Category Archives: Genome

More variants will undoubtedly emerge over time, and it is unclear how much these variants will complicate, or even set back, efforts to bring the pandemic to an end. Ongoing genomic sequencing is key in identifying the emergence of vaccine escape variants, Moi said. This makes it all the more troubling that most nations have failed to even come close to the levels of genome sequencing that may be needed.

The state of the genomic surveillance situation is grimmest in 38 countries with reported COVID-19 infections but no sequencing data shared with Gisaid. These make up some of the poorest countries in the world, such as Chad and Burundi. The African continent, as of June 27, has reported more than 5.3 million infections (3.9 million of these are confirmed), but its countries have sequenced and released only about 22,700 genomes, or at best only 0.6% of its cases. More than 40% of those genome sequences (about 9,600) come from just one country, South Africa.

The consequences of the paucity of data on Africa could be serious for people everywhere. Africa, given its human population variation, is a candidate to becoming the source of ever more pathogenic and refractory strains, said Muntasar Ibrahim, a Sudanese geneticist and professor of molecular biology at the University of Khartoum, where he leads its Institute of Endemic Diseases.

Shortfalls in sequencing cannot be blamed simply on a lack of money. (Sequencing costs about $120 per SARS-CoV-2 genome, but the costs can be significantly lowered by sequencing the genomes in large batches, according to Haussler.) Some of the poorest countries have sequenced more of their cases than some of the richest countries, so wealth cannot be the only determining factor. Gambia, for instance, at 7.8%, has sequenced more than Germany (3.6%), a country with 60 times its gross domestic product per capita.

Nor do low rates merely reflect how hard countries have been hit by the pandemic. About 10% of the U.S. population has had COVID-19, resulting in a low sequencing rate (1.7%) even though the U.S. has sequenced the most SARS-CoV-2 genomes. But the U.K., where about 7% of the population has had the disease, has sequenced more than 10% of its caseload: It has only the 13th-highest rate of sequencing in the world, but it has sequenced more virus genomes than all the countries ahead of it put together.

What really seems to have determined the genome-sequencing performance of countries during the pandemic is a combination of their strategic choices and biomedical infrastructure.

Tom Maniatis, chief executive officer of the New York Genome Center (NYGC), noted that COVID-19 surveillance in the U.S. has been compromised by a systemic lack of connections between facilities that have samples of the virus hospitals, public health laboratories and commercial testing facilities and facilities with the capacity to sequence them. Though the situation has improved, there have been persistent logistical challenges, he said.

Maniatis and Soren Germer, who leads the sequencing and analytics teams at NYGC, said that obtaining samples had been the biggest challenge in the U.S. During the early days of the pandemic when New York was particularly hard hit, even the most research-focused hospitals often did not have the resources to collect samples for research, they explained by email. We have heard stories of truly heroic efforts to save some of these samples for research and surveillance, but the severely strained hospitals had to prioritize treating patients and protecting staff. Maniatis and Germer also pointed to a lack of coordinated funding in the U.S., which has been uneven at the state and local level and has only recently begun at the federal level.

Rolf Apweiler, director of the European Bioinformatics Institute, says that the nations depositing SARS-CoV-2 sequences into the dedicated genome data platform that his organization operates also vary substantially in their ambitions. While some countries aim low or have no genomic surveillance of SARS-CoV-2, he said, countries like Denmark, Iceland, Australia and the U.K. aim to sequence between 10% of all positive samples in times of high infection rates and all positive samples technically feasible in times of low infection rates.

The genome sequencing effort may already be bearing fruit for some of the countries engaging in it most vigorously. COG-UK is a consortium of genomic experts working to track, trace and control the SARS-CoV-2 virus in the U.K. It formed when the countrys scientists took steps early in the pandemic to ensure genomic sequencing at scale, aided by 20 million from the government. Within weeks of its formation in March 2020, the consortium had made the first sampled genomes publicly available; it has now sequenced more than 450,000 virus genomes.

OGrady credits that work with helping to contain the pandemic in the U.K. Genome sequencing identified the B.1.1.7 variant, providing us with an answer as to why case numbers were increasing dramatically towards the end of 2020 and enabling us to implement successful control measures, he said. When other variants were discovered in South Africa and elsewhere, U.K. authorities increased the testing and contract tracing efforts and curtailed the spread of the variants into the country.

Many countries are now working to scale up their sequencing programs. In February, the CDC pledged $200 million as a down payment for genome surveillance. In April, the Biden administration dedicated $1.7 billion to boosting sequencing efforts and fighting variants of SARS-CoV-2. The U.S. is now investing heavily in sequencing with the realization that the gains weve made are fragile and could be upended by viral variants, OConnor said.

In January, the Indian government set up the Indian SARS-CoV-2 Genomics Consortium to expedite the gene sequencing effort through a growing network of institutions. The nationally coordinated genome-sequencing program has sequenced more than 15,000 genomes in about three months, said Anurag Agrawal, a senior scientist with the consortium and director of the CSIR-Institute of Genomics and Integrative Biology in New Delhi, one of the participating institutions. I expect the numbers to keep getting better, he said.

The situation is improving in Africa, too. Segun Fatumo, an assistant professor of genetic epidemiology and bioinformatics at the Medical Research Council/Uganda Virus Research Institute, said that African governments urgently need to provide funding for relevant research and infrastructure. But he also noted that Africa has been moderately successful in the fight against the coronavirus, and genome sequencing has greatly contributed to this.

The WHO has established a network of COVID-19 genomic sequencing laboratories across Africa in 18 countries, he said. Africa is central to human origin and disease susceptibility, so large-scale genomic study in populations of African descent might yield potential therapeutic strategies.

Apweiler feels that a pandemic can be successfully managed only if it is tackled at a global level with as much coordination and collaboration as possible. A problematic new lineage of SARS-CoV-2 in one country may become a worldwide problem very quickly, he said. Our response to the pandemic will be globally only as strong as the weakest part of the global efforts.

Moi agrees about the importance of sequencing, but also suggests that it will always be necessary to balance that effort against other local priorities to ensure the best public health impact. Particularly during large outbreaks, sequencing large numbers of virus [genomes] may not be practical and could increase the burdens on laboratories and medical facilities that are already under pressure, she said. But she is also confident that with optimal sequencing strategies in place, powerful insights can still be achieved with well-planned sampling and testing.

Had the pandemic happened even five years ago, it would have been a lot more difficult to implement genomic surveillance programs at scale, OConnor said. The technologies to democratize sequencing and make it available to small laboratories and public health authorities simply werent available.

The infrastructure and technology developed to map the virus could also be beneficial beyond COVID-19. Our next hope is that the detailed observation of viral evolution during the pandemic and the research will help with the more rapid development of targeted therapeutics in future pandemics, Maniatis said.

To him, the real question is whether the informational networks and infrastructure will enable viral surveillance to become routine, so that the discovery of the next potential pandemic virus can be a normal part of the public health system. The WHO has called the integration of genome sequencing into the regular practices of the global health community a must in preparations for future threats.

Haussler agreed that building global pathogen sequencing and genome sharing capability could help prevent future viral outbreaks. It is one of the most important investments the world can make at this point, he said. It is likely to save many lives and many trillions of dollars in the long run.

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A Lack of COVID-19 Genomes Could Prolong the Pandemic Quanta Magazine - Quanta Magazine

Drugs that slow tumor growth by targeting genetic abnormalities in the cancer itself are now well established in oncology. But what if doctors could treat cancer by altering gene activity throughout the body, tricking it into fighting off the disease?

A handful of startups and academics are working towardthat goal. Its not gene therapy in the traditional sense, because theyre not removing or replacing disease-causing genes. Rather, theyre using novel drugs to turn the expression of certain genes up or down to achieve an anti-cancer effect.

Were unlocking new biological pathways so we can go after undruggable targets and treat diseases in very new ways, said Robert Habib, chief executive officer of London-based MiNA Therapeutics, in an interview. MiNA is developing a pipeline of small activating RNAs (saRNAs), which are short, double-stranded oligonucleotides designed to enter cells and boost the activity of target genes to achieve a therapeutic effect.

In September, MiNA raised about $30 million to advance its lead asset, MTL-CEBPA, into a phase 2 trial. The saRNA drug is designed to target the gene CEBPA, which encodes a transcription factor thats key to the bodys production of cancer-fighting myeloid cells. These cells can be depleted in the tumor microenvironment, contributing to drug resistance in cancer.

MTL-CEBPA enters the cell nucleus and uses RNA activation to boost levels of the CEBPA protein. MiNAs drug is in testing alongside Bayers Nexavar, initially in liver cancer, though Habib and his colleagues believe its unique mechanism of action could prove useful across a range of solid tumors.

Myeloid cells are a problem in liver cancer but also in many other solid tumors, he said.

MiNA is now planning a second clinical trialin combination with Mercks immuno-oncology blockbuster Keytruda in a broader set of solid tumors, Habib said.

RELATED: MiNA raises $30M to take small activating RNA into phase 2

A related technology called small interfering RNAs (siRNAs) has long been of interest for its potential to shut down cancer-promoting genes, but translating it into therapies has been challenging. It has been hindered by tissue bio-accumulationmaking sure the delivery system is safe and provides a wide enough therapeutic window in tissues beyond the liver, said Anna Perdrix Rosell, Ph.D., co-founder and managing director of London-based Sixfold Biosciences, in an interview.

Sixfold is working on a siRNA technology called Mergo, which it aims to prove can deliver siRNAs to cancer cells within specific organs while leaving healthy tissues alone. The companys preclinical testing issupported by an Innovate UK Smart Grant, and the companyis now working to define its lead cancer targets, with the goal of moving into clinical trials in 2022, Rosell said.

Gene-silencing specialistSirnaomicsis working on several RNA-interfering drugs to treat solid tumors. Its lead asset, STP705, uses a polypeptide nanoparticle to deliver two siRNAs targeting the genes TGFB1 and COX-2.

Suppressing those genes inhibits cancer-associated fibroblasts, which are cells in the tumor microenvironment that promote tumor growth, the Gaithersburg, Maryland, company has set out to show. It is in early clinical trials in solid liver tumors, squamous cell carcinoma and basal cell carcinoma.

Another gene-directed approach involves injecting DNA into tumors with the goal of making them more responsive to immunotherapyor turning them from cold tumors to hot ones. One company working on this technology is Pennington, New Jersey-based OncoSec Immunotherapies. Its lead technology, called tavokinogene telseplasmid (TAVO), uses electrical pulses to temporarily open cancer cell membranes, after which DNA is injected into them.

The DNA makes IL-12, a naturally occurring, immune-stimulating protein that the companys scientists believe could help overcome resistance to checkpoint inhibitors like Keytruda, a PD-1 blocker. Its a common problem in cancer care: An estimated 60% to 80% of melanoma patients, for example, do not respond to PD-1 blockade. And IL-12 cant be given systemically because it causes toxic side effects.

OncoSecs DNA-delivery system is designed to prompt the body to make more of its own IL-12. The DNA essentially co-opts the cells function to cause it to make IL-12, explained Daniel OConnor, CEO off OncoSec, in an interview.

OncoSec has partnered with Merck to test TAVO in combination with Keytruda in advanced melanoma and triple-negative breast cancer. TAVO is given every six weeks as an injection into tumors, though not every tumor has to be medicated, OConnor said. We see shrinkage in the tumors that are treated, but also in those that are untreated, he said. In April, OncoSec presented interim data from the melanoma trial, reporting an overall response rate of 30%, with some complete responses and no serious side effects.

RELATED: Omega grabs $126M to bring 'genome-tuning' cancer treatment into the clinic

Investors continue to show enthusiasm for the idea of manipulating gene activity to achieve an anti-cancer response. One recent beneficiary of their largesse was Omega Therapeutics, a Cambridge, Massachusetts-based company that raised $126 million in March to advance its genome-tuning drugs, including its lead treatment for liver cancer, OTX-2002.

Omega refers to the drug as an epigenetic controller, because its designed to control the expression of the cancer-promoting gene C-MYC. The companys technology tunes gene expression up or down without permanently changing DNA, and it does so by targeting regulatory factors in loops of DNA known as Insulated Genomic Domains (IGDs), CEO Mahesh Karande explained to Fierce Biotech in March.

We treat diseases created by functional or structural changes in IGDs, Karande said at the time.

Meanwhile, in academia, researchers are continuously searching for new technologies to make the process of adjusting gene activity safer and applicable to a wider variety of tumor types.

In May, for example, researchers at MUSC Hollings Cancer Centerdescribed a peptide theyre designing that can deliver aSiRNA into cells by adhering to antennae-like protrusions on cell surfaces known as filopodia. The researchers are initially developing the technology to target oral cancers, which typically have high levels of filopodia.

The MUSC researcher leading the effort, Andrew Jakymiw, Ph.D., an associate professor of oral health sciences, said in an interview that if the filopodia-targeted SiRNA delivery system pans out, it could prove applicable to a range of cancers.

Many invasive carcinomashave high levels of filopodia, while normal cells typically have very few, Jakymiw said. So this could potentially be used as a strategy to target more aggressive forms of this type of cancer.

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Outsmarting cancer with RNA, 'genome-tuning' drugs and other gene-altering therapies - FierceBiotech

WHEN THE Mutambaras first son was a about 18 months old they began to worry about his hearing. The toddler did not respond when asked to come to Mama. He was soon diagnosed as deaf, though no doctor could tell the Zimbabwean couple the cause. Several years later their second son was also born deaf.

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This time a doctor referred them to Hearing Impairment Genetics Studies in Africa (HI-GENES), set up in 2018 by Ambroise Wonkam, a Cameroonian professor of genetics now at the University of Cape Town. The project is sequencing the genomes of Africans with hearing loss in seven countries to learn why six babies in every 1,000 are born deaf in Africa, a rate six times that in America. In Cape Town, where Mr and Mrs Mutambara (not their real names) live, a counsellor explained that the boys deafness is caused by genetic variants rarely found outside Africa.

What is true of deafness is true of other conditions. The 3bn pairs of nucleotide bases that make up human DNA were first fully mapped in 2003 by the Human Genome Project. Since then scientists have made publicly available the sequencing of around 1m genomes as part of an effort to refine the reference genome, a blueprint used by researchers. But less than 2% of all sequenced genomes are African, though Africans are 17% of the worlds population (see chart). We must fill the gap, argues Dr Wonkam, who has proposed an initiative to do just thatThree Million African Genomes (3MAG).

The evolutionary line leading to Homo sapiens diverged 5m-6m years ago from that leading to chimpanzees, and for almost all that time the ancestors of modern humans lived in Africa.

Only about 60,000 years ago did Homo sapiens venture widely beyond the continent, in small bands of adventurers. Most of humanitys genetic diversity, under-sampled though it is, is therefore found in Africa. Unfortunately, that diversity is also reflected in the greater variety of genetic illnesses found there.

The bias in sequencing leads to under-diagnosis of diseases in people of (relatively recent) African descent. Genetic causes of heart failure, such as the one that caused the ultimately fatal collapse of Marc-Vivien Fo, a Cameroonian football player, during a game in 2003, are poorly understood. The variation present in most non-Africans with cystic fibrosis is responsible for only about 30% of cases in people of African origin. This is one reason, along with its relative rarity, that the illness is often missed in black children. Standard genetic tests for hearing loss would not have picked up the Mutambara boys variations. And such is the diversity within the continent that tests in some countries would be irrelevant in others. In Ghana HI-GENES found one mutation responsible for 40% of inherited deafness. The same variation has not been found in South Africa.

Bias also means that little is known about how variations elsewhere in the genome modify conditions. With sickle-cell disease, red blood cells look like bananas rather than, as is normal, round cushions. About 75% of the 300,000 babies born every year with sickle-cell disease are African. The high share reflects a bittersweet twist in the evolutionary tale; sickle-cell genes can confer a degree of protection against malaria. Other mutations are known to lessen sickle-cells impact, but most knowledge of genetic modifiers is particular to Europeans.

Quicker and more accurate diagnosis would mean better treatment. The sooner parents know their children are deaf, the sooner they can begin sign language. Algorithms that incorporate genetic information, such as one for measuring doses of warfarin, a blood-thinner, are often inappropriately calibrated for Africans.

Knowing more about Africans genomes will benefit the whole world. The continents genetic diversity makes it easier to find rare causes of common diseases. Last year researchers investigating schizophrenia sequenced the genomes of about 900 Xhosas (a South African ethnic group) with the psychiatric disorder. They found some of the same mutations that a team had discovered in Swedes four years earlier. But those researchers had to analyse four times as many of the homogeneous Scandinavians to find it. Research by Olufunmilayo Olopade, a Nigerian-born oncologist, into why breast cancer is relatively common in Nigerian women, has revealed broad insights into tumour growth.

Dr Wonkams vision for 3MAG, as outlined in Nature, a scientific journal, is for 300,000 African genomes to be sequenced per year over a decade. That is the minimum needed to capture the continents diversity. He notes that the UK biobank is sequencing 500,000 genomes, though Britains population is a twentieth the size of Africas. The plummeting cost of technology makes 3MAG possible. Sequencing the first genome cost $300m; today the cost of sequencing is around $1,000. If data from people of African descent in similar projects, like the UK biobank, were shared with 3MAG, that would help. So too would collaboration with genetics firms, such as 54Gene, a Nigerian start-up.

The 3MAG project is building on firm foundations. Over the past decade the Human Heredity and Health in Africa consortium, sponsored by Americas National Institutes of Health and the Wellcome Trust, a British charity, has supported research institutes in 30 African countries. It has funded local laboratories for world-class scientists such as Dr Wonkam and Christian Happi, a Nigerian geneticist.

There are practical issues to iron out. One is figuring out how to store the vast amounts of data. Another is rules around consent and data use, especially if 3MAG will involve firms understandably keen to commercialise the findings. Dr Wonkam wants to see an ethics committee set up to review this and other matters.

At times he has wondered whether his plan is too big, too crazy and too expensive. But similar things were said about the Human Genome Project. Its researchers used the Rosetta Stone as a metaphor for the initiative and its ambition. In a subtle nod, Dr Wonkam has a miniature of the obelisk on a shelf in his office. It is also a reminder of how understanding African languages, whether spoken or genetic, can enlighten all of humanity.

This article appeared in the Middle East & Africa section of the print edition under the headline "The African genome project"

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The promise of the African genome project - The Economist

Jun 26th 2021

RACISM MAY often run deep, but one of the most depressing things about it is how superficial it really is. In most parts of the world it is literally a matter of black and white. A persons skin colour, however, has little biological significance. It is merely a balance between defending the lower layers of the dermis from cancer-causing ultraviolet light (which favours dark skin) and promoting the beneficial role of ultraviolet in the synthesis of vitamin D (which favours light skin). The farther someones ancestors lived from the equator, the paler their skin evolved to be.

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Go back far enough, though, and everyones ancestors lived in Africa, the continent where Homo sapiens originated. Most non-Africans alive today trace the bulk of their ancestry to Africans who burst forth on an unprepared world about 60,000 years ago. Indeed, the oldest representative of the species yet found in Britain retained the dark skin of his African forebears. Africa is where humanity grew upand where the bulk of human genetic diversity is found to this day.

Only now is a serious effort beginning to explore Africas genetic richness. Better late than never. The Three Million African Genomes (3MAG) project, a continent-wide endeavour, proposes to do for the place what has already been done for Europe, North America and parts of Asianamely to catalogue and analyse the genetic diversity of those who live there. That will be scientifically fascinating, for it will help elucidate how H. sapiens evolved. But it will be medically important, too. It may even help erode that black-and-white excuse for racism.

Genetic diversity brings with it diversity of genetic disease. Cystic fibrosisin any case rarer in Africa than in Europeis often caused there by a different mutation from the one involved in the European version, and is thus missed by tests developed in the West. A mutation responsible in Ghana for 40% of inherited deafness is unknown in South Africa. And so on. It also brings a diversity of genetic response to disease. Some of the molecular details of the immune system, for example, vary with geography. Understanding that variation in Africa will improve understanding of immunity to infection, helping Africans and non-Africans alike.

More genetic information will also cast light on evolution. Early H. sapiens migrants from Africa encountered other species of human being on their travels. These were descendants of previous migrations out of Africa of archaic members of the genus. At least two of these other types of human, the Neanderthals and the Denisovans, interbred with the newcomers, and some of their genes are still found in modern Asians and Europeans, doing various jobs including protecting them from disease. Preliminary analysis suggests that those who remained behind in Africa similarly interbred with yet another species of humanbut one of which no fossil record remains.

There is an irony in all this. Xenophobia has probably existed for as long as people have. But racist attitudes were reinforced in the 19th century by an enthusiasm for physical anthropology and eugenics. The former attempted to classify human beings on the basis of visible characteristics, such as skin colour, head shape and facial features, that are genetically inherited. If this had been a neutral analysis, it would have been unexceptional. But often it was not neutral. It not only classified, but ranked. White-skinned Europeans put themselves at the topand black-skinned Africans at the bottom. Add eugenics to that mix and the result was toxic.

The 3MAG project will not, alone, overthrow the legacy of these misadventures and the prejudices they reinforced. The thinking that gave rise to them is still too deeply ingrained in too many minds for it to do that by itselfeven, probably, for it to come close. But to those whose minds are open, a group of 21st-century African scientists revealing that the true, glorious genetic diversity of human beings lies in their own continent more abundantly than in any other will be a superb rebuttal to the doctrines of those misguided Victorian European gentlemen.

This article appeared in the Leaders section of the print edition under the headline "Know thyself"

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Why the African genome project is so useful - The Economist

28 June 2021

The UK Government should review regulation of direct-to-consumer genetic and genomictests, according to a report by the House of Commons Science and Technology Committee.

The 'Direct-to-consumer genomic testing' report outlined the possible risks posed by the increasing availability and scope of consumer genetic and genomic testing. Such tests have become increasingly popular in recent years by2020 at-home testing company 23andMe had sold over 250,000 genetic testing kits in the UK, and as the cost of whole genome sequencing comes down more genomic tests are likely to be marketed. However, there are concerns that medical information gleaned from such tests can be misleading (see BioNews 1020).

'We recommend that the Government should require direct-to-consumer tests to be subject to greater pre-market assessment by an external body' said the report. 'We suggest that any such external assessment should cover the test's clinical performance (the extent to which a test can provide information about diagnosis, treatment, management or prevention of disease that will lead to an improved outcome), as well as its analytical performance (how well a test predicts the presence or absence of a particular gene or genetic change)'.

Further recommendations included the development of technical standards to allow such results to be integrated with NHS data, This could reduce the burden on the NHS from having to re-test individuals following consumer tests, but also allow commercial test results to support research. There are also recommendations around limiting some test types to professional use, and banning tests that would fall outside clinical guidelines such as testing children for late-onset conditions.

The committee also specifically addressed that the Government must improve the UK's data protection framework for genomic testing, including implementing more effective data safeguards, given the risks and opportunities presented by technological developments and growing numbers of direct-to-consumertests.

'This is timely, and an innovative attempt to regulate a market that has grown primarily for commercial purposes to date,' said Moin Saleem, Professor of Paediatric Renal Medicine at the University of Bristol. 'In the context of the public having growing access to individual genetic information, and therefore deeply personal data, it is absolutely necessary.'

'We welcome this report, and hope that it will stimulate wider public discussion of direct-to-consumer genetic and genomic tests,' said Sarah Norcross, director of the Progress Educational Trust (the charity that publishes BioNews). 'Government would be well advised to pay attention to this area if the UK is to maintain its position as a leader in genomics. Top-tier science and technology require top-tier regulation.'

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Policymakers advised to address problems with direct-to-consumer genomic testing - BioNews

Why is genome sequencing so important?

Sequencing provides proof how much the sample under study has changed from the original Wuhan virus strain, and whether mutation at a particular gene will result in higher infectivity. That informs policy, for example, whether or not to lock down, and it is also vital for designing more effective vaccines.

What is global best practice on sequencing?

The best strategy is to sequence 5% of all Covid-positive samples. Britain leads the race here. The Covid-19 Genomics UK Consortium website says 5,69,877 viruses have been sequenced so far and its daily graphs clearly capture the emergence of the Delta (B.1.617.2) variant as the most dominant one. Other developed countries such as the US and Australia too have sequenced 5% or more of the positive samples.

How does India fare?

The Indian Covid-19 Genome Surveillance, maintained by the CSIR Institute of Genomics & Integrative Biology (IGIB) in Delhi, says that until June 21, 30,483 genomes were sequenced mainly for INSACOG Covid surveillance project and the Kerala governments GENESCoV2 project. This is a tiny fraction of the 3-crore Covid cases detected in India since March 2020. To be fair to scientists involved, India began its surveillance in earnest from December 2020.

IGIB director Anurag Agrawal says the 5% rule hasnt been INSACOGs plan for quite a while. He said the idea is to carry out fixed sampling by time and geography plus strategic deeper sampling when needed. He has often been quoted as promoting smart sequencing.

What are the shortcomings in Indias approach?

Some in the scientific community fear positive samples are picked up randomly for sequencing, instead of undertaking scientific sampling. Mumbai-based Foundation for Medical Researchs Nerges Mistry, credited with pioneering work in TB genomics, advocates cluster-based testing: If there are a number of cases in a small geographic area say, a school or a residential building then the need is to pick up 80% of all the positive samples from this area for sequencing to find out if a variant is at work.

Another concern is that there is no special focus on the most vulnerable subgroup of patients those who contract Covid while under treatment for major diseases. Experts say such patients would be the best reservoir for the SARS-CoV-2 virus to linger longer and start mutations to enhance its survival.

Plus, there has been widespread criticism from the scientific community on the failure to rope in the private sector to speed up the sequencing project.

What holds us back? Funding? Infrastructure?

Funding is a problem. When the INSACOG consortium of 10 elite research labs was set up around six months ago to do Covid sequencing, the Centre earmarked Rs 115 crore for a six-month period. The allocation did not take place and the Department of Biotechnology was asked to fund the consortium from its own resources.

Genome sequencing is expensive. In the private sector, genomic tests to check for disorders (including for lifestyle diseases and cancer) run into tens of thousands of rupees. Newer technologies that allow huge numbers of samples to be sequenced at once could bring down the costs to under Rs 5,000, but such upgrades havent yet taken place. Recurring costs of reagents (some of them have to be imported) are also a concern.

The second problem is capacity. While the IGIB has the capacity to do 10,000 sequences a month, there is no clarity on the capacity of other labs. Sequences are relatively speaking simple to do, but given Indias size and huge volume of patients, scientists believe the private sector should be roped in for the purpose.

Logistics are also a nightmare. How quickly can samples from rural areas be transferred to sequencing labs is an issue.

What are the fastest ways to improve Indias efforts?

One solution will be to rope in other labs with the technical knowhow to sequence more. Another suggestion is to create linkages: Smaller local labs can start off the process and transport a stabler sample to bigger labs for the final workup and diagnosis.

Then there is the option of starting wastewater monitoring, which is cheaper than individual sequencing, at least in high-load cities such as Mumbai and Delhi. IIT Gandhinagar recently showcased its wastewater surveillance for Covid through the detection of the genetic material of SARS-CoV-2.

Views expressed above are the author's own.

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Detecting Covid Variants: Sequence of Missteps: Quick genome sequencing is vital. Why are we so bad at it, and - The Times of India Blog

NEW YORK Eli Lilly this week said that Health Canada has issued a "notice of compliance with conditions" for selpercatinib (Retevmo) in three molecularly defined lung and thyroid cancer indications. The health regulator granted conditional marketing authorization for selpercatinib as a treatment for RET fusion-positive, adult non-small cell lung cancer patients; for RET-mutant, metastatic or unresectable medullary thyroid cancer patients who are at least 12 years old; and for RET fusion-positive, metastatic thyroid cancer in adults who have received sorafenib (Bayers Nexavar) or lenvatinib (Eisais Lenvima) and stopped responding to radioactive iodine therapy.

Health Canada relied on data from the LIBRETTO-001 Phase I/II trial, which the US Food and Drug Administration also used to approve the drug for these indications last year. The conditional authorization pathway in Canada facilitates expedited access to drugs for life-threatening illnesses based on early data from clinical trials, but sponsors must submit confirmatory safety and efficacy evidence to maintain access. Lilly is conducting two Phase III trials to establish the clinical activity of selpercatinib.

The US Food and Drug Administration this week granted breakthrough therapy designation to Mirati Therapeutics' investigational KRAS inhibitoradagrasib for patients with previously treated non-small cell lung cancer whose tumors harbor KRAS G12C mutations. The FDA's designation is based on preliminary results of Mirati's ongoing multi-cohort Phase I/IIKRYSTAL-01 trial, which is evaluatingadagrasibas a treatment for a variety of KRAS G12C-mutated advanced cancers in addition to NSCLC. According to a statement from Mirati CEO Charles Baum, the firm plans to submit a new drug application foradagrasibduring the second half of this year.

Legend Biotech said this week that it will establish a cell therapy manufacturing facility in Belgium. The new facility, where Legend will manufacture ciltacabtagene autoleucel (Janssen's cilta-cel), is part of a collaboration and licensing agreement between Legend and Janssen focused on developing and commercializing the anti-BCMA CAR T-cell therapy. Regulatory agencies in the US and Europe, among other countries, are currently reviewing cilta-cel as a treatment for relapsed or refractory multiple myeloma. Legend, which has previously established cilta-cel manufacturing facilities in China and New Jersey, expects the Belgium facility to be operational by 2023.

The Access to Comprehensive Genomic Profiling Coalition this week added Personal Genome Diagnostics and Sema4 to its coalition of diagnostics companies, laboratory service providers, and comprehensive genomic profiling industry stakeholders to advocate for broad US health insurance coverage of CGP for patients living with advanced cancer.

In Brief This Week is a selection of news items that may be of interest to our readers but had not previously appearedinPrecision Oncology News.

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In Brief This Week: Eli Lilly, Mirati Therapeutics, Legend Biotech, Personal Genome Diagnostics, Sema4 - Precision Oncology News

Abstract

The Qinghai-Tibet Plateau endemic Chinese mountain cat has a controversial taxonomic status, whether it is a true species or a wildcat (Felis silvestris) subspecies and whether it has contributed to cat (F. s. catus) domestication in East Asia. Here, we sampled F. silvestris lineages across China and sequenced 51 nuclear genomes, 55 mitogenomes, and multilocus regions from 270 modern or museum specimens. Genome-wide analyses classified the Chinese mountain cat as a wildcat conspecific F. s. bieti, which was not involved in cat domestication of China, thus supporting a single domestication origin arising from the African wildcat (F. s. lybica). A complex hybridization scenario including ancient introgression from the Asiatic wildcat (F. s. ornata) to F. s. bieti, and contemporary gene flow between F. s. bieti and sympatric domestic cats that are likely recent Plateau arrivals, raises the prospect of disrupted wildcat genetic integrity, an issue with profound conservation implications.

The domestic cat (Felis catus or Felis silvestris catus), one of the most popular pets today, has an estimated worldwide population of over 600 million, including probably more than 100 million free-ranging feral cats (1). The origin and history of cat domestication have attracted wide public attention, as well as scientific interest (2). The first genetic study of the origin of domestic cats, based on a mitochondrial and nuclear DNA assessment of nearly 1000 specimens of domestic cats and their wildcat progenitors, Felis silvestris, revealed a single domestication event from the African wildcat (F. s. lybica) in the Near East (3). Ancient cat domestication coincided with the rise of both early agriculture and civilization in the Fertile Crescent and subsequently expanded across the world. Ancient DNA analysis of archeological cat remains reinforced this conclusion by showing that African wildcats from both the Near East and Egypt contributed to the modern domestic cats gene pool at different historical times (4). Nevertheless, uncertainty remains as to whether multiple, independent cat domestication centers might exist, particularly given the lack of sampling in previous studies from East Asia.

The wildcat, F. silvestris, from which domestic cats arose, is widely distributed in the Old World and classified by controversial taxonomic systems, ranging from a monotypic taxon with multiple lineages to a species complex comprising at least two species (5). According to the most recent genetic study of wildcat samples collected worldwide (3), F. silvestris is resolved as a polytypic wild species including five distinct interfertile subspecies: F. s. silvestris, the European wildcat; F. s. lybica from the Near East and northern Africa; F. s. cafra from southern Africa; F. s. ornata, the Asiatic wildcat from central Asia east of the Caspian Sea; and F. s. bieti, the Chinese mountain cat endemic to the Qinghai-Tibet Plateau. However, the Felidae taxonomy by Kitchener et al. (5) merged F. s. cafra, F. s. lybica, and F. s. ornata into F. lybica to unify wildcats from Africa to central Asia, while maintaining F. silvestris in Europe and F. bieti in China their own species statuses.

Two wildcat taxa, the Chinese mountain cat and Asiatic wildcat, are found in China. The Asiatic wildcat (F. s. ornata) occurs from the eastern Caspian Sea north to Kazakhstan to western India, western China, and southern Mongolia. Its spotted coat pattern distinguishes it from other, usually striped, wildcat lineages. The Chinese mountain cat (F. s. bieti), also known as the Chinese desert cat or Chinese steppe cat, was first described as an independent species, F. bieti, in 1892 (6). With a restricted distribution on the Qinghai-Tibet Plateau, it is the only wild felid endemic to China and is characterized by a sand-colored fur with faint dark stripes, a thick tail, ear tufts, and light blue pupils (7). Molecular genetic studies suggested a reconsideration of the Chinese mountain cat as a conspecific of the wildcat based on its close association with other wildcat subspecies (3, 8), but this taxonomic revision has not been unanimously accepted. Arguing against it, Kitchener et al. (5, 9) wrote, F. bieti is morphologically distinct and is supposedly sympatric with F. l. ornata, which would also preclude its recognition as a subspecies of F. silvestris/lybica. However, the presumed reproductive isolation between the Chinese mountain cat and Asiatic wildcat was based on their morphological divergence and possible overlapping distribution, either of which might not hold true given the two taxas poorly defined ranges and possible misidentification or mislabeling of specimens in previous studies (7, 9).

Recent advances in genomic studies of exotic species have demonstrated that hybridization between closely related taxa is common in nature and is important in shaping the genomes of modern animals (1012). Intertaxa hybridization has also been documented in various Felidae lineages, such as the big cats (genus Panthera) and neotropical small cats (genus Leopardus) (8, 13, 14). In Northwest China, observations of cats possibly derived from interbreeding between Chinese mountain cats and domestic cats are occasionally reported, leading to the postulation that local wildcats may have contributed to the gene pools of domestic cats in China. As one of the worlds oldest civilization centers, China has been involved in or has given rise to numerous domesticated animal varieties, including those of the dog and the pig (15, 16). Also, the earliest evidence of a commensal relationship between human and cat, in this case, the Asian leopard cat (Prionailurus bengalensis), was unearthed from a Neolithic site in Northwest China (17, 18), casting light on the existence of an environment conducive to a human-cat commensal process at that time in the East Asia.

On the other hand, genetic introgression from domestic species into their wild congeners has been documented in many taxa and, by introducing deleterious traits, it could threaten those wild populations by compromising their fitness in the wild (19, 20). In some regions of Europe, the anthropogenic spread of domestic cats has caused the expansion of feral cats range and the subsequent hybridization with the European wildcats (2123). Such widespread genetic infiltration from F. s. catus into F. s. silvestris is a substantial threat to the survival, distinctiveness, and genetic integrity of those sympatric European wildcat populations. On the Qinghai-Tibet Plateau where the Chinese mountain cat is endemic, most local domestic cats are free ranging, whose effect on wild conspecifics is a concern. However, the circumstance and the extent of genetic admixture between those two remain unknown, let alone its potential conservation impacts on local wildlife.

To resolve the phylogeny of one of the least studied felids in the world and to elucidate the evolutionary dynamics of the wildcats and domestic cats in East Asia, we assembled thus far the most comprehensive set of samples of the Chinese mountain cat over its entire range in the Tibetan region, the Asiatic wildcat from Xinjiang, and domestic cats across China, especially from those regions sympatric, parapatric, and allopatric with the Chinese mountain cat. Our data from both whole-genome sequencing (WGS) and uniparental mitochondrial DNA (mtDNA) and Y chromosome haplotype sequencing jointly showed that the Chinese mountain cat F. s. bieti and Asiatic wildcat F. s. ornata are equidistant and conspecific within the wildcat (F. silvestris). We also revealed an ancient introgression between F. s. bieti and F. s. ornata and a complex pattern of contemporary gene flow from F. s. bieti into domestic cats across, but not beyond, its range. Last, Chinas domestic cats share a Near Eastern origin with worldwide domestic cats, thus suggesting a single, not multiple, domestication event of cats arising from the African wildcat (F. s. lybica).

We sampled from a wide distribution of domestic cats in China and two of its wildcat congeners in Northwest China. From domestic cats, we collected blood, tissue, or saliva samples from 239 outbreed, unrelated individuals from 23 sites throughout China, including three locations within, three on the periphery of, and 17 distant from the Chinese mountain cat core distribution (Table 1). The wildcat collection included four Asiatic wildcats F. s. ornata from Xinjiang and 27 Chinese mountain cats F. s. bieti across their full range on the Qinghai-Tibet Plateau spanning Qinghai, Gansu, and Sichuan Provinces. Twelve of the 27 F. s. bieti were tissues or blood from road kills or zoo animals, and 15 were pelts or bones from museums or local villages (Fig. 1A and data file S1).

MtDNA, mitochondrial DNA.

(A) Sampling localities and range of wildcats and domestic cats in China. Labels at each sampling site indicate the numbers of individuals in each of the following categories (separated by slashes): total sample size, with that of F. s. catus proportional to the circle area; samples with mtDNA (Mt) fragment data; samples with Y chromosome (Ychr) fragment data; and samples with WGS (autosome) data. Individual animal codes are in parentheses. Morphologically confirmed F. s. bieti individuals with F. s. ornata mtDNA haplotype and F. s. catus individuals with F. s. bieti Ychr haplotype are underlined in red and blue, respectively. The lower left panel summarizes sample sizes from each taxon and their population genetic backgrounds based on mtDNA, Ychr, and autosomal single-nucleotide variants (SNVs). Sympatry refers to domestic cats either from or not from the F. s. bieti range and gray, blue, and red correspond to the F. s. catus, F. s. bieti, and F. s. ornata clades, respectively. (B) Morphology of representative individuals from a purebred F. s. bieti (left), an F. s. bieti with an F. s. ornata mtDNA haplotype (middle), and an F. s. catus with an F. s. bieti Ychr haplotype (right). Photo credit: Shu-Jin Luo and Hao Meng, Peking University.

