture-based platforms and by qPCR to quantify microbial DNA. All samples were then subjected to next-generation sequencing and metagenomics analysis to enumerate the bacteria and fungi present before and after growth on culture-based media. Results: Several pathogenic or toxigenic bacterial and fungal species were identified in proportions of >5% of classified reads on the samples, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Ralstonia pickettii, Salmonella enterica, Stenotrophomonas maltophilia, Aspergillus ostianus, Aspergillus sydowii, Penicillium citrinum and Penicillium steckii. Samples subjected to culture showed substantial shifts in the number and diversity of species present, including the failure of Aspergillus species to grow well on either platform. Substantial growth of Clostridium botulinum and other bacteria were frequently observed on one or both of the culture-based TYM platforms. The presence of plant growth promoting (beneficial) fungal species further influenced the differential growth of species in the microbiome of each sample. Conclusions: These findings have important implications for the Cannabis and food safety testing industries.

The chloroplast genome hidden in plain sight, open access publishing and anti-fragile distributed data sources.

We sequenced several cannabis genomes in 2011 of June and the first and the longest contigs to emerge were the chloroplast and mitochondrial genomes. Having been a contributor to the Human Genome Project and an eye-witness to the real benefits of immediate data release, I have first hand experience with the potential mal-investment of millions of dollars of tax payer money narrowly averted due to the adopted global rapid data release policy. The policy was vital in reducing duplication of effort and economic waste. As a result, we felt obligated to publish the Cannabis genome data in a similar spirit and placed them immediately on a cloud based Amazon server in August of 2011. While these rapid data release practices were heralded by many in the media, we still find some authors fail to find or reference said work and hope to compel the readership that this omission has more pervasive repercussions than bruised egos and is a regression for our community.

Hurt, tired and queasy: Specific variants in the ATPase domain of the TRAP1 mitochondrial chaperone are associated with common, chronic "functional" symptomatology including pain, fatigue and gastrointestinal dysmotility.

Functional disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. There are bidirectional associations between functional disorders and mitochondrial dysfunction. In this study, provided clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were retrospectively reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients. Functional symptomatology is strongly associated with specific variants in the ATPase binding pocket. In particular, the combined presence of all three functional categories is strongly associated with p.Ile253Val (OR 7.5, P = 0.0001) and with two other interacting variants (OR 18, P = 0.0005). Considering a 1-2% combined variant prevalence and high odds ratios, these variants may be an important factor in the etiology of functional symptomatology.

Cannabis microbiome sequencing reveals several mycotoxic fungi native to dispensary grade Cannabis flowers.

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

Expanded genetic codes in next generation sequencing enable decontamination and mitochondrial enrichment.

We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA.

Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human embryonic stem cells.

Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.

An integrated semiconductor device enabling non-optical genome sequencing.

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

MicroRNAs and their isomiRs function cooperatively to target common biological pathways.

BACKGROUND: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules. RESULTS: To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs. CONCLUSIONS: Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.

Development of personalized tumor biomarkers using massively parallel sequencing.

Clinical management of human cancer is dependent on the accurate monitoring of residual and recurrent tumors. The evaluation of patient-specific translocations in leukemias and lymphomas has revolutionized diagnostics for these diseases. We have developed a method, called personalized analysis of rearranged ends (PARE), which can identify translocations in solid tumors. Analysis of four colorectal and two breast cancers with massively parallel sequencing revealed an average of nine rearranged sequences (range, 4 to 15) per tumor. Polymerase chain reaction with primers spanning the breakpoints was able to detect mutant DNA molecules present at levels lower than 0.001% and readily identified mutated circulating DNA in patient plasma samples. This approach provides an exquisitely sensitive and broadly applicable approach for the development of personalized biomarkers to enhance the clinical management of cancer patients.

Whole methylome analysis by ultra-deep sequencing using two-base encoding.

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.

A small-cell lung cancer genome with complex signatures of tobacco exposure.

Cancer is driven by mutation. Worldwide, tobacco smoking is the principal lifestyle exposure that causes cancer, exerting carcinogenicity through >60 chemicals that bind and mutate DNA. Using massively parallel sequencing technology, we sequenced a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. A total of 22,910 somatic substitutions were identified, including 134 in coding exons. Multiple mutation signatures testify to the cocktail of carcinogens in tobacco smoke and their proclivities for particular bases and surrounding sequence context. Effects of transcription-coupled repair and a second, more general, expression-linked repair pathway were evident. We identified a tandem duplication that duplicates exons 3-8 of CHD7 in frame, and another two lines carrying PVT1-CHD7 fusion genes, indicating that CHD7 may be recurrently rearranged in this disease. These findings illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer.