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The investing game is rarely plain sailing. While no doubt investors would like the choices that make up their portfolio to always go up, the reality is more complicated. There are periods when even shares of the worlds most successful companies have been on a downward trajectory for one reason or another. While its no fun watching a stock you own drift to the bottom, any savvy investor knows that if the companys fundamentals are sound to begin with, the pullback is often a gift in disguise. This is where the chance for strong returns really comes into play. Buy the Dip is not a clich without reason. With this in mind, we scoured the TipRanks database and picked out 3 names which have been heading south recently, specifically ones pinpointed by those in the know as representing a buying opportunity. Whats more, all 3 are rated Strong Buys by the analyst consensus and projected to rake in at least 70% of gains over the next 12 months. Here are the details. Flexion Therapeutics (FLXN) Lets first take a look at Flexion, a pharma company specializing in the development and commercialization of therapies for the treatment of musculoskeletal pain. The company has two drugs currently in early-stage clinical trials but one which has already been approved by the FDA; Zilretta is an extended-release corticosteroid for the management of osteoarthritis knee pain. The drug was granted regulatory approval in 2017, and Flexion owns the exclusive worldwide rights. FLXN stock has found 2021 hard going and is down by 30% year-to-date. However, the recent weakness, says Northland analyst Carl Byrnes has created a unique buying opportunity. Like many biopharmas, Flexions marketing efforts took a hit during the height of the pandemic last year, as shutdowns and restrictions impacted its operations. However, Byrnes anticipates Zilretta to exhibit stellar growth in 2021 and beyond. We remain highly confident that the demand for ZILRETTA will continue to strengthen, bolstered by product awareness and positive clinical experiences of both patients and HCP, augmented by improvements in HCP interactions and deferral of total knee arthroplasty (TKA) surgical procedures, the analyst said. Byrnes expects Zilrettas 2021 sales to surge by 45% year-over-year to $125 million, and then increase by a further 50% to $187.5 million the following year. That revenue growth will go hand in hand with massive share appreciation; Byrnes price target is $35, suggesting upside of ~339% over the next 12 months. Needless to say Byrnes rating is an Outperform (i.e. Buy). (To watch Byrnes track record, click here) Barring one lone Hold, all of Byrnes colleagues agree. With 9 Buys, FLXN stock boasts a Strong Buy consensus rating. While not as optimistic as Byrnes objective, the $20.22 average price target is still set to yield returns of an impressive 153% within the 12-month time frame. (See FLXN stock analysis on TipRanks) Protara Therapeutics (TARA) Staying in the pharma industry, next up we have Protara. Unlike Flexion, the cancer and rare disease-focused biotech has no therapies approved yet. However, the picture should soon become clear regarding the timing of a BLA (biologics license application) for TARA-002, the companys investigational cell therapy for a rare pediatric indication - lymphatic malformations (LM). TARA-002 is based on the immunopotentiator OK-432, currently approved as Picibanil in Japan and Taiwan for the treatment of multiple cancer indications as well as LM. Currently, Protara is seeking to get the FDAs acceptance that TARA-002 is comparable to OK-432. If everything goes according to plan, the company anticipates potential BLA filing in H2:2021 and potential approval in H1:2022. Protara shares have tumbled 40% year-to-date. That said, Guggenheim analyst Etzer Darout believes the stock is significantly undervalued. We estimate risk-adjusted peak sales of ~$170M (75% PoS) in the US alone (biologics exclusivity to 2034-2035), the 5-star analyst said. The company has outlined a no additional study scenario that estimates a US launch in 2022 and an additional registration study scenario that estimates a 2023 launch and we see current levels as a buying opportunity ahead of regulatory clarity on LM. Furthermore, Tara is expected to submit an IND (investigational new drug) for a Phase 1 trial for TARA-002 in 2H21 for the treatment of non-muscle invasive bladder cancer (NMIBC). Darout notes 80% (~65K) of all newly diagnosed bladder cancer patients suffer from this specific condition including ~45% that are high grade with high unmet need. The company also owns IV Choline, a Phase 3-ready asset, for which the FDA has already granted both Orphan Drug Designation and Fast Track Designation for IFALD (intestinal failure-associated liver disease). Based on all of the above, Darout rates TARA a Buy and has a $48 price target for the shares. The implication for investors? Upside of a strong 225%. (To watch Darouts track record, click here) Overall, with 3 recent Buy ratings under its belt, TARA gets a Strong Buy from the analyst consensus view. The stock is backed by an optimistic average price target, too; at $43.67, the shares are anticipated to appreciate by ~198% in the year ahead. (See TARA stock analysis on TipRanks) Green Thumb Industries (GTBIF) Last but not least is Green Thumb, a leading US cannabis MSO (multi state operator). This Chicago-based company is one of the stalwarts of the rising cannabis sector, boasting the second highest market-cap in the industry and exhibiting impressive growth over the last year. In 2020, revenue increased by 157% from 2019, to reach $556.6 million. That said, despite delivering another excellent quarterly statement in March, and being well-positioned to capitalize on additional states legalizing cannabis, the stock has pulled back recently after the company was hit by a damning Chicago Tribune article. According to Chicago Tribune, the company is being investigated by the fed over "pay to play" payments regarding the procurement of cannabis licenses in Illinois. Countering the claims, GTBIF management said the allegations are unfounded and that there is no factual evidence to support them. Furthermore, the company pointed out it has not even been contacted by the authorities regarding the matter. Who to believe, then? Its an easy choice, according to Roth Capitals Scott Fortune. We believe these tenuous claims create an opportunity to own the best-in-class operator currently off 25% from recent highs, the 5-atar analyst opined. In our view, the GTI business and track record of execution is not at risk in terms of the seemingly baseless accusations. We will continue to monitor any new additional incremental evidence potentially surfacing but believe the allegations are unfounded. We believe the upside opportunity remains compelling at these levels. Going by Fortunes $45 price target, shares will be changing hands for a 70% premium a year from now. Fortunes rating remains a Buy. (To watch Fortunes track record, click here) The negative news has done little to dampen enthusiasm around this stock on Wall Street. The analyst consensus rates GTBIF a Strong Buy, based on a unanimous 12 Buys. The average price target, at $47.71, suggests an upside of 79% over the next 12 months. (See GTBIF stock analysis on TipRanks) To find good ideas for stocks trading at attractive valuations, visit TipRanks Best Stocks to Buy, a newly launched tool that unites all of TipRanks equity insights. Disclaimer: The opinions expressed in this article are solely those of the featured analysts. The content is intended to be used for informational purposes only. It is very important to do your own analysis before making any investment.

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Bionano Genomics Announces Presentations on Optical Genome Mapping at the American Association for Cancer Research (AACR) Annual Meeting 2021 - Yahoo...

SAN DIEGO, April 06, 2021 (GLOBE NEWSWIRE) -- Bionano Genomics, Inc. (Nasdaq: BNGO) announced today a publication in the peer-reviewed journal Genes describing an analysis by optical genome mapping (OGM) of representative examples of common genetic syndromes that are the basis of prenatal genetic testing recommended by medical associations in the US and around the world. The papers authors have outlined a potential framework for OGM to be developed as the basis of a new standard of care in prenatal genetic analysis because of its ability to identify all types of structural variants (SVs).

What we are seeing now, on a consistent basis, is the demonstration of utility for OGM in a variety of applications. Development and validation of OGM assays for pre-natal analysis is an area where the field could transform itself and help physicians and their patients, commented Erik Holmlin, PhD, CEO of Bionano Genomics. Over the last year alone, we have seen a great number of publications and presentations that demonstrate Saphyrs prowess in cytogenetic analysis of genetic diseases, leukemias and solid tumors. While there remains significant work for these proof-of-concept studies to translate into broader, mainstream adoption, we believe the momentum is strong.

The publication is presented as a commentary in the special issue of Genes titled "Advances in Prenatal Genetic Screening and Diagnosis Technologies", which addresses the increasingly complex decisions physicians and couples face about the quality and quantity of genetic information they wish to access as the technology for genetic testing advances. As non-invasive prenatal screening tests (NIPT) become the standard screening tool for pregnancies around the world, OGM could provide a high-throughput, comprehensive and high-resolution follow up genome analysis in case of a positive NIPT screen or for high-risk pregnancies following an abnormal ultrasound. The study authors led by Bionano chief medical officer Dr. Alka Chaubey and Augusta University professor Dr. Ravindra Kolhe state that development and validation of assays based on OGM would enable clinicians to accurately detect all types of genetic disorders with a single technology, resulting in a workflow that is both cost-effective and has a fast turn-around time.

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Global medical associations including ACOG, ISUOG, ACMG recommend prenatal testing for the detection and prevention of genetic disorders using cytogenetic methods such as karyotype analysis, fluorescent in situ hybridization (FISH), and chromosomal microarray (CMA). Additionally, multiple molecular methods are used to identify repeat expansion and contraction disorders in routine clinical laboratories.

In this study OGM with Saphyr was used to detect a variety of genetic abnormalities in amniotic fluid and chorionic villus samples collected during pregnancy. The paper shows examples of how different OGM assays identified cases of trisomy 21 or Down syndrome, sex chromosome aneuploidy where additional copies of the X chromosome were present, DiGeorge syndrome, the most common microdeletion syndrome affecting about 1 in 4,000 births, a repeat expansion causing fragile X syndrome, and an unbalanced translocation in the fetus caused by a balanced translocation in the parent. The site-to-site reproducibility of the OGM prenatal workflow was also evaluated by analyzing 5 samples in replicates at two different sites, on different instruments, and with different operators, showing concordance across both sites.

The publication is available at https://www.mdpi.com/2073-4425/12/3/398/htm

About Bionano GenomicsBionano is a genome analysis company providing tools and services based on its Saphyr system to scientists and clinicians conducting genetic research and patient testing, and providing diagnostic testing for those with autism spectrum disorder (ASD) and other neurodevelopmental disabilities through its Lineagen business. Bionanos Saphyr system is a research use only platform for ultra-sensitive and ultra-specific structural variation detection that enables researchers and clinicians to accelerate the search for new diagnostics and therapeutic targets and to streamline the study of changes in chromosomes, which is known as cytogenetics. The Saphyr system is comprised of an instrument, chip consumables, reagents and a suite of data analysis tools. Bionano provides genome analysis services to provide access to data generated by the Saphyr system for researchers who prefer not to adopt the Saphyr system in their labs. Lineagen has been providing genetic testing services to families and their healthcare providers for over nine years and has performed over 65,000 tests for those with neurodevelopmental concerns. For more information, visit http://www.bionanogenomics.com or http://www.lineagen.com.

Forward-Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as may, will, expect, plan, anticipate, estimate, intend and similar expressions (as well as other words or expressions referencing future events, conditions or circumstances) convey uncertainty of future events or outcomes and are intended to identify these forward-looking statements. Forward-looking statements include statements regarding our intentions, beliefs, projections, outlook, analyses or current expectations concerning, among other things: the potential for OGM with Saphyr to serve as a foundation for a new standard of care in prenatal genetic analysis; our expectations or beliefs regarding past and potential future publications and presentations that demonstrate Saphyrs capabilities; the potential for OGM with Saphyr to improve outcomes for physicians and patients, including in the field of prenatal analysis; our beliefs regarding the potential benefits of Bionanos Saphyr technology; Saphyrs capabilities in comparison to and in conjunction with other genome analysis technologies, including in prenatal genetic testing; the potential for Saphyr to reduce or eliminate sequential and confirmatory assays and expedite patient treatment; expectations that Saphyr can allow clinicians to accurately detect all types of genetic disorders with a single technology; and the execution of Bionanos strategy. Each of these forward-looking statements involves risks and uncertainties. Actual results or developments may differ materially from those projected or implied in these forward-looking statements. Factors that may cause such a difference include the risks and uncertainties associated with: the impact of the COVID-19 pandemic on our business and the global economy; general market conditions; changes in the competitive landscape and the introduction of competitive products; changes in our strategic and commercial plans; our ability to obtain sufficient financing to fund our strategic plans and commercialization efforts; the ability of medical and research institutions to obtain funding to support adoption or continued use of our technologies; the loss of key members of management and our commercial team; and the risks and uncertainties associated with our business and financial condition in general, including the risks and uncertainties described in our filings with the Securities and Exchange Commission, including, without limitation, our Annual Report on Form 10-K for the year ended December 31, 2019 and in other filings subsequently made by us with the Securities and Exchange Commission. All forward-looking statements contained in this press release speak only as of the date on which they were made and are based on management's assumptions and estimates as of such date. We do not undertake any obligation to publicly update any forward-looking statements, whether as a result of the receipt of new information, the occurrence of future events or otherwise.

CONTACTSCompany Contact:Erik Holmlin, CEOBionano Genomics, Inc.+1 (858) 888-7610eholmlin@bionanogenomics.com

Investor Relations andMedia Contact:Amy ConradJuniper Point+1 (858) 366-3243amy@juniper-point.com

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Publication Outlines Potential for Optical Genome Mapping with Bionanos Saphyr System to be the Foundation of a New Workflow in Prenatal Genetic...

Rare, incurable conditions call for the most innovative treatmentsand thats exactly the approach the National Institutes of Health is taking to develop a new test for patients with methylmalonic acidemia, or MMA.

A study publishedin the journal Genetics in Medicine describes a breath test that not only measures the severity of a patients MMA, but can also assess whether genomic therapies targeting the liver would be beneficial. It can alsomonitor the bodys reaction to liver and kidney transplants,which are treatment options in extreme cases.

The genomic disorder,diagnosed in about one of every 80,000 newborns in the U.S., prevents certain proteins and fats from metabolizing, causing them to build up andtrigger kidney diseases, pancreatitis and other complications.

RELATED: The next generation of gene therapy for rare diseases forges ahead as developers weather hurdles

Rather than attempting to track metabolic protein levels in MMA patients, the team of NIH scientistsrepresenting the National Human Genome Research Institute, National Institute of Diabetes and Digestive and Kidney Diseases and National Institute of Mental Healthfocused instead on the actual process of metabolism. Their breath test measures the amount of carbon dioxide exhaled, since a common form of MMA interrupts the oxidation process of metabolism.

Vast fluctuations in metabolic substances in the bodies of patients make it difficult for us to tell if treatments like genome editing and transplants are likely to be successful, senior author Charles Venditti, an investigator in the NHGRI Medical Genomics and Metabolic Genetics Branch, said in a statement. Instead of looking at levels, we decided to measure metabolism itself.

To do so, patients were given a drink or feeding tube containing a commercially available food additive with a slightly heavier form of carbon than the version typically found in exhaled carbon dioxide, making it easier for the scientists to track that specific metabolic process.

The breath test found that healthy patients and those who had undergone organ transplants for MMA exhaled higher levels of the added carbon than untreatedpatients. The results highlight the tests ability to effectively measure oxidation levels and therefore determine a patients suitability for transplant.

Next, the researchers will see how well the test performs when measuring oxidation in MMA patients who have undergone gene therapies or mRNA treatments, Venditti said. Theyre also hoping to make the test available for widespread clinical and research use.

RELATED: Singapore startup develops 60-second COVID-19 breathalyzer test

Several similar diagnostic breath tests are already in use, though none for the treatment of MMA.

An FDA-approved test developed by Advanced Breath Diagnostics is used to diagnose gastroparesis, a stomach disorder, while Owlstone Medicals breath-based biopsy system has been shown to detect signs of liver disease in a single exhale. And in November, Singapore startup Breathonix said its 60-second breathalyzer test achieved 90% accuracy in screening for COVID-19.

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NIH finds success in capturing breaths to assess and treat rare genomic disease - FierceBiotech

Mercks CRISPRi whole-genome Libraries and Pools Pave the Way for Breakthroughs in Identifying Gene Pathways

Merck, a leading science and technology company, has launched its CRISPR inhibition (CRISPRi) whole-genome libraries and pools to complement its industry-leading suite of genome-editing products and technologies. CRISPRi is a useful tool for screening an entire genome, smaller gene family or individual targets. The CRISPRi libraries are fully customizable, providing the same flexibility as the entire family of Merck screening products.

Instead of imprecise DNA repair to knock out a gene, CRISPRi modulates gene expression, allowing for robust loss-of-function studies, even in essential genes, with fewer off-target effects. CRISPRi provides unique insights into the underlying biology often missed when only using gene knock out or overexpression studies. Using CRISPRi as a discovery tool, researchers can make scientific breakthroughs in idenfying new disease pathways or drug targets.

The whole CRISPRi libraries and pools were created by the University of California, San Francisco, USA, and further enhanced by the Life Science business of Merck, resulting in superior algorithm design and improved scaffolding that has been proven to increase gene knock down, even in difficult-to-repress targets. The CRISPRi offerings feature:

Mercks comprehensive gene modulation offering also includes CRISPR activation (CRISPRa) whole-genome libraries and pools, often used in conjunction with CRISPRi.

The Life Science business provides a full suite of screening tools for genome-editing applications including CRISPR knock out, RNAi knock down and open reading frame gain-of-function studies. Genome-editing and gene modulation technologies complement each other and used together, they can fully elucidate gene pathways and identify drug targets.

With 17 years of experience in the genome-editing field, the Life Science business of Merck is a global leader in developing innovative CRISPR tools, products and services to support every step of genome engineering-facilitated research, from basic research to therapeutic delivery.

Learn more about Mercks new CRISPRi whole-genome libraries and pools.

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Merck's CRISPRi whole-genome Libraries and Pools Pave the Way for Breakthroughs in Identifying Gene Pathways - PharmiWeb.com

An international research team has sequenced the genomes of the oldest securely dated modern humans in Europe who lived around 45,000 years ago in Bacho Kiro Cave, Bulgaria. By comparing their genomes to the genomes of people who lived later in Europe and in Asia the researchers from the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, show that this early human group in Europe contributed genes to later people, particularly present-day East Asians. The researchers also identified large stretches of Neandertal DNA in the genomes of the Bacho Kiro Cave people, showing that they had Neandertal ancestors about five to seven generations back in their family histories. This suggests that mixture with Neandertals was the rule rather than the exception when the first modern humans arrived in Europe.

Last year, a research team led by researchers from the National Institute of Archaeology with Museum at the Bulgarian Academy of Sciences and the Max Planck Institute for Evolutionary Anthropology, Germany, reported the discovery of modern human remains found in direct association with the Initial Upper Palaeolithic stone tools at the site of Bacho Kiro Cave in Bulgaria. The oldest individuals found in the cave were directly radiocarbon dated to between 43,000 and 46,000 years ago. They are thus the earliest known dispersal of modern humans across the mid-latitudes of Eurasia.

Mateja Hajdinjak and colleagues have now sequenced the genomes of five individuals found at the Bacho Kiro Cave. Four individuals are between 43,000 to 46,000-years-old and were found together with stone tools belonging to the Initial Upper Palaeolithic, the earliest culture associated with modern humans in Eurasia. An additional individual found in the cave is around 35,000-years-old and found with stone tools of a later type. It was previously thought that bearers of the Initial Upper Palaeolithic died out without contributing genetically to modern humans arriving later. However, the researchers now show that the oldest Bacho Kiro Cave individuals, or groups closely related to them, contributed genes to present-day people. Surprisingly, this contribution is found particularly in East Asia and the Americas rather than in Europe where the Bacho Kiro Cave people lived. These genetic links to Asia mirror the links seen between the Initial Upper Palaeolithic stone tools and personal ornaments found in Bacho Kiro Cave and tools and ancient jewelry found across Eurasia to Mongolia.

The earliest people at Bacho Kiro Cave lived at a time when Neandertals were still around. The researchers therefore scanned their genomes for fragments of Neandertal DNA. We found that the Bacho Kiro Cave individuals had higher levels of Neandertal ancestry than nearly all other early humans, with the exception of a 40,000-year-old individual from Romania. Crucially, most of this Neandertal DNA comes in extremely long stretches. This shows that these individuals had Neandertal ancestors some five to seven generations back in their family trees says Mateja Hajdinjak.

Although only a handful of genomes from modern humans who lived at the same time in Eurasia as some of the last Neandertals have been recovered, nearly all of them have recent Neandertal ancestors. The results suggest that the first modern humans that arrived in Eurasia mixed frequently with Neandertals. They may even have become absorbed into resident Neandertal populations. Only later on did larger modern human groups arrive and replace the Neandertals says Svante Pbo, who coordinated the genetic research.

Reference:Hajdinjak M, Mafessoni F, Skov L, et al. Initial Upper Palaeolithic humans in Europe had recent Neanderthal ancestry. Nature. 2021;592(7853):253-257. doi:10.1038/s41586-021-03335-3

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Ancient Genomes of the Earliest Europeans - Technology Networks

Abstract

Escherichia coli and other Enterobacteriaceae are diverse species with open pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553 Escherichia and 274 non-Escherichia spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.

Enterobacteriaceae are a family of Gram-negative bacteria that can cause clinical infections (1, 2) and persist environmentally (3, 4) across diverse environmental niches (5). Antimicrobial resistance (AMR) in Enterobacteriaceae has emerged as a major problem in the past decade (6, 7). Dissemination of AMR genes often occurs via mobile genetic elements (MGEs), which can transfer genes within and between species both locally (8) and globally (9). Freshwater-, wastewater-, and livestock-associated strains of Enterobacteriaceae have been proposed as reservoirs for AMR genes in clinical isolates (1013), but the links between these remain cryptic (14).

Species within Enterobacteriaceae are well-known examples of open pangenomes (15, 16) containing substantial genetic diversity, with movement of genes via horizontal gene transfer (HGT) (17). Current understanding of the ecology and evolution of pangenomes is incomplete (18), with ongoing debate about the roles of niche adaptation and selection (1922). However, published Enterobacteriaceae genomes are biased toward clinical isolates (23, 24), and sampling frames reflecting truly interlinked communities are limited. Much remains unknown about the population genetics of Enterobacteriaceae (25) and the role of plasmids in nonclinical contexts (26).

Genomic studies of Enterobacteriaceae have predominantly used short-read whole-genome sequencing. AMR genes and their flanking regions are frequently fragmented in short-read assemblies due to repetitive elements and structural rearrangements (8, 27). Combining short and long reads (hybrid assembly) produces complete, high-quality genomes (28), allowing accurate structural resolution. Here, we report a study of the multispecies pangenome within nonclinical Enterobacteriaceae using hybrid sequencing. We used hybrid assembly of 827 sympatric Enterobacteriaceae (including Citrobacter, Enterobacter, Escherichia, and Klebsiella spp.) to characterize the pangenome of these genera, considering both niche [cattle, pig, sheep, or wastewater treatment work (WwTW) associated] and geography (sampling location).

We collected samples from 19 locations 5 km apart (maximum distance: 60 km) in South Central England (United Kingdom) in 2017, namely, 14 livestock farms (four pig, five cattle, and five sheep) and water sources around five WwTWs over three seasonal time points (TPs) (Fig. 1A). We pooled samples within each location and TP and then cultured isolates from these pooled samples. A selected subset of 832 of 2098 cultured isolates underwent short- and long-read sequencing and hybrid genome assembly (Fig. 1B; see Materials and Methods), resulting in 827 high-quality genomes (table S1; n = 495 from livestock farms, n = 332 from WwTWs), from four genera: Citrobacter (n = 127), Enterobacter (n = 71), Escherichia (n = 553), and Klebsiella (n = 76). Most farm isolates were Escherichia spp. (451 of 495, 91.1%), with WwTW isolates having roughly even proportions of genera (fig. S1). Isolates contained a median of one AMR gene (range: 0 to 23), with variation by genus: Klebsiella isolates carried a median of 4 (range: 1 to 18).

(A) Relative sampling locations of the farms (cattle, pig, and sheep) and wastewater treatment plants (WwTWs) in this study, sampled at three different TPs. (B) Schematic illustration of the sampling, culture, and sequencing workflow, resulting in high-quality genome assemblies with a median of one circularized chromosome and two circularized plasmids per assembly. (C) Mid-point rooted core genome phylogeny of E. coli isolates (n = 488), with tips colored by phylogroup and ring colors showing sampling niche. Inset panel at center of phylogeny shows phylogroup abundances (as proportion of isolates) from different sampling niches.

Isolates were highly diverse, containing diversity not present in published genomes (fig. S2). Escherichia diversity included all main E. coli phylogroups (Fig. 1C), as well as 53 Escherichia fergusonii, and 13 isolates from clades I, II, III, and V (table S1). Phylogroup B2 was strongly associated with WwTWs compared with livestock (34.3% versus 5.3% of Escherichia isolates in each category respectively; 2 = 70.4, P < 0.001), particularly in influent and effluent samples (Fig. 1C). This observation is in line with Ludden et al. (29), who found that phylogroup B2 was rare in livestock-associated E. coli sampled in England (4 of 431 genomes). Pigs had a greater proportion of phylogroup A isolates (Fig. 1C). Of 187 identified E. coli multilocus sequence types (STs), 56.1% (105 of 187) were seen only once, similar to the 61% observed by Touchon et al. (30) in a study of nonclinical E. coli. Only 12 Escherichia STs were seen in both livestock and WwTW isolates, with phylogroup B1 as the most represented (5 of 12 STs). ST10 was the most prevalent ST (n = 45), seen in 10 of 14 farms and 3 of 5 WwTWs. This is in agreement with recent studies in England: Ludden et al. (29) also found ST10 to be the most prevalent E. coli ST in livestock-associated isolates, and AbuOun et al. (13) found that the ST10 clonal complex was the most prevalent in isolates from pig farms. Our observations and good agreement with recent work in this setting suggest that our dataset, although sampled from a relatively small geographical region, is representative of broader nonclinical E. coli populations.

Considering only livestock E. coli isolates, over time, there was a persistent phylogroup signature of both livestock host and farm, with individual farm explaining slightly more variance than livestock type (R2 = 28.1% versus 25.8%; fig. S3). In other words, between-farm differences in E. coli phylogroup abundance were of the same order as differences between livestock species. However, livestock type explained less variance for STs than phylogroups (R2 = 8.5%), with only 39 of 131 STs (29.8%) seen on more than one farm. This suggests that associations between livestock species and E. coli population structure are more important at the higher level of phylogroup, and clear associations between ST and livestock species are not the norm. There were only 26 instances where an E. coli ST was observed over time on the same farm (involving 16 STs), and most of these (22 of 26 instances, 12 of 16 STs) were STs also seen across farms (fig. S4); these could therefore represent more generalist STs.

However, STs can encompass large genomic variation. We therefore also considered E. coli strain clusters using a core genome distance of <100 single-nucleotide variants (SNVs) (maximal diversity observed across sampled E. coli: 211,251 SNVs; median pairwise distance: 46,144 SNVs). There were 280 isolate pairs with <100 SNVs, of which 181 (64.6%) were isolates cultured from the same pooled sample (i.e., same farm and same TP) (fig. S5A). Overall, 10.5% of all isolate pairs from the same pooled sample had <100 SNVs between them, compared with 1.4% (n = 52) of isolate pairs from different TPs on the same farm and 0.2% (n = 44) between different farms of the same animal (fig. S5B). Of the latter, 41 of 44 were between cattle farms, and 36 involved a single cattle farm (RH06). There were only three isolate pairs with <100 SNVs between farms of different animals (fig. S5A). All of these were between farms in close geographic proximity (two instances from pig farm RH03 and cattle farm RH10, and one instance from cattle farm RH07 and sheep farm RH12; see Fig. 1A for distances), suggesting local strain movement. There were no isolate pairs with <100 SNVs between WwTW and livestock niches, and only three isolate pairs occurred across TPs at WwTWs (all at a single WwTW).