A multilocus screening, based on partial mtDNA and Y chromosome sequencing, was performed in all samples (270 specimens for mtDNA and 103 for Y chromosome analysis) for an initial understanding of the genetic diversity patterns in the wildcat and domestic cat populations. A statistical parsimony network based on both markers revealed three distinct clusters that corresponded to the domestic cat F. s. catus, the Asiatic wildcat F. s. ornata, and the Chinese mountain cat F. s. bieti (Fig. 2A). Beginning with modern samples (N = 255), we amplified a 2620-bp (base pair) mtDNA fragment spanning ND5, ND6, and CytB to distinguish 106 variable sites and 52 unique mtDNA haplotypes in 250 samples, including five haplotypes from 12 Chinese mountain cats, three from four Asiatic wildcats, and 44 from 234 domestic cats (data file S2). One-third (4 of 12) of the modern Chinese mountain cat specimens carried two mtDNA haplotypes that aligned with those of Asiatic wildcats, while the other eight individuals had three haplotypes exclusively found in the F. s. bieti clade. For degraded DNA extracted from museum samples (N = 15), we separately amplified four short fragments within the 2.6-kb mtDNA haplotype and concatenated them into 400 to 1000base pair (bp) sequences (table S1). This yielded a similar proportion of individuals with the admixed genetic background, as 3 of the 11 succeeded Chinese mountain cat museum specimens were different from the rest and contained the Asiatic wildcat diagnostic variants (data file S2).

(A) Statistical parsimony networks of Felis silvestris and F. s. catus based on mtDNA and Ychr fragments. The larger a haplotypes circle, the more individuals share that haplotype. Colors represent the morphology-based taxonomic classifications of the animals (red, F. s. ornata; blue, F. s. bieti; gray, F. s. catus). (B) Bayesian phylogenies of Felis spp. based on the mitochondrial genome (excluding the control region) and the Ychr single-copy region. The branches are color-coded to coordinate with the morphology-based taxonomic classification of the taxon with the same-colored name. The shaded boxes are color-coded to correspond to the genetic affiliations of the three clades of interest in this study. Asterisks mark individuals with morphological appearances that disagree with their genetic affiliation based on certain genetic markers. (C) Phylogeny of Felis spp. based on genome-wide autosomal neutral SNVs reconstructed using the neighbor-joining method and a distance matrix calculated following Gronaus method, with the bootstrap support values marked on major nodes. The branches are color-coded as in (B) and the asterisks on certain branches correspond to the same individuals marked in (B).

We assembled two Y chromosome fragments from DBY7 and SMCY7 from 103 male cats and, based on six indels and single-nucleotide variants (SNVs) from the concatenated 1015-bp sequences, found three distinctive Y haplotypes from 91 succeeded samples, each representing one of the three Felis taxa in the study (table S1 and data file S3). The Y chromosome haplotype network revealed patrilineal introgression between wildcats and domestic cats. Three domestic cats, from Qinghai and Sichuan, which are within the Chinese mountain cat F. s. bieti core range, shared the signature F. s. bieti Y haplotype, and one Asiatic wildcat F. s. ornata showed a Y chromosome haplotype typical for domestic cats F. s. catus (Fig. 2A).

On the basis of adequate DNA quality, we selected 55 representative samples (8 Chinese mountain cats, 1 Asiatic wildcat, and 46 domestic cats) for Illumina paired-end sequencing, subsequently generating mitogenome and WGS data for 51 of these samples (including four Chinese mountain cats F. s. bieti, B1, B2, B3, and B4) at 6.8 to 15.1 times coverage per individual. Only the mitochondrial genome was reconstructed for the other four Chinese mountain cats (B5, B6, B7, and B8) because of sample quality constraints (data file S4). Raw sequencing reads from 20 domestic cats and two black-footed cats (Felis nigripes) were downloaded from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (NCBI, Bethesda, MD). Our final dataset of 73 nuclear genomes had an average of 16 coverage and 20,425,451 biallelic autosomal single-nucleotide variants (SNVs) after quality filtering and masking. We also retrieved 66 different mitogenomes from 77 individuals, including six from eight F. s. bieti specimens (B1, B2/B5, B3/B8, B4, B6, and B7), and combined them with seven published genomes of Felis genus (one from F. s. bieti) (8) for downstream analyses.

Phylogenies reconstructed from mitogenome, Y chromosome, and the autosomal neutral region illuminated the evolution and taxonomy of the Chinese mountain cat in relation to other wildcats and domestic cats. Phylogenetic inference based on mitochondrial sequences, excluding the control region, clusters all taxaincluding the Asiatic wildcat (F. s. ornata), European wildcat (F. s. silvestris), Chinese mountain cat (F. s. bieti), and a haplogroup containing all domestic cats (F. s. catus) and African wildcats (F. s. lybica)into a single F. silvestris clade, with F. s. ornata situated as the basal lineage within the clade, although support of the node was not strong (Fig. 2B and fig. S1).

The patrilineal genealogy of the 929-kb Y chromosome single-copy region assembled from all available Felis spp. sequencing data also clustered F. s. catus/F. s. lybica, F. s. bieti, and F. s. ornata into one monophyletic group that is distinct from outgroup F. nigripes, despite an unresolved internal phylogeny among the three (Fig. 2B). In the neighbor-joining tree based on autosomal SNVs and average genomic divergence matrices (24), F. s. bieti (B1 to B4) and F. s. ornata (O1) formed a clade that diverged early before the domestic cat radiation (Fig. 2C). Notably, all phylogenomic inferences placed the Chinese mountain cat within the F. silvestris subspecies clade, distinguishing it from other congeneric outgroup species: the black-footed cat (F. nigripes), the sand cat (F. margarita), and the jungle cat (F. chaus). These genome-wide phylogenetic patterns provided robust evidence for a close association of the Chinese mountain cat with the other F. silvestris taxa and corroborated the previously suggested reclassification of F. bieti as a subspecies of F. silvestris (3, 8).

The phylogenies based on mtDNA or Y chromosome data showed that domestic cats from China were indistinguishable from those of other regions of the world, thus supporting a single domestication event for all domestic cats arising from the African wildcat (F. s. lybica). Nevertheless, all domestic cats from East Asia, including those from China and one from South Korea (W9; see data file S4), formed a monophyletic group in the autosomal phylogeny, thus indicating a recent association among East Asian domestic cats (Fig. 2C).

The discordant phylogenies inferred from maternal, paternal, and biparental genetic markers likely resulted from incomplete lineage sorting and/or hybridization among lineages (8). Consistent with the patterns from partial mtDNA and Y chromosome genealogies, mitogenomes from three voucher Chinese mountain cats F. s. bieti (B4, B6, and B7; Fig. 1) clustered within the Asiatic wildcat F. s. ornata, and two domestic cats (C8 and C12; Fig. 1) carried F. s. bieti signature Y chromosome haplotypes (Fig. 2B). Genome-wide autosomal phylogeny (Fig. 2C) illustrated robust monophyly of individuals from F. s. catus, F. s. bieti, and F. s. ornata, with no apparent interlineage genetic admixture (thus excluding errors of morphological misidentification). In addition, both Bayesian coalescence analyses based on mitogenome and Y chromosome sequences estimated the time to the most recent common ancestor of F. s. catus, F. s. bieti, and F. s. ornata at around 1.5 million years (Ma) ago during the Middle Pleistocene (fig. S1), consistent with estimations from earlier studies (8, 25). Such a relatively rapid and recent divergence of these lineages may have led to the phylogenetic discordance observed in different genealogies.

Principal components analysis (PCA) of autosomal neutral SNVs detected strong signal partitioning among the three F. silvestris clades (Fig. 3A). The first PC (PC1), which maximized 36% of the variance, distinguished black-footed cats from the other F. silvestris taxa, thus indicating a species-level divergence. The Chinese mountain cat, Asiatic wildcat, and domestic cat were separated along PC2, which explained 10% of the variance and suggested a subspecies-level divergence. PC3 revealed the intraclade genomic diversity within domestic cats that segregated Chinese domestic cats from other, worldwide cat populations. Also, alternative pairwise population genetic difference estimates also revealed a similar hierarchical variance partitioning among the five groups (table S2), with the FST between F. nigripes and the other four groups larger than 0.7, the FST between F. s. bieti, F. s. ornata, and F. s. catus markedly lower (0.3 to 0.7), and the FST between Chinese and worldwide domestic cat populations as low as 0.1.

(A) Principal components analysis (PCA) of 73 individuals showing only the first three PCs. Two black-footed cats (F. nigripes) were separated from the others with the first PC. The two wildcats, F. s. bieti and F. s. ornata, and F. s. catus (domestic cats) were separated with the second PC. F. s. catus individuals were partitioned along the third PC, with a moderate differentiation between domestic cats from China and those worldwide. (B) Population structure of 72 domestic and wildcat individuals estimated in ADMIXTURE with K = 4. The four clusters correspond to F. nigripes, F. s. bieti, F. s. catus from China, and F. s. catus worldwide, with 10 F. s. catus (C6 to C15) carrying about 10% genetic admixture from F. s. bieti. (C) Genomic admixture between F. s. bieti and F. s. catus from China (C1 to C46) estimated by D statistics, the f4 ratio test, percentage of F. s. bieti diagnostic sites in the genome of F. s. catus, and f statistics. The D statistics results are summarized as boxplots showing the z-scores distribution of each F. s. catus and a significance level of z > 2 (red dotted line). Plotted percentages of diagnostic sites, f4 ratio, and f statistics reveal the genomic admixture levels from F. s. bieti to each F. s. catus in the dataset.

