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We describe the use of a Decentralized Autonomous Organization (DAO) to crypto-fund the single molecule sequencing and publication of a Type II Cannabis plant. This resulted in the construction of the most contiguous Cannabis genome assembly to date. The combined use of the Dash cryptocurrency, DAOs, and Pacific Biosciences sequencing delivered a 1.03 Gb genome with a N50 of 665Kb in 77 days from funding to public upload. This represents a 230 fold improvement in the contiguity of the first cannabis assemblies in 2011 and a 4 fold improvement over all cannabis assemblies to date. 34Gb of additional sequencing pushed the assembly to a N50 of 3.8Mb. Hi-C data from Phase Genomics further scaffolded the assembly to 35 contigs at an N50 of 74Mb but requires additional curation. The genome is partially phased and larger than previously reported (2N = 1.33Gb). The CBCA, THCA and CBDA synthase gene clusters have been phased onto respective contigs demonstrating tandem repeat expansions.

The chemical expression of THCA and CBDA has been previously attributed to copy number gains and losses in THCAS and CBDAS. Here we describe an alternative mechanism to ablate THCAS activity in 5 CBGA dominant cannabis varietals. Five cannabis cultivars from three unique cultivators surprisingly contain a THCAS gene but also share homozygous Pro333Arg and Ser355Asn mutations in THCAS. Pro333Arg is commonly found in THCAS dominant varietals but is uniquely found in a homozygous state in CBGA varietals in conjunction with homozygous Ser355Asn missense mutations. These results hint at convergent evolution in cannabinoid synthesis where selection for and against THCA synthesis has been applied., https://www.kannapedia.net

This is a slide deck that accompanied a PANDA open sciences presentation.

Background:: After submitting our review report on Corman et al. (referred hereinafter as CD-report) and republishing it on a scientific preprint server (http://doi.org/10.5281/zenodo.4298004) and Researchgate.net we offered the report for public discussion at cormandrostenreview.com on 27th November 2020. The scientific community provided additional literature, references, and analyses concerning the CD-report and the Corman et al. manuscript. Several “advocatus diaboli” confronted us with correct or assumed problems in our report. The most common critique of the CD-report was the lack of “wet lab” experiments to support our concerns over the technical flaws in the PCR protocol. Aim: This vibrant debate on our CD report has provided additional information worthy of further public documentation to address these critiques. We summarize the current published knowledge of “wet lab testing”, routine diagnostic use and validation of the original PCR-Protocol described by Corman et al. Further, this addendum highlights that independent research groups (some of them with Corman and/or Drosten as author) also pointed out important concerns with the original manuscript and Corman PCR protocol distributed by the WHO. Many of these references were already provided by the authors of the original CD-report but it is worth underscoring their relevance to the formation of our critiques of the CD manuscript. Methods: We searched the literature for ‘SARS-CoV-2 qPCR’ and ‘Corman’ or ‘Charité’. Then we combined these references with those provided by other scientists working in relevant Life Sciences/data analysis fields. In the first section of the addendum, the publications will be discussed point by point, highlighting their findings in relation to the CD-report. In a second section, additional aspects about the Corman et al . publication are discussed. This spans a meta-analysis of the unusual peer-review process, timeframes, and further technical vulnerabilities of the Corman et al . PCR-protocol. An additional concern was raised about the CD-report regarding the discussion of appropriate controls. We cite several studies that underscore the importance of internal controls in assessing viral load and the lack of such internal controls in the Corman qPCR method. These internal controls are required for normalizing swab sampling variance and they are critical for interpreting viral load. They are notably absent from the Corman PCR protocol. Several people also expressed confusion regarding the NCBI submissions provided by Corman et al . The sequences provided lack two of the target gene sequences Corman et al. claim to target. The only sequences referenced in the manuscript are listed ( KC633203, KC633204, KC633201, GU190221, GU190222, GU190223) and none of these have sequences that match their N and E gene primers. This not only brings their validation into question but also prevents others from reproducing the work presented in Corman et al. Results: We present 20 scientific publications providing ‘wet lab’ evidence of the performance of the Corman et al. PCR protocol. Of those, 17 found problems with incorrect primer design (mismatches, dimer formation, melting temperature) in the SARS-CoV-2 specific “confirmatory” test named RdRp-PCR for “RNA-dependent RNA-polymerase” or the E-gene assay. These documented problems include: ● Documented primer dimers and False Positives in non-template controls (NTCs) ● Documented poor sensitivity and False Negatives compared to other assays ● No internal control to normalize the sample preparation variability and its impact on viral load estimation ● No defined Ct for calling samples “Positive cases” ● Poorly documented positive controls and sequences used in their study Conclusions: We believe the references provided in this addendum itemize the scientific consensus evident in the literature regarding the flaws in the original PCR detection method for SARs-CoV-2 published by Corman et al. . Further, since several important flaws were published in peer-reviewed journals, the lack of correction of the original PCR protocol by either Eurosurveillance or as an update in the Charité-WHO protocol brings into question the scientific integrity of the authors of Corman et al. These references settle any remaining debate that the Corman et al. manuscript should be retracted on technical grounds alone. The rapidity of the peer-review and conflicts of interest are even more troubling., This is an addendum to our report also published on Zenodo: 10.5281/zenodo.4298004

In the publication entitled “Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR” (Eurosurveillance 25(8) 2020) the authors present a diagnostic workflow and RT-qPCR protocol for detection and diagnostics of 2019-nCoV (now known as SARS-CoV-2), which they claim to be validated, as well as being a robust diagnostic methodology for use in public-health laboratory settings. In light of all the consequences resulting from this very publication for societies worldwide, a group of independent researchers performed a point-by-point review of the aforesaid publication in which 1) all components of the presented test design were cross checked, 2) the RT-qPCR protocol-recommendations were assesses w.r.t. good laboratory practice, and 3) parameters examined against relevant scientific literature covering the field. The published RT-qPCR protocol for detection and diagnostics of 2019-nCoV and the manuscript suffer from numerous technical and scientific errors, including insufficient primer design, a problematic and insufficient RT-qPCR protocol, and the absence of an accurate test validation. Neither the presented test nor the manuscript itself fulfils the requirements for an acceptable scientific publication. Further, serious conflicts of interest of the authors are not mentioned. Finally, the very short timescale between submission and acceptance of the publication (24 hours) signifies that a systematic peer review process was either not performed here, or of problematic poor quality. We provide compelling evidence of several scientific inadequacies, errors and flaws. Considering the scientific and methodological blemishes presented here, we are confident that the editorial board of Eurosurveillance has no other choice but to retract the publication., More information on this paper can be found here: https://cormandrostenreview.com/

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.

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.