Together, these results indicate that different livestock hosts have a stable balance of E. coli phylogroups and that each farm setting can harbor substantial strain-level diversity, which, in our dataset, is unique to that farm and can persist over time. In contrast, isolates from locations proximal to WwTWs do not have a stable population of strains, reflecting the more transitory nature of this setting. We did not recover any transmission links at the strain level between WwTWs and livestock, although the vast diversity of natural E. coli populations means this should not be interpreted as evidence that these links do not exist.

We recovered 2292 circularized plasmids across all Enterobacteriaceae, ranging in size from 1240 bp to 824 kbp (median: 43 kbp; table S2). There were 297 of 2292 (13.0%) with no identifiable plasmid replicon, and most of these were from WwTW isolates (192 of 297, 64.6%). Multiple replicons were carried by 723 of 2292 (31.5%), and these plasmids tended to be larger (median length: 106,811 bp versus 6669 bp for single replicon plasmids). Of E. coli isolates with complete genomes, over two thirds (70.4%, 245 of 348) carried a plasmid with an IncFII replicon. Forty-three percent of circularized plasmids (986 of 2293) had at least one match with >99% identity to other publicly available plasmid sequences (fig. S2B). However, 12.3% (282 of 2293) had a top identity score of <95% to a previous known sequence (fig. S2B), and 17 plasmids with no match were identified, suggesting that sampling recovered previously undescribed plasmid diversity. We grouped circularized plasmids into 611 distinct plasmid clusters using alignment-free distances (see Materials and Methods), which closely matched their gene content (fig. S6A). A recent analysis by Redondo-Salvo et al. (31), clustering over 10,000 plasmids from prokaryotes using average nucleotide identity, found that plasmids within the same cluster contained a common genomic backbone. We also found that the synteny of shared genes was strongly conserved within plasmid clusters (fig. S6B), supporting this concept of common backbones that remain stable while allowing genes to be gained by insertion.

A median of 3.3% of genes were on plasmids (range: 0 to 16.5%), with substantial variation by genus and niche (fig. S7A). This is a comparable value to the 3% finding of Touchon et al. (30) in E. coli with variation over a similar range [Fig. 4A of (30)]. We also considered the effect of plasmid copy number (i.e., multiplying plasmid lengths by their inferred copy number) to calculate the total DNA in both chromomes and plasmids within isolates (fig. S7B). E. coli isolates had a median of 5.7% of DNA present on plasmids, which was substantially higher in pig farm isolates (median: 10.1%; fig. S7B), linked to the presence of large plasmids. We analyzed the variation of gene content with phylogeny and niche (Fig. 2). Chromosomal genes were highly genus specific (R2 = 55.0%); the plasmid-borne pangenome was far more variable but still had a weak association with genus (R2 = 6.5%) (Fig. 2). Within E. coli, plasmid gene content was linked to niche (R2 = 5.6%) and phylogroup (R2 = 5.2%), with a stronger interaction between niche and phylogroup (R2 = 7.9%) (Fig. 2).

Plots are shown for isolates (A) across Enterobacteriaceae and (B) within E. coli, for both the chromosomal component of the pangenome and the plasmid-borne component analyzed separately. Color indicates (A) genus within Enterobacteriaceae and (B) phylogroup within E. coli. Stacked bar charts in the center of each show the variance in gene content explained by niche, phylogeny (genus or phylogroup), and their interaction. The plasmid-borne component has greater residual variance than the chromosomal component, with a comparatively stronger niche-phylogeny interaction (darkest shaded bar).

Plasmids were predicted to be conjugative, mobilizable, or nonmobilizable (see Materials and Methods), and we explored whether this affected their distribution. Nonmobilizable plasmid clusters were less commonly shared between different phylogroups within farms compared with mobilizable or conjugative plasmids (fig. S8). Although AMR genes were predominantly found in conjugative or mobilizable plasmid clusters, consistent with their frequent acquisition and movement between strains, plasmid clusters with AMR genes were not more commonly distributed across multiple phylogroups (chi-square test 2 = 0.64, P = 0.42; fig. S8). However, on pig farms, most of the conjugative plasmid clusters seen across multiple phylogroups carried AMR genes, suggesting that they play an important role within this niche.

Since many isolates had multiple plasmids in their genome, we also considered the possibility of plasmid-plasmid interactions. Positive epistasis between large (>10 kbp) and small plasmids has been suggested to promote plasmid stability in Enterobacteriaceae, based on analyses of genomes in public databases (32). In E. coli isolates with complete genomes (n = 348), we observed a significant association between small and large plasmid presence (chi-square test 2 = 4.44, P = 0.035), with 45.7% carrying at least one large (>10 kbp) and one small plasmid and only 3.7% carrying a small plasmid without a large plasmid. Thus, previous results that support the existence of positive large-small plasmid epistasis are reproducible in this natural population, suggesting that this is an important feature of Enterobacteriaceae plasmid distributions. We also found evidence for specific plasmid-plasmid associations. For example, cattle E. coli isolates showed co-occurrence of a ColRNA plasmid (cluster 37: median length, 4.6 kbp) and an IncFII plasmid cluster (cluster 279: median length, 106 kbp), with 14 of 16 isolates with the ColRNA plasmid also carrying the larger IncFII plasmid. Isolates were from three phylogroups (A: n = 2, B1: n = 5, and E: n = 9) and four farms, suggesting a robust association that reflects plasmid epistasis independent of chromosomal background.

Plasmids carried more diverse and less genus-restricted genes, as expected from their role as the more flexible component of the genome. Despite carrying just 3.3% of total gene content, plasmid-borne genes accounted for 11.5% of unique genes (8.9 to 17.0% considering each genus; fig. S9), and 40.1% were seen in more than one genus (19.6 to 55.6% considering each genus; table S3). Plasmids are important vehicles of AMR genes in clinical contexts; similarly, plasmids had a much greater burden of AMR genes in the niches studied here. Considering isolates with circularized chromosomes (see Materials and Methods), 901 of 1876 AMR genes (48.0%) were found on plasmids, i.e., a 14.5 relative burden in plasmids. They also had a higher burden of insertion sequences (ISs), which are linked to the movement of genes by HGT. Of 26,565 ISs, 3695 (21.7%) were found on plasmids (6.6 relative burden). There was a weak correlation between the number of plasmid- and chromosome-associated AMR genes within an isolate (Spearmans = 0.11, P = 0.004) but a strong positive correlation for the number of ISs (Spearmans = 0.40, P < 0.001) (fig. S10A) seen across genera (fig. S10B).

We observed different patterns of ISs across chromosomes and plasmids (fig. S11). Some ISs were strongly associated with plasmids, with the strongest association being for IS26. However, 27.5% of isolates carrying IS26 on a plasmid also carried it on their chromosome, consistent with its characteristically active behavior. It has been shown that IS26 can drive the reorganization of plasmids in clinical settings by replicative transposition (33), as well as amplify AMR genes (34) and create within-plasmid heterogeneity in a single isolate (35). Its high prevalence in Enterobacteriaceae plasmids here suggests that it may play a similar role outside the context of clinical AMR. The most prevalent IS on both chromosomes and plasmids was ISKpn26, with 50.2% of ISKpn26-positive isolates having it both chromosomally and plasmid borne.

Considering Escherichia, WwTW isolates showed a greater diversity of ISs, with 65% of ISs found in a higher proportion of WwTW isolates compared with those from farms (fig. S12), including IS30 which has been proposed as a marker for naturalized wastewater populations of E. coli (36). Touchon et al. (30) suggest that water-borne E. coli strains are adapted to this niche rather than being fecal contaminants and can therefore adapt by acquiring genetic material from not only gut bacteria (as in, e.g., a livestock or human host) but also other diverse environmental bacteria. The observation of greater IS diversity in WwTW isolates here would be consistent with this hypothesis.

We also investigated the overall patterns of co-occurrence of ISs to see whether they were strongly linked on plasmids. Overall, ISs had random levels of co-occurrence on Escherichia plasmids (upper tail P = 0.85 from null model simulations of checkerboard score; see Materials and Methods; fig. S13A), suggesting that ISs frequently move independently between plasmid backgrounds. In contrast, applying the same method to AMR genes, we found they significantly co-occurred (upper tail P = 0.02; fig. S13B), suggesting coselection and underlining the tendency of some AMR genes to co-occur in specific regions of plasmids.

Plasmids fell into two broad classes across genera: small multicopy plasmids (<10 kbp, 10 to 100 copy number inferred from coverage relative to chromosome) and large low-copy plasmids (>10 kbp, <10) (Fig. 3A). AMR plasmids were almost all large low-copy plasmids (172 of 183, 94.0%). While small multicopy plasmids are of interest in facilitating evolutionary innovation (37, 38), this finding suggests that they do not play a major, direct role in AMR in livestock- and WwTW-associated Enterobacteriaceae.