The ADMIXTURE Bayesian analysis of autosomal neutral SNVs clustered 72 cats into four groups whose primary genomic affiliations correlated with black-footed cats, Chinese mountain cats, Chinese domestic cats, or worldwide domestic cats (Fig. 3B and fig. S2) (F. s. ornata was excluded due to its limited sample size). Notably, domestic cats from the Chinese mountain cats core range in Sichuan and Qinghai (N = 10, C6 to C15 in Fig. 1A) carried about 10% genomic ancestry from F. s. bieti, indicating an extensive introgression from Chinese mountain cats to their sympatric domestic cats.

D statistics further assessed the extent of genetic admixture between Chinese mountain cats and Chinas domestic cats while using the worldwide domestic cat data as a baseline. We quantified the level of wildcat genetic introgression in each domestic cat by determining the fraction of diagnostic sites, f statistics, and f4 ratio test results (Fig. 3C and table S3). All 10 domestic cats sympatric with Chinese mountain cats displayed significant admixture signals in D statistics, with the average z score ranging from 6.9 to 11.4, and the fraction of introgression between 4 and 12%, a result consistent with the estimated ancestry proportion in population clustering analysis (Fig. 3B). We also detected introgression signals in five domestic cats (C1 to C5) collected from northern Qinghai and Gansu, a region peripheral to the Chinese mountain cats range (see Fig. 1A). The exact proportion of F. s. bieti introgression in those individuals varied between 0.5 and 7% depending on the analysis method, but nevertheless significantly higher than that of domestic cats not from the Chinese mountain cats geographic range (Figs. 1A and 3C and table S3). Overall, genetic introgression from Chinese mountain cats to domestic cats is restricted to the sympatric area in Qinghai, Sichuan, and Gansu, and the proportion of wildcat admixture in domestic cats decreases with increasing distance away from the Chinese mountain cats core distribution. Notably, because none of the domestic cats with F. s. bieti genetic introgression appeared morphologically different from other domestic cats, morphological characters are likely not reliable diagnostic markers to identify hybrids.

We further examined the fine-scale distribution of the putative introgression regions within the genomes of the admixed domestic cats (C6 to C15) to determine whether the observed introgression in the sympatric domestic cats was introduced recently or was from ancient signals preserved in the population. On the basis of diagnostic SNVs, large continuous genomic segments (each more than 30 Mb) with Chinese mountain cat ancestry were identified in 6 of the 10 domestic cats (fig. S3). This indicated possible recent hybridization events between Chinese mountain cats and domestic cats because long-range linkage disequilibrium (LD) across the wildcat chromosomes had not been completely disrupted by recombination. Individual C8, a domestic cat from eastern Qinghai that carried a Chinese mountain cat-like Y chromosome haplotype (Fig. 1B), displayed an extended, more than 30 Mb, homozygous region with both alleles from the Chinese mountain cat, a pattern consistent with a recent hybridization event that was reinforced by possible further interbreeding between the fertile hybrid offspring.

We dated the unidirectional introgression from the Chinese mountain cat F. s. bieti to its sympatric domestic cat population in the Tibetan area based on the extent of LD decay computed in ALDER (26). Domestic cats C6 to C15 from the core F. s. bieti range (namely, hybrid1) and C1 to C5 from the F. s. bieti distribution periphery (namely, hybrid2) were referred to as two admixed populations. The genomic introgression in the hybrid1 population was well supported (P = 8.30 1016), with an exponential fit starting at 2 centimorgan (cM) (table S4), and was estimated to have occurred about 7.42 generations earlier (Fig. 4A). Using a generation time of 2 years for the domestic cat, hybridization between Chinese mountain cats and domestic cats on the Qinghai-Tibet Plateau occurred about 15 years ago. We also detected a significant admixture signal (P = 8.90 105) in the hybrid2 population, estimated to be about 30.72 generations or about 62 years ago (Fig. 4B).

Weighted LD curves with putative pure Chinese domestic cats and Chinese mountain cats as two reference populations for the (A) hybrid1 population with an exponential fit starting at 2.0 cM and (B) hybrid2 population with an exponential fit starting at 0.5 cM.

We used the pairwise sequential Markovian coalescent (PSMC) model to understand the demographic histories, dispersals, and divergences of the wildcat and domestic cat clades within China (Fig. 5A). Both the Chinese mountain cat F. s. bieti and the Asiatic wildcat F. s. ornata displayed a moderate population expansion 1 to 2 Ma ago, followed by a constant, gradual decline. The effective population size (Ne) of the African wildcat (F. s. lybica), as represented by the genomic diversity of the domestic cat (F. s. catus), experienced a drastic rise around 100 to 400 (Ka) ago during the Middle to Late Pleistocene, which may reflect an ancient range expansion and/or population growth.

(A) Population dynamics estimated in a PSMC model of five individuals representing F. nigripes, F. s. bieti, F. s. ornata, F. s. catus from China, and F. s. catus worldwide, with 100 bootstrap replicates. The results from F. s. catus individuals represent the dynamics of their wild ancestor, F. s. lybica. g, average generation time; , mutation rate. (B) Demographic model with divergence and migration among F. nigripes, F. s. bieti, F. s. ornata, and F. s. catus, with the effective population sizes (in thousands), divergence times (in million years), and total migration rates estimated by the Generalized Phylogenetic Coalescent Sampler. Within the graph, the values and ranges outside and within the parentheses represent the average and combined 95% Bayesian credible intervals, respectively, of each estimated parameter, and the branch widths are proportional to effective population sizes.

We used the coalescent-based Generalized Phylogenetic Coalescent Sampler (G-PhoCS) to estimate the population divergence times and migration scenarios among F. s. bieti, F. s. ornata, and F. s. catus/F. s. lybica using F. nigripes as the outgroup for time calibration (Fig. 5B). Using a given topology based on the autosomal phylogeny (Fig. 2C), we performed 12 independent analyses using all the combinations between one of the three domestic cats (C20, C25, and W19) and one of the four Chinese mountain cats (B1 to B4; fig. S4 and table S5). The coalescent time of F. s. catus and F. s. bieti/F. s. ornata lineages was estimated to be around 1.87 Ma ago [95% highest posterior density (HPD) at 1.76 to 1.97 Ma ago], and then F. s. bieti and F. s. ornata coalesced around 1.27 Ma ago (95% HPD at 1.19 to 1.37 Ma ago). We detected four significant interlineage migration bands, indicating the presence of gene flow from F. s. catus to F. s. ornata and from F. s. ornata to F. s. bieti, with a total migration rate of about 0.1, and from the F. s. bieti/F. s. ornata lineage to F. s. catus, with a total migration rate of about 1.5 (Fig. 5B). The total migration rate from F. s. catus to F. s. bieti was minor and varied among different analyses from 0 to 0.09, with only one analysis that included B2 showing a significant level of gene flow. This observation confirmed that the hybridization between domestic cats and Chinese mountain cats was recent, and hence, the extent of genetic influence in F. s. bieti varied by individual. The effective population sizes estimated by G-PhoCS and PSMC were well correlated, with the Ne of the ancestor of F. s. catus/F. s. bieti/F. s. ornata lineages around 157,000, the Ne of the common ancestor of F. s. bieti and F. s. ornata lineages increasing to about 269,000, and the current population sizes of F. s. bieti and F. s. ornata shrinking to about 20,000 and 23,000, respectively.

Using WGS data generated from 46 domestic cats sampled across China, we found that both phylogenomic and population structure analyses clustered domestic cats from China and worldwide into one panmictic group (Figs. 2 and 3A and table S2), thus supporting the single-origin scenario of all domestic cats being derived from the Near Eastern wildcat (3, 4). However, autosomal phylogeny (Fig. 2C) and ADMIXTURE (Fig. 3B) also revealed a close genetic association among domestic cats from China and South Korea that distinguished them from other populations in the world. This pattern implies a certain degree of isolation of domestic cats in East Asia after they dispersed to or were introduced into this region. Although our sampling of domestic cats in China specifically targeted local cats and avoided cat breeds originally from regions outside China, we also detected genetic introgression from the worldwide cat population in several individuals from southeastern China, which could be due to recent genetic interactions with introduced cats from other countries into the gene pool of local feral cats.

There is no statistical evidence suggesting a significant contribution from local wildcats into the genetic ancestry of modern domestic cats in East Asia, yet a complex hybridization scenario among domestic cat and wildcat lineages in the area was revealed. Genomic analyses indicate ancient admixture events between the Chinese mountain cat and the Asiatic wildcat, introgression from the domestic cat to the Asiatic wildcat (Figs. 2A and 5B), and recent genetic interaction between the Chinese mountain cat and its sympatric domestic counterparts (Figs. 2 and 3) on the Qinghai-Tibet Plateau.

First, an ancient, unidirectional introgression from the Asiatic wildcat F. s. ornata to the Chinese mountain cat F. s. bieti was evident, as we observed only the misplacement of F. s. ornata mtDNA haplotype in F. s. bieti but not vice versa (Fig. 2), and only the migration band from F. s. ornata to F. s. bieti was significant in the G-PhoCS analysis (Fig. 5B). The signal of F. s. ornata admixture in F. s. bieti was apparent only in the maternally inherited mitochondrial lineages, while a genome-wide autosomal phylogeny and clustering algorithm supported monophyly for all morphologically distinguishable F. s. bieti. This cytonuclear discrepancy is consistent with an ancient admixture scenario in which a female F. s. ornata mated with a male F. s. bieti, and then their offspring backcrossed with F. s. bieti for a long period of time. Such asymmetric hybridization has been reported in various mammalian lineagesincluding Neotropical wild cats (Leopardus spp.), canids in North America, and African savannah and forest elephants (13, 27, 28)and was generally associated with population size contrasts and mating preferences when two lineages met (29). Likewise, the asymmetric introgression between the two wildcat lineages in Asia could be explained by the larger body size of F. s. bieti relative to F. s. ornata. Perhaps, larger males were preferred by female F. s. ornata, thus giving male F. s. bieti a mating advantage. When the ancient F. s. bieti population overlapped with the range of F. s. ornata, whose population size was supposedly large, such admixture could have occurred, leaving the signal in the contemporary F. s. bieti genome.