(A) Plasmid length (x axis) and inferred copy number (y axis) of all circularized plasmids (n = 2292), faceted by genus. Plasmids with 1 AMR gene (colored points) tended to be larger and present in lower copy numbers. (B) Relative GC content of all plasmids to their host chromosome for all circularized plasmids present in an assembly with a circularized chromosome (n = 1753 plasmids across 616 isolates), split by predicted plasmid mobility. Boxplots are additionally shown classifying plasmids within predicted mobility types by the number of AMR genes carried: those 1 AMR gene (red) or no AMR genes (black). Comparisons with P values are shown for all plasmids within a predicted mobility class. (C) Length distributions of plasmid clusters (see Materials and Methods).

Experimental evidence shows that selective advantages favor lower GC content in intracellular elements (39), and this has been proposed as an explanation for the tendency of plasmids to typically have lower GC content than their host chromosome. Under this hypothesis, the more dependent on the bacterial host the plasmid is, the lower its relative GC content should be. We investigated this in our dataset. Overall, plasmids had a lower relative GC content than their host chromosomes (median difference, 2.5%; Fig. 3B). There was an approximate gradient of relative GC content with predicted plasmid mobility (Fig. 3B), with plasmids predicted to be mobile having a smaller relative difference. Furthermore, this difference was less marked for AMR plasmids (median, 0.3%) across mobility categories (Fig. 3B). Nearly half had a higher GC content than their host chromosome (46.7% versus 17.7% of non-AMR plasmids). Together, this suggests that AMR plasmids are being selected for in these environments, which counteracts the usual selective advantages for lower GC content. Alternatively, this may be a signal of their relatively recent acquisition by their host.

We identified 2364 potential HGT events involving transfers of sequence >5000 base pairs (bp) between isolates of different genera (see Materials and Methods). These represent possible instances of the recent movement of genetic material across species boundaries. Isolates from the same farm were ~10 more likely to show evidence of cross-genera HGT than would be expected (chi-square test 2 = 1159, P < 0.001; fig. S14), and 12.3% of these cross-genera HGT events involved at least one AMR gene, with most of these AMR HGT events between pig isolates (37 of 48, 77.0%).

The movement of genes can also occur within individual genomes. We therefore also investigated occurrences where the same gene was present on both the chromosome and plasmid(s) within an E. coli genome. We observed distinct differences between niches, with increased amounts of chromosome-plasmid sharing in pig and WwTW isolates compared with cattle and sheep (fig. S15). This may be a signature of increased selection for AMR in these niches, such that usually transitory gene movements and duplications are retained in genomes for long enough to be detected.

To understand the strength of different factors shaping the pangenome, we analyzed the pangenome of a single species, E. coli, in more detail. Isolates recovered from the same location spanned total E. coli diversity (Fig. 4A). Interisolate core genome distances were strongly correlated with chromosomal gene repertoire relatedness (GRR) (Fig. 4A). Core genome distance explained the majority of variance in chromosomal GRR (Fig. 4B), but there was a consistent contribution from geography and time: isolates from the same pooled sample sharing more genes than would be expected (+1.2%), as did isolates from the same farm at different time points (+0.5%) (Fig. 4B). There was no such effect for isolates from different farms of the same livestock, suggesting that this reflects local geography rather than adaptation to livestock host. Although the variance explained was much lower, local geography effects were also observed for plasmid GRR (Fig. 4C), but core genome distance was uncorrelated with plasmid GRR apart from for near-identical strains (Fig. 4D). Isolates from different STs from different farms of the same livstock could still have high plasmid GRR (Fig. 4E), suggesting that host-specific plasmids may facilitate niche adaptation.

(A) Pairwise comparisons of GRR for chromosomal genes show that chromosomal GRR falls off rapidly at small patristic distances, followed by an approximately linear decrease. Fits show intra-ST comparisons (thick black line), all comparisons (thin black line), and a linear model (dashed black line). Violin plots above show the distribution of patristic distances depending on the relative sample source of the two isolates in the pairwise comparison (white boxplot: median and IQR; black point: mean), showing that even isolates cultured from the same sample (same farm and same TP) span equivalent diversity to isolates cultured from different locations. (B) Coefficients from a linear model for chromosomal GRR with an interaction term with patristic distance (excluding intra-ST comparisons). (C) Variance explained by phylogeny and geography for chromosomal and plasmid GRR. (D) GRR for plasmid-borne genes with patristic distance. Fits show intra-ST comparisons (thick red line), all comparisons (thin red line), and a linear model (dashed red line). Inset panel shows left-hand region of the plot with only intra-ST comparisons, with chromosomal GRR relationship also shown (gray points, black line). (E) Plasmid GRR comparisons shown by isolate sources, excluding intra-ST comparisons. Colors on the x axis are the same as in (A). Plots include all E. coli isolates with a circularized chromosome (n = 363).

We have investigated the pangenome of major genera of sympatric Enterobacteriaceae from locations within a 30-km radius, using a diverse set of nonclinical isolates cultured from the same samples and focusing in detail on E. coli. Despite high overall diversity, with most of the strains only observed once in the dataset, we observed the persistence of strains and plasmids on farms over the course of a year. Our results highlight the combination of persistence and dynamism that characterizes Enterobacteriaceae genomes at multiple scales, with relevance both for understanding the population structure of species within Enterobacteriaceae and for managing AMR. The existence of farm-level differences in E. coli populations that persists over time, with a small number of possible interfarm transfers, suggests that livestock farms function as distinct but linked niches. It could be that everything is everywhere (frequent movement of strains and genes between farms), but the environment selects (different farms have different selective pressures). However, the observation of persistent strains over the course of a year on farms, despite presumably varying selective conditions, and the overrepresentation of putative cross-genera HGT events in isolates at the same location suggest that geographical effects or intrinsic properties of certain bacterial/MGE lineages could affect the evolution of AMR on these time scales. Future modeling work and investigation will be required to distinguish these hypotheses. Overall, our findings underline the importance of local control strategies for the emergence and spread of AMR beyond clinical settings.

Here, we have focused on genome dynamics across different niches but have not attempted to integrate our findings with detailed contextual information from the participating sites. This will be crucial to give further insight into how differences in antimicrobial usage on farms and other management practices can affect AMR in multispecies pangenomes, such as the decline in mcr-1 prevalence in Enterobacteriaceae on pig farms after a ban on colistin in feed, as observed in both China (40) and the UK (41). Other factors beyond antimicrobial usage could include herd size, herd management practices, and cleaning and disinfection practices. Investigating these is part of our ongoing work. Similarly, differences in management between WwTWs may affect the prevalence of AMR genes in effluent, and this is also the subject of ongoing work.

Resource limitations meant that we were unable to sequence and genetically evaluate all isolates that were cultured, and despite detailed sampling, we will not have captured all the persistence, HGT, and strain-sharing events across niches: The instances of sharing that we did identify remain fairly anecdotal. This is a problem faced by even intensive sampling efforts, which can only capture a tiny fraction of the population diversity and so are unlikely to retrieve close links in transmission chains (42) (of strains or MGEs). Future analyses could still investigate the links between such sharing and possible transmission routes between both farms and WwTWs, including the land application of manure (43), the land application of sewage sludge (44), and groundwater flow (45). Older studies have established that specific AMR genes can be more prevalent in groundwater closer to manure storage (46), but working out how to do equivalent genomic analyses for strains and MGEs across larger geographic distances with such diversity is difficult. Although this study is unprecented in evaluating four genera in such detail, AMR gene dissemination and important structural associations of AMR genes and MGEs may also be occurring within other genera not studied here. Furthermore, we did not investigate the relationship between isolates in this study and clinical human compartments in the same study area. Ludden et al. (29, 47) previously reported limited direct overlap for E. coli or Klebsiella pneumoniae strains from livestock and humans in a different region of England but highlighted that 5% of human E. coli isolates potentially shared closely related AMR-associated MGEs with those found in livestock. We intend to see whether this finding holds in our ongoing work.

In conclusion, our study highlights the plastic and dynamic nature of AMR gene dissemination within the pangenome of major Enterobacteriaceae in several important nonclinical niches. It also demonstrates how robustly evaluating the flow of AMR genes and MGEs across highly diverse and dynamic niches is challenging even with extensive sampling. The implications of this for adequately understanding dissemination and selection of AMR genes in a One Health context should not be underestimated.

Isolates were sequenced from samples collected as part of the The environmental REsistome: confluence of Human and Animal Biota in antibiotic resistance spread (REHAB) study in 2017, which aimed to characterize nonclinical Enterobacteriaceae populations in four different niches within a defined study area of South Central England: cattle farms, pig farms, sheep farms, and water environments linked to WwTWs. Sampling occurred at each location at three separate TPs: January to April 2017 (TP1), June to July 2017 (TP2), and October to November 2017 (TP3).

Five cattle farms, five sheep farms, and four pig farms were recruited from the study area following a defined recruitment process. Briefly, we aimed to recruit the five largest farms for each livestock type within the area using local APHA databases, progressively inviting the next largest farm if a farm declined. All participating farmers provided written consent for farm sampling for research purposes, and farm samples were taken between January and November 2017 on three separate visits (TPs) for each farm. Each farm was divided in five or fewer epidemiological groups, defined as a group of animals expected to share similar characteristics and managed in the same way. Ten pooled samples were collected from each of these groups, with each sample composed of small pinches of fresh feces from the floor combined into a small composite sample around 5 cm in diameter. Each groups 10 samples were pooled, diluted up to 105 in phosphate buffer solution (pH 7.2), and plated onto CHROMagar ECC (CHROMagar Microbiology, Paris, France) and CHROMagar ECC plates containing cefotaxime (1 mg/liter) as a marker for multidrug resistance. Up to 10 colonies were collected from cefotaxime (1 mg/liter)supplemented plates and 14 colonies from CHROMagar ECC plates; where 10 colonies were not recovered, additional colonies were taken from the CHROMagar ECC plates, resulting in 24 isolates per farm. Pure isolate subcultures were subsequently stored at 80C in MicroBank beads (Pro-Lab Diagnostics, Neston, Cheshire, UK), and the bacterial species were confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) (Bruker, Coventry, UK) or 16S rRNA sequencing (48). The median number of sequenced isolates for a farm TP combination was 12 (range: 9 to 14), with 495 farm isolates in total: cattle (n = 178), pig (n = 143), and sheep (n = 174).

Five WwTWs were selected on the basis of a defined recruitment process including the following: geographic location within the study area, wastewater treatment configuration, wastewater population equivalent served, consented flow, and the accessibility of the effluent receiving river for sampling both upstream and downstream. Sampling took place in 2017 over three sampling rounds: February to March (TP1), June to July (TP2), and October to November (TP3). Sewage influent samples were collected after WwTW coarse screens, and effluent samples were collected at the last sampling point before entering the river. For each sampling round, ~6 repeated 200-ml samples of influent and effluent were collected between 9:00 a.m. and 12:00 p.m. using a sampling pole and sterile Whirl-Pak collection bags. Repeat samples in each round were pooled before processing to reduce the impact of temporal variability in wastewater flows and composition. Sediment samples were collected from 100 m upstream and 250 m downstream of the effluent entry point into the river. Sediment samples were collected using a custom sampling pole that held a removable 50-ml plastic centrifuge tube (Sigma-Aldrich, UK). Using a fresh sterile 50-ml tube each time, sediment from the riverbed was collected from the surface layer at three points at each sampling site; near bank, the center of the river, and the far bank. These samples were pooled before analysis to account for spatial variability in sediment composition. Influent, effluent, and sediment samples were stored in an insulated box at ~4C until getting back to the laboratory (<6 hours). Influent, effluent, 100 m upstream, and 250 m downstream environmental samples collected from each sewage treatment works were transferred to the laboratory on ice and processed within 24 hours of collection. Each sample was vortexed briefly, serial diluted to 103 in nutrient broth containing 10% glycerol (Oxoid, Basingstoke, UK), and plated onto CHROMagar Orientation agar (Chromagar, Paris, France) and CHROMagar Orientation agar supplemented with cefotaxime (1 g/ml) (Cambridge Biosciences, Cambridge, UK). Colonies with putative morphology for species of interest were subcultured from dilution plates with suitably isolated growth. A total of up to 20 colonies was picked per sample: Up to 10 colonies were picked from the cefotaxime (1 mg/liter)supplemented plates, and the remainder were picked from the nonsupplemented plates. Pure isolates subcultured on Columbia blood agar (CBA) (Oxoid, Basingstoke, UK) were subsequently stored at 80C in nutrient broth containing 10% glycerol, and bacterial species were confirmed using MALDI-TOF (Bruker, Coventry, UK).

A subset of isolates were selected for sequencing to represent diversity within the four major genera within each niche, including the use of third-generation cephalosporin resistance as a selective marker to identify a subgroup of multidrug-resistant isolates within each genus. A total of 832 isolates were each sequenced with both a short-read (Illumina HiSeq 4000) and a long-read sequencing approach [four isolates selected for sequencing failed subsequent hybrid assembly and were not included in further analyses; one further isolate was removed after assembly (see Genome assembly)]. For the first TP, the latter involved sequencing using either PacBio SMRT (n = 192) or Oxford Nanopore Technologies (ONT) methodologies (n = 127). The results of a pilot study comparing sequencing and assembly approaches using a subset of REHAB isolates (28) were used to inform the choice of ONT as the long-read sequencing approach for all isolates from the second (n = 255) and third (n = 254) TPs.

Isolate stocks from 80C storage were cultured onto CBA and supplemented with cefpodoxime (Thermo Fisher Scientific, USA) 10-g discs for isolates not sensitive to cefotaxime during original sample isolation. DNA was extracted using the Qiagen Genomic tip/100G (Qiagen, Venlo, The Netherlands) according to the manufacturers instructions. DNA concentration was quantified by Qubit 2.0 fluorimeter (Invitrogen, UK), and quality and fragment size distribution were assessed by TapeStation 2200 (Agilent, Santa Clara, USA). ONT sequencing libraries were prepared by multiplexing six to eight DNA extracts per flow cell using kits SQK-RBK004, SQK-LSK108, and EXP-NBD103 according to the manufacturers protocol. Libraries were loaded onto flow cell versions FLO-MIN106 R9.4 (0.1) SpotON and sequenced for 48 hours on a GridION (ONT, Oxford, UK).

We used the hybrid assembly and sequencing methods described in our pilot study (28) to produce high-quality Enterobacteriaceae genomes from short and long reads. Briefly, we used Unicycler (v0.4.7) (49) with normal mode, --min_component_size 500, --min_dead_end_size 500, and otherwise default parameters. Our pilot study (28) explored the accuracy of assemblies from this method (and others) in detail using multiple metrics, including the following: the mapping of Illumina short reads back to assemblies, the mapping of long reads back to assemblies, the comparison of assemblies from the same isolate, using known marker genes to assess overall quality, the assemblys overall circularity, and the presence of indel errors. We found excellent concordance in structural agreement of circular structures from different assemblies [see, e.g., fig. S4 of (28)], giving us confidence that circular structures from hybrid assemblies (chromosomes and plasmids) are accurate. One possible concern could be that chimeric reads in long-read datasets could lead to incorrect structures in the final hybrid assembly. However, an independent investigation (50, 51) using data from our pilot study alongside simulated data showed that long readonly assemblies with Unicycler are only affected by the presence of chimeric reads at rates of ~15% of reads and are thus not a concern for real datasets, which typically have rates of <2%.

Final assemblies from all isolates had a median of four contigs [interquartile range (IQR): 3 to 8; range: 1 to 391], with a median of two circularised plasmids (IQR: 1 to 4, range: 0 to 14). One Citrobacter isolate from TP1 was removed from the dataset after we identified a sample mixup, meaning that its metadata were unreliable. The majority (616 of 827, 74.5%) of the assemblies had a circularized chromosome, and 558 of 827 (67.4%) were complete, i.e., chromosome and all plasmids circularized (table S1).

We assigned species and ST from assembled genomes using mlst (v2.16.4) (52). We also validated species assignments by downloading all National Center for Biotechnology Information (NCBI) Refseq complete genomes for the four genera under study as of 4 June 2020 and using fastANI (v1.3) (53) to compute average nucleotide identity scores against reference genomes for each assembled genome. We took the species assignment of the top hit for each assembled genome. Furthermore, we manually checked genus assignments using a t-distributed stochastic neighbor embedding (t-SNE) plot of isolate genomes against a collection of reference genomes and made corrections to the assignment if necessary. We used ClermonTyping (v1.4.1) (54) to assign phylogroup to n = 553 Escherichia isolates. Considering the genus Escherichia, there were 553 isolates, 410 with circularized chromosomes, and of these, 379 were complete genomes containing 961 complete plasmids in total. Considering only E. coli, there were 502 E. coli isolates, 372 with circularized chromosomes, and of these, 348 were complete genomes containing 878 complete plasmids in total. A minority of genomes were E. fergusonii (n = 51), from clades I to V (n = 14), or could not be typed (n = 7), with n = 481 genomes from within the principal E. coli phylogroups (A: n = 131; B1: n = 193; B2: n = 59; C: n = 11; D: n = 25; E: n = 50; F: n = 6; and G: n = 6).

Sequenced isolates from three other Enterobacteriaceae genera included the following: Citrobacter (n = 128: 81 Citrobacter freundii and 46 unassigned Citrobacter sp.), Enterobacter (n = 71: 59 Enterobacter cloacae and 12 unassigned Enterobacter sp.), and Klebsiella (n = 76: 40 K. pneumoniae, 30 Klebsiella oxytoca, 2 Klebsiella aerogenes, and 4 unassigned Klebsiella sp.). The majority of the farm-associated isolates were E. coli, whereas WwTW-associated isolates had roughly equal numbers of genera (fig. S1). This reflects both the diversity present in each niche and the selection strategy to sequence equal numbers across genera where feasible.