Another unidirectional F. s. lybica/F. s. catus to F. s. ornata introgression was revealed via Y chromosome genealogy and G-PhoCS analysis (Figs. 2A and 5B). That interlineage gene flow could also be explained by the difference in the population sizes of ancient African and Asiatic wildcat populations (Fig. 5A) and/or the dispersal of postdomestication F. s. catus into Central Asia during the last millenniums. However, we were unable to investigate further because of both our small sample size and the uncertainty regarding the exact geographic origin of the specimens. Further study may reveal whether the introgression occurred between the historical African and Asiatic wildcats before cat domestication or between free-ranging domestic cats and Asiatic wildcats, a scenario resembling the genetic infiltration of feral domestic cats into the native European wildcat population in Scotland (30).

Because the Chinese mountain cat is the only wildcat endemic to the Qinghai-Tibet Plateau of China, its genetic integrity has been a subject of scientific interest and conservation concern. Genomic introgression from the Chinese mountain cat to its sympatric domestic cats was widespread (Fig. 4) and was estimated to be a contemporary, not an ancient, event. We observed a gradual decrease of F. s. bieti genetic contribution in domestic cats and older hybridization incidences as we progressed from the center (e.g., Aba in western Sichuan and Golog in eastern Qinghai) to the margins (e.g., Jiuquan in western Gansu and Xining in northern Qinghai) of the F. s. bieti range. The noticeable genetic admixture in domestic cat populations in the western Sichuaneastern Qinghai boundary (F. s. bieti core range) and western Gansunorthern Qinghai area (peripheral range) dated back to 7 and 30 generations ago, respectively, corresponding to the beginning of the 21st century and mid-20th century. Because the signals of the earlier interbreeding could have likely been concealed by later events if multiple waves of population admixture recurred (26), the contrast between the timing of admixture in domestic cats from different locations may reflect a continuous gene flow from Chinese mountain cats to domestic cats during the last century. This scenario is also consistent with a pattern of more recent introgression in the areas occupied by abundant Chinese mountain cats that are in constant contact with domestic cats, whereas relatively older hybridization signals have been preserved in cats located in the peripheral F. s. bieti distribution.

Since the 1950s, Chinese population census data have recorded a marked increase in the numbers of households and residents on the Qinghai-Tibet Plateau (31), a trend that coincides with the earliest F. s. bieti to F. s. catus admixture in Qinghai as documented in this study. Unlike dogs, cats are not generally associated with the traditional pastoral nomadic Tibetan lifestyle, and it is likely that the arrival and establishment of domestic cats on the Plateau is relatively recent. Regional socioeconomic development, immigration into the highlands, and alterations in local livelihoods may have facilitated an expansion of free-ranging domestic cats, setting the stage for their close contact, frequent interaction, and possible interbreeding with the sympatric Chinese mountain cat. An exact population status of the Chinese mountain cat in the wild is unknown, but, nevertheless, it is sparse and at a low density (32). Therefore, the Chinese mountain cat could possibly face a similar crisis as that of the European wildcat and lose its genetic integrity and evolutionary adaptation to the local environment because of introgression from an increasingly dominant local domestic cat population (22, 33).

Gene flow from the domestic cat to the Chinese mountain cat F. s. bieti was detected in the G-PhoCS analysis (Fig. 5B), despite a large variance in the estimates of total migration rates when different pairs of domestic cats and Chinese mountain cats were tested. Such fluctuation across individuals is consistent with recent introgression events in which the extent of introgression varies by individual within the Chinese mountain cat population (fig. S3). Unlike the abovementioned admixture analysis, no significant gene flow signals were detected from Chinese mountain cats to domestic cats in the G-PhoCS analysis. As the domestic cats used in G-PhoCS were from areas far from the Chinese mountain cat range, this scenario mostly likely resulted from a contemporary admixture that was restricted to the local sympatric cats and it exerted minor or no effect on domestic cat populations elsewhere.

The Felidae taxonomy by Kitchener et al. (5) considers the Chinese mountain cat its own species while maintaining the Asiatic wildcat as a subspecies. In our population genomic analysis, the Chinese mountain cat, the Asiatic wildcat, and the domestic cat are equidistant, corroborating a subspecies-level recognition of these groups. The WGS of the Chinese mountain cat, Asiatic wildcat, and domestic cat from China and worldwide, together with publicly available partial genomic data for the European wildcat and African wildcat, provide support to the classification of the Chinese mountain cat as a wildcat subspecies, F. s. bieti. Phylogenetic analyses based on mitogenome, Y chromosome, and genome-wide autosomal markers (Fig. 2) demonstrated a monophyletic placement of the Chinese mountain cat and other wildcat subspecies (F. s. catus, F. s. ornata, and F. s. silvestris) within one clade, rather than a species-level distinctiveness between them (34). The estimated divergence time between those F. silvestris subspecies is around 1.5 Ma ago, which agrees with previous estimates based on nuclear sequence fragments and SNP arrays (8, 25) and is more recent than the divergence between the accepted Felis species (i.e., F. chaus, F. margarita, F. nigripes, and F. silvestris) at around 3 Ma ago. PCA results also reflect a threefold smaller genetic distance among F. silvestris subspecies compared to their species-level divergence with F. nigripes. Nevertheless, we do not exclude an alternative to resolve the conflict between the genomic pattern and current taxonomic nomenclature, which is to elevate all wildcat lineages, including the Asiatic wildcat (F. s. ornata) and African wildcat (F. s. lybica), to independent species statuses, or F. ornata and F. lybica, respectively, thus retaining the Chinese mountain cat as F. bieti to be consistent. This would however require a comprehensive analysis including whole-genome data from all wildcat taxa, especially those from F. s. silvestris, F. s. ornata, and F. s. lybica.

Evidently, this study shows that interlineage admixture of the Chinese mountain cat F. s. bieti and its closely related taxa further supports the inclusion of all lineages as wildcat conspecifics based on the biological species concept, which considers interbreeding as the prerequisite for a species (35). The key argument from the proponents for the species status of the Chinese mountain cat lies on its distinctive morphological characters, a presumed sympatric distribution with the Asiatic wildcat, and an absence of gene flow between free-ranging Chinese mountain cats and Asiatic wildcats (9). However, recent surveys in Northwest China showed that the range attributed to the Asiatic wildcat may have been overestimated and that its presumed presence on the Qinghai-Tibet plateau in northeastern Qinghai (36) may not be true. That assertion, if proven, would dispute the supposed sympatry of the two lineages. In addition, extensive genetic exchange between those two lineages was revealed through the presence of F. s. ornatalike mitochondrial lineages in voucher Chinese mountain cats (Fig. 2). A significant migration band (total migration rate of 0.09) from the Asiatic wildcat to the Chinese mountain cat was also detected in demographic analysis with G-PhoCS analysis (Fig. 5B). Such interbreeding could diminish the morphological distinctions between the taxa, as we observed when a Chinese mountain cat with an F. s. ornatalike mtDNA haplotype did not have the typical thick and fluffy tail (Fig. 1B). Answers to the remaining questions require more surveys and studies to fine map the Asiatic wildcat and Chinese mountain cat distribution in Northwest China; to delineate the subspecies boundaries or hybrid zones; to elucidate the ancestry, adaptation, and evolution of these taxa; and to resolve the historical and current patterns of gene flow among the wildcat and domestic cat lineages in the region.

In conclusion, this study examined the genetic ancestry, population structure, and demographic history of wildcat and domestic cat lineages in East Asia from a whole-genome perspective. Phylogenomic and population genomic analyses based on voucher specimens verified that the Chinese mountain cat, a traditionally delineated felid species endemic to the eastern Qinghai-Tibet Plateau of China, is equidistant with other currently recognized wildcat lineages such as the Asiatic wildcat (F. s. ornata) and hence should be recognized as a conspecific, F. s. bieti. We revealed ancient introgression between F. s. bieti and F. s. ornata as we found two deeply divergent mtDNA lineages within F. s. bieti. Domestic cats (F. s. catus) in China clustered with other cat populations worldwide, supporting the single, Near Eastern origin of cat domestication from the African wildcat (F. s. lybica), followed by the domestic cats subsequent global spread. Contemporary genetic introgression from F. s. bieti into sympatric domestic cats is evident across, but not beyond, the range of F. s. bieti. The timing of admixture coincided with large-scale socioeconomic changes in the Tibetan area during the mid-20th century. That process likely led to an expansion of domestic cats into the region and suggests that domestic cats arrived rather late to the Plateau and thus had not encountered F. s. bieti until recently. The increasingly abundant local domestic cat population may pose a threat to the Chinese mountain cat and jeopardize its genetic integrity and evolutionary adaptation to high altitude, an issue with profound conservation implications and worth further study.