All genomes were annotated with Prokka (v1.14.0) (55). We performed a multispecies pangenome analysis by clustering genes into gene groups using Roary (v3.12.0) (56) across all isolates at various sequence identity thresholds with the maximum number of clusters set to 300,000 (-g 300,000) and without splitting paralogs (-s). At a 95% identity for blastp, there were 139,788 gene groups across all genera. Further to this analysis, genes were also clustered at a higher sequence identity (>99% identity threshold) to identify recent HGT events, which gave 214,743 gene groups across all genera. (These pangenome analyses included the subsequently removed Citrobacter isolate with unreliable metadata.) For n = 616 isolates with circularised chromosomes, we split the genome into chromosomal and plasmid-borne components (i.e., all other contigs) to analyze the genomic location of genes. We excluded isolates without circularized chromosomes from this analysis. For within-species pangenome analyses, the more recently developed Panaroo gives lower annotation error rates and a more accurate core genome than Roary or other methods by using gene adjacency (i.e., synteny) information (57), although it is not suitable for cross-species analyses where no core genome is expected. Therefore, for a higher-resolution within-species analysis of n = 488 E. coli isolates (excluding E. fergusonii and clades I to V), we used Panaroo (v0.1.0) (57) to extract a core genome alignment based on 2915 concatenated core genes (Fig. 1C). The phylogeny was produced using iqtree (v1.6.11) (58), with branch lengths not corrected for recombination, and plotted with ggtree (v2.0.1) (59).

We searched all plasmids against PLSDB (version: 2020-03-04) (60), which contains 20,668 complete published plasmids, using screen in mash (v2.0) (61) and keeping the top hit. All plasmids had a match apart from 17 small plasmids predicted to be nonmobilizable (median length: 4.8 kbp; range: 2.9 to 20.7 kbp), from Escherichia (n = 11), Enterobacter (n = 2), and Citrobacter (n = 4). We clustered plasmids using mob cluster and assigned replicon types with mob typer, both part of the MOB suite (v1.4.9) (62). Mob cluster uses single linkage clustering with a cutoff of a mash distance of 0.05 [corresponding to 95% average nucleotide identify (ANI)], resulting in 611 clusters (table S2). In total, there were 134 different combinations of replicons observed on plasmids (replicon haplotypes). The most abundant replicon was IncFIB (n = 459), which was seen across all niches [pig (n = 80), cattle (n = 113), sheep (n = 78), and WwTWs (n = 188)]. Only nine small multicopy plasmids (~6 kbp) carried AMR genes, all of which had a ColRNAI replicon; these ColRNAI plasmids have been proposed to be sources of evolutionary innovation (37, 38).

We considered the relationship between such distance-free clustering and plasmid gene content. On the basis of gene clustering with Roary (see above), we compared the structure of circularised plasmids using all connecting edges between two genes. We defined the resemblance for both gene content (gene presence/absence) and gene structure. The gene content resemblance between two plasmids with n1 and n2 genes, respectively, with N genes in common, was defined as rcontent = 2 N/(n1 + n2). The edge structure resemblance between two plasmids with g gene-gene edges in common was defined as redge = 2 g/(n1 + n2). Typically redge < rcontent, but this definition does allow for the case where repeated genetic elements produce redge > rcontent (e.g., fig. S6B).

To visualize cross-genera pangenomes (e.g., Fig. 2), we used t-SNE. We used the Rtsne function with a perplexity of 30 on gene presence/absence matrices in the Rtsne R package (v0.15) (63). To conduct permutational analyses of variance, we used the adonis function from the vegan R package (v2.5-6) (64) on the matrix of pairwise Jaccard distances, which was calculated using the vegdist function. For between-genera analyses, we used the formula dist~niche*genus. For within-Escherichia analyses, we used the formula dist~niche*phylogroup.

We searched assemblies using ABRicate (v0.9.8) (65) for acquired resistance genes (i.e., excluding mutational resistance) in the NCBI AMRFinder Plus database (PRJNA313047). We used a minimum identity threshold of 90% and a minimum coverage threshold of 90% (table S4). Isolates that cultured selectively from cefotaxime-supplemented plates carried more AMR genes than nonselectively cultured isolates (median of 7.5 versus 1.0), as expected. We also searched for ISs using the ISFinder database (66) as a database in ABRicate with the same identity and coverage thresholds (table S5).

We performed an all-against-all comparison of assemblies with mummer (v3.23-2) (67) using the -maxmatch option to identify shared sequences of length >5000 bp between genomes of different genera (these could include both transfer of whole plasmids or partial sequences). For comparing the observed distribution of cross-genera HGT events to the expected, we assumed a random distribution drawn from all possible cross-genera comparisons from livestock isolates.

We constructed the bipartite presence/absence network of ISs and replicon haplotypes for the 34 replicon haplotypes, which were observed on 10 or more plasmids. We simulated null models of co-occurrence patterns using the cooc_null_model with null model sim9, which fixes the row and column sums of the presence/absence matrix, in the R package EcoSimR (v0.1.0) (68). Simulations used n = 10,000 iterations with a burn-in of 500 iterations.

We selected a subset of E. coli genomes with a circularized chromosome (n = 363) and used the core genome tree constructed with iqtree (Fig. 1; dropping other E. coli isolates) to calculate patristic distances between isolates. We calculated chromosomal and plasmid GRR for all pairwise comparisons using output from roary (95% identity threshold, as above) and fit linear models for GRR (Fig. 4).

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Niche and local geography shape the pangenome of wastewater- and livestock-associated Enterobacteriaceae - Science Advances

Congenica and Camtech Diagnostics agree strategic partnership to support genomic analysis platform expansion in Asia

Cambridge, United Kingdom and Singapore 13 April 2021- Congenica, the digital health company enabling rapid and accurate analysis of complex genomic data to transform peoples lives, today announces a strategic partnership with Camtech Diagnostics (Camtech), a technology company providing total solutions to the biodefence, food and clinical diagnostics sectors. The partnership covers the distribution of Congenicas platform in Asia-Pacific.

Under the partnership agreement Camtech will be responsible for the marketing, distribution and sales of Congenicas clinical decision support platform in Singapore, Malaysia, Japan and South Korea. Congenicas leading technology platform is proven to enable rapid analysis of genomic data, allowing medical labs to deliver personalised medicine at scale. Congenica has an existing presence in China and this latest agreement will support continued expansion in the rare disease market in the Asia-Pacific region.

Camtech has significant experience in the healthcare diagnostics industry, having launched novel laboratory-based and point-of-care diagnostic tests to the market. It also has expertise in Next Generation Sequencing, laboratory based and digital health solutions, which will be complementary to Congenicas solution.

Muthu Meyyappan, PhD, Chief Commercial Officer, Congenica, said: Camtech benefits from a strong reputation in Southeast Asia with an established network through which our platform can be rapidly distributed and integrated into rare disease diagnostic procedures. Global expansion remains a priority for Congenica and entry into these key territories signifies further strategic progress whilst opening up significant new markets for Congenicas leading genomic analysis platform.

Kuok Meng-Han, PhD, Managing Director, Camtech, said: The advanced healthcare systems of Asia Pacific have been undergoing rapid development and growth. They have a clear need for fast, reliable tools to support the diagnosis of rare diseases. We believe that Congenicas market leading genomic analysis platform has the potential to make a significant impact in the region. We are excited to partner with Congenica to rollout this platform and move closer to genomic medicine being more widely incorporated into standard clinical practice.

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**ENDS**

About Congenica

Congenica is a digital health company enabling the rapid analysis and interpretation of genomic data, empowering researchers to provide life-changing answers that improve wellbeing and disease management.

Congenicas world-leading software enables rapid genomic data analysis at scale, performing 20x faster than industry averages and providing a 30% higher analytical yield, reducing genomic interpretation costs by up to 95%.

Born out of pioneering research from the Wellcome Sanger Institute and the NHS, Congenica has a global footprint supporting leading international laboratories, academic medical centres and biopharmaceutical companies and is the exclusive Clinical Decision Support partner for the NHS Genomic Medicine Service.

For more information visit http://www.congenica.com.

About Camtech Diagnostics

Camtech Diagnostics aims to enable ideal human health and wellness through technology and innovation in testing.

Founded in 2013, its vision is to provide novel diagnostic tests and digital solutions to obtain the most accurate real-time data and actionable insights for personalised health

It develops and commercialises a range of testing solutions for biodefence, food and clinical diagnostics applications, that aim to disrupt current lab-based testing approaches and has built significant R&D and Manufacturing capabilities to support these developments.

For more information visit http://www.camtechdiagnostics.com

Contact details for editorial enquiries:

Chris Gardner, Angela Gray, David Daley

Email: Congenica@consilium-comms.com

Tel: +44 (0)20 3709 5700

Noah Konig, Head of Marketing at Congenica

Email: noah.konig@congenica.com

Alvin Liew, Head of Corporate Affairs at Camtech

Email: alvin@camtech.com.sg

Read the original post:
Congenica and Camtech Diagnostics agree strategic partnership to support genomic analysis platform expansion in Asia - Yahoo Finance

More News13 Apr 2021 | 3:54 PM

Pune, Apr 13 (UNI) Actor-activist Vira Sathidar, best known for featuring in the National Award-winning film Court, died on Tuesday due to COVID-19 related complications, filmmaker Chaitanya Tamhane confirmed.

Panaji, Apr 13 (UNI) Goa Forward Party (GFP) President Vijay Sardesai on Tuesday announced party's decision to formally withdraw from National Democratic Alliance (NDA).

Mr Sardesai said, Enough is enough, the people are fed up and want the BJP out.He alleged that under Chief Minister Pramod Sawant, development of Goa quickly turned into corruption and benefits galore for a handful of individuals who looked to make their quick buck with Sawant as CM.

Mumbai, Apr 13 (UNI) State Deputy Chief Minister Ajit Pawar on Tuesday wished the people of the state a happy Gudi Padwa and Marathi New Year, saying that one of those high Gudhis of success should be the victory of Gudi 'Coronamukti'.

Nagpur, April 13 (UNI) The Maharashtra Association of Resident Doctors (MARD) of GMCH and IG GMCH members has called on Divisional Commissioner Dr Sanjeev Kumar and apprised him of the suffering through which the patients are going through.

Continued here:
Genome sequencing lab will be set up to track new Covid-19 strains: Vishwajit Rane - United News of India

DUBAI: The International Prize for Arabic Fiction has announced its six shortlisted authors who are competing to receive a $50,000 award when the winner is revealed on May 25. Read on to learn more about the writers and their novels.

Abdulatif Ould Abdullah - The Eye of Hammurabi

In the Algerian writers book The Eye of Hammurabi, he opens with the interrogation of a man in a military encampment after he fled from the angry inhabitants of Douar Sidi Majdoub.

As the story unfolds, the main character revisits his past to explore the roots of his present dilemma and tells stories which blend imagination and reality, illusion and the truth.

Jalal Bargas - Notebooks of the Bookshop Keeper

Notebooks of the Bookshop Keeper is by Jordanian poet and novelist Jalal Bargas. Set between 1947 and 2019, this novel is based on several notebooks of stories about people facing different hardships, such as losing their homes or not knowing who their family are.

The main character is Ibrahim, a bookshop keeper, a cultured man and voracious reader of novels. However, due to his isolation, loneliness and maltreatment by a cruel world, he suffers mental illness and descends into full schizophrenia. He attempts suicide, before meeting the woman who will change his life.

Amira Ghenim- The Calamity of the Nobility

The Tunisian writer and academics book The Calamity of the Nobility relates an untold story from Tunisias contemporary history about renowned author, scholar and reformer El-Taher El-Haddad.

Although historical references do not mention anything about his relationship with women, except for his desperate defense of them, the author adds an imaginary love affair with a woman called Lella Zubaida to her fictional retelling of his life.

The novel gives prominence to the voices of female narrators, as custodians of memory who contradict a distorted, patriarchal version of history.

Dunya Mikhail- The Bird Tattoo'

The Iraqi authors The Bird Tattoo is a painful novel about the sale of Yazidi women in Iraq by Daesh.

It focuses on Helen and Elias, who fall in love and marry, and their experiences with the group. Alongside this tragedy, the novel sheds light on aspects of Yazidi folklore, rich in customs and legends.

Abdelmajid Sebbata - File 42

File 42, by Moroccan author Abdelmajid Sebbata, follows two parallel storylines. In the first, Christine Macmillan, a successful American novelist, and Rasheed Benaser, a young Moroccan researcher and doctoral student, embark on an investigation to find the unknown author of a forgotten Moroccan novel from 1989, in which Christines father, Steve, appears as one of the characters.

The second plot line is narrated by Zuheir Belqasem, a rich and delinquent Moroccan teenager who assaults Al-Ghalia, an underaged maid. His mother uses her influence as a prominent lawyer to close the case and send him to Russia to pursue his university studies. However, horrors await him there which no-one had foreseen.

Habib Selmi- Longing for the Woman Next Door

The Tunisian authors book tells the story of two neighbors who have nothing in common, apart from both being Tunisian and living in the same apartment building.

He is in his sixties, educated and married to a Frenchwoman. She is several years younger and from a lower social stratum, and married to an eccentric man. At first, he is cautious and patronizing. But later, the rules of the game change. The novel explores a rich, turbulent and extraordinary relationship, which celebrates life in its simplest and most beautiful manifestations but is also tinged with darkness and tragedy.

Go here to see the original:
What We Are Reading Today: The Genome Odyssey by Euan Angus Ashley - Arab News

Technological developments in next-generation sequencing (NGS) approaches and mass spectrometry (MS)-based methods have advanced the landscape of molecular biology research. Scientists are now able to identify and characterize various constituents of a cell, tissue or organism and analyze them in their totality their "ome" be it the entire expression of genes (genome), proteins (proteome) or metabolites (metabolome), at any given time.Piecing together this information helps us to understand the molecular journey from genotype to phenotype and where it can go wrong, in the case of disease phenotypes. As an increasing quantity of omics information becomes accessible to researchers, it has become clear that working with the data sets in segregation can limit their utility. Connectivity between the omics "worlds" is fundamental, but it has been challenging until now.

In January, theEuropean Molecular Biology Laboratorys European Bioinformatics Institute (EMBL-EBI) announced that it had launched the Genome Integrations with Function and Sequence, or GIFTS, platform. This novel platform enables scientists that are using Ensembl and UniProt to access all of the up-to-date genomic and protein data for human and mouse genomes. Technology Networks spoke with Beth Flint, Ensembl applications project leader at EMBL-EBI, Maria Martin, team leader in protein function development at EMBL-EBI and Daniel Zerbino, team leader in genome analysis at EMBL-EBI, to learn more about GIFTS and how it will be used to help the research community.

Molly Campbell (MC): Please can you talk to us about the rationale behind the Genome Integrations with Function and Sequence (GIFTS)?Daniel Zerbino (DZ): GIFTS aims to provide a clear and unambiguous bridge between two flagship data resources at the EMBLs European Bioinformatics Institute (www.ebi.ac.uk), namely Ensembl and UniProt. Together, they offer a wealth of information on protein synthesis: Ensembl describes the upstream sequences of nucleotides that encode protein-coding genes, whose transcripts are transcribed into downstream protein isoforms, documented in UniProt. Each resource already points to the other, thus connecting genes to proteins and vice-versa, but because of differences in release cycle calendars these links were not 100% consistent. GIFTS now details our shared understanding of which gene maps to which protein.

MC: Who is behind the development of GIFTS?Beth Flint (BF): GIFTS has been developed at EMBL-EBI as a collaboration between Ensembl and UniProt. The project brought together the expertise from these two groups and allowed us to build a tool that will allow people to easily explore the relationships between the data these groups produce. Collaborative projects of this nature take full advantage of the breadth of knowledge and diverse range of skills at EMBL-EBI. The GIFTS project was possible due to the input of curators, annotators, database and API experts, user interface developers and pipeline automation specialists.

MC: Why is it important to connect the genome and proteome worlds?Maria Martin (MM): The genome is the storehouse of the genetic material needed for an organism to function. Proteins are the primary effectors of the instructions encoded in our genomes and they and their products ultimately shape our cells, tissues, organs and bodies in response to our environment. Proteins provide an essential link between genome sequence and the eventual phenotype. The functional analysis of genomic and other large-scale biomedical datasets requires integrated information about many distinct types of biological entity, including individual genes, transcripts and proteins.

MC: Why has this been difficult previously?MM: Ensembl focuses on the annotation of transcripts in reference genomes using available cDNA, EST and RNA-seq data, while UniProt focuses on annotating protein sequences using experimental evidence from the literature, homologs in other species and proteomics experiments. The study of genomes and proteomes requires a very specialized scientific knowledge which needs to be combined to effectively map them.

MC: What do you hope the outcome of launching GIFTS will be?BF: The mappings that GIFTS pipelines produce will help the Ensembl and Uniprot teams update the data they present via their main sites. Using the GIFTS data will present a uniform view of mappings between the two domains and ensure that consistent information is presented. This provides an enormous benefit to those who use these mappings. Behind the public facing interface of GIFTS are tools used by the annotators and curators in the Ensembl and Uniprot groups. These tools enable them to review and improve mappings. As this process continues, it is hoped that over time canonical UniProt isoforms will be selected for all human genes and that these will match the MANE transcripts from Ensembl.

MC: Are there any intentions to launch a similar platform for other "omics" data?DZ: EMBL-EBI resources strive to be interoperable with each other, and this new bridge is merely strengthening a very tight network of data resources. For example, other EMBL-EBI resources, such as the Reactome pathway database or the Gene Expression Atlas already link directly and unambiguously to UniProt proteins or Ensembl genes. A sister project, Structure Integration with Function, Taxonomy and Sequence (SIFTS) now connects UniProt protein sequences to their 3D structures, stored in PDBe. All these interconnections are enabling researchers to make sense of all the resources at EMBL-EBI, as exemplified by our unified search utility.

Daniel Zerbino, Beth Flint and Maria Martin were speaking to Molly Campbell, Science Writer for Technology Networks.

Read more here:
New Platform Connects the Genome and Proteome Worlds - Technology Networks