We collected samples from 27 Chinese mountain cats (F. s. bieti), 4 Asiatic wildcats (F. s. ornata), and 239 domestic cats (F. s. catus), all with known geographic locations. F. s. bieti specimens included feces, blood, skin tissues, dry pelt, and skulls from zoos, museums, or local villages in Qinghai, Sichuan, and Gansu; a collection effort that represents the largest ever range-wide sampling of this taxon (data file S1). F. s. ornata samples included blood or dry skin from southern Xinjiang. Last, we sampled buccal swab, blood, or skin tissues from outbred, unrelated F. s. catus from 23 sites across China, particularly areas that are sympatric, parapatric, or allopatric with the Chinese mountain cat (Fig. 1 and data file S1). All samples were recruited in compliance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora through permissions issued to the School of Life Sciences (principal investigator: S.-J.L.), Peking University, by the State Forestry Administration of China.

We extracted genomic DNA from blood or skin tissues using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA) and from fecal samples using the QIAamp DNA Stool Mini Kit (QIAGEN), both following the manufacturers protocols. We collected DNA from buccal swab samples using a PERFORMAgene PG-100 collection kit (DNA Genotek, Ottawa, ON, Canada) and extracted DNA using the buffer and protocol provided by the kit. DNA concentrations and quality were examined with the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and diluted to working solutions for further analysis.

Genomic DNA extraction from museum samples was performed in a dedicated ancient DNA laboratory and followed a modified silica-based spin column method and standard ancient DNA criteria while maintaining strict precautions to minimize contamination risk from modern DNA samples and facilities (37). For each specimen, 10 to 30 mg of skin tissue were pulverized in liquid nitrogen, washed twice with ddH2O, and digested at 55C overnight with 600 l of ATL buffer from the DNeasy Blood and Tissue Kit (Qiagen), 24-mAU proteinase K (Qiagen), and 7 l of 1 M dithiothreitol. After digestion, we purified the DNA using a silica column from a QIAquick polymerase chain reaction (PCR) purification kit (Qiagen) and kept the products at 4C before subsequent downstream analysis.

We used PCR primers, redesigned based on published F. silvestris mtDNA sequences (3), to amplify a 2.7-kb mtDNA fragment spanning ND5, ND6, and CytB, and then selected four short fragments (200 to 400 bp each) within this region to amplify highly degraded DNA from museum samples. Two Y chromosome DNA fragments encompassing the DBY7 and SMCY7 intronic regions that had been used previously in mammals and Felidae (38, 39) were amplified in all male individuals to examine their patrilineal ancestry.

The 2.7-kb mtDNA and the Y chromosome fragments were separately amplified in a 15 l of PCR reaction system containing 1 GC buffer I, 1.0 mM deoxynucleotide triphosphates (dNTPs), 1 U of TaKaRa LA Taq DNA polymerase (Takara Bio, Shiga, Japan), 0.4 M each of forward and reverse primers, and 10 to 20 ng of genomic DNA. For DNA extracted from museum specimens, we set up PCR reactions in an ancient DNA laboratory room and followed a previously published protocol (37), optimizing each step to amplify the short mtDNA fragments in a 25-l PCR reaction system containing 1 PCR buffer II, 5.0 mM MgCl2, 0.8 mM dNTPs, 10 g of bovine serum albumin, 1 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Waltham, MA, USA), 0.2 M each of forward and reverse primers, and 5 l of genomic DNA. PCR products were cleaned and sequenced on an ABI 3730XL sequencing system (Applied Biosystems) as described previously (37). DNA sequences were inspected in Sequencher v5.0 (Gene Codes Corporation, Ann Arbor, MI, USA) and concatenated into haplotypes for downstream analyses.

We constructed Illumina sequencing libraries with 300- to 500-bp inserts from 55 genomic DNA extracts, following the manufacturers protocols (Illumina, San Diego, CA, USA). The sample set included eight F. s. bieti, three of which carried F. s. ornata mtDNA haplotypes; one F. s. ornata; and 46 F. s. catus across China, two of which carried F. s. bieti Y chromosome haplotype (Fig. 1A). The libraries were sequenced on an Illumina HiSeq X Ten platform at Novogene Co. (Beijing, China) to generate 150-bp paired-end reads. For four Chinese mountain cats with either low DNA quality or endogenous DNA content, we produced 2-Gb sequencing data per individual for mitochondrial genome assembly. For the remaining 51 samples including four Chinese mountain cats, one Asiatic wildcat, and all domestic cats, 30- to 40-Gb sequencing data per individual were generated for WGS. For additional comparison, we downloaded WGS data of 20 domestic cats representing a worldwide distribution and two black-footed cats (F. nigripes) from the NCBI SRA and included that data in our analyses.

To exclude nuclear mtDNA segment (Numt) interference while assembling the mitogenomes, the sequencing reads from each individual were first mapped to a domestic cat mitogenome reference sequence (accession no. U20753) using the Burrows-Wheeler Aligner (BWA) minimum essential medium (MEM) algorithm (40). We then assembled the mapped reads into mitogenomes without the control region via a de novo genome assembly approach in Geneious v.9.1.5 (www.geneious.com). From the 77 individuals sequenced from this and previous studies, mitogenomes of 66 Felis spp. were assembled and identified for further analysis.

For genome-wide SNV identification and genotyping, we mapped the WGS reads of 73 individuals to the domestic cat reference genome assembly felCat8 [downloaded from the UCSC (University of California, Santa Cruz) genome browser (UCSC, CA, USA)] and the domestic cat Y chromosome reference sequence (accession no. KP081775.1) using a BWA-MEM algorithm with default parameters. After removing PCR duplications and multitargeted reads with SAMtools (41), the local realignment of the uniquely mapped reads were performed via RealignerTargetCreator and IndelRealigner in GATK v3.7 (42). The reads realigned to autosomes and the X chromosome were piled up using SAMtools for SNV calling in BCFtools (43). The raw dataset of autosomal and X chromosome SNVs was filtered for downstream analysis, retaining only those biallelic SNVs with Phred-scaled quality scores of more than 20, raw read depths between 400 and 1600, genomic distances of more than 5 bp to the nearest indel, and no missing data across all individuals. We further excluded SNVs within repetitive regions, CpG island regions, and protein-coding regions of the domestic cat felCat8 reference genome annotations, resulting in a dataset of 20,425,451 variable sites from the putative neutral regions of the genome, for downstream phylogenetic and population genomic analyses. The statistics of the WGS reads of each sample are summarized in data file S4.

The realigned Y chromosome reads from 40 malesincluding 2 Chinese mountain cats, 36 domestic cats, 1 Asiatic wildcat, and 1 black-footed catwere piled up in SAMtools, and genotypes were called as haploid using BCFtools. We filtered the initial dataset to keep only those biallelic SNVs with Phred-scaled quality scores of more than 20, raw read depth between 100 and 400, and more than 5-bp distances to the nearest indel. To eliminate X chromosome interference in this paternal genealogy analysis, we identified Y chromosome regions with X homologs by mapping sequencing reads from two female domestic cats to the cat Y chromosome. After filtering, only those SNVs located in the 929-kb single-copy Y chromosome region (44), with no mapped sequencing reads from females, were included in downstream Y-haplotype analysis.

We aligned mtDNA and Y chromosome haplotypes with Clustal X v2.0.10 (45) and identified variable sites with MEGA v6.06 (46) (data files S2 and S3). Statistical parsimony networks were constructed using TCS v1.1.3 (47) to infer the phylogenetic relationships among domestic cats and wildcats (Fig. 2A).

The 66 mitogenomes assembled from high-throughput sequencing data were aligned along with published mitogenome sequences from the domestic cat (accession no. U20753) and other Felis spp. (accession no. KP202273.1 to KP202278.1) for phylogenetic reconstruction. We selected the best fit nucleotide substitution model using jModelTest v2.1.4 (48). A Bayesian approach with two parallel Markov chain Monte Carlo runs were performed in MrBayes v3.2.6 (49) for 1,000,000 generations, with sampling every 500 generations. Phylogenetic analyses based on maximum parsimony, maximum likelihood (ML) with a TrN (Tamura-Nei) + I + G model, and neighbor joining constructed from Kimura two-parameter distances were performed in PAUP v4.0b10 (50), and the statistical reliability of each node was assessed by 100 bootstrap replicates.

We reconstructed the Y chromosome phylogeny of 40 male cats following the same ML procedure with an HKY (Hasegawa-Kishino-Yano) + G model in PhyML v3.1 (51). The Bayesian trees based on mitochondrial genome and Y chromosome were illustrated with Figtree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) (Fig. 2B), and the bootstrap support or posterior probability of those tree topologies is marked in fig. S1.

We used BEAST v2.4.4 (52) to estimate the coalescence times of different wildcat lineages based on our mtDNA and Y chromosome SNVs. Mitogenome analysis was performed with TN93 + I + G as the substitution model, the lognormal relaxed clock model, the Yule tree prior, and the coalescence times of genus Felis (4 Ma ago) and F. silvestris subspecies (1.5 Ma ago) as two calibrations (8, 25). We performed Y chromosome analysis with HKY + G as the substitution model, the lognormal relaxed clock model, the Yule tree prior, and the coalescence times between F. nigripes and F. silvestris (3 Ma ago), and among F. silvestris subspecies as calibrations (8, 25). In both mitochondrial and Y chromosome coalescence analyses, we performed four parallel runs for 50,000,000 generations, with parameters sampled every 1000 generations, and then log files and tree files were combined with the first 10% of the generations as burn-in to achieve our final parameter estimations (fig. S1).

To reconstruct the genome-wide autosomal phylogeny, we computed a pairwise p-distance matrix based on the 73 individuals autosomal SNVs following Gronau et al. (24) and used that matrix to build a neighbor-joining tree in the Ape package v4.0 in R v3.2.3 (53). We ran 100 bootstrap replicates by resampling the autosomal variants from 1-Mb nonoverlapping windows and summarized the support values using the method implemented in the Ape package of R.

We performed PCA based on biallelic autosomal variants from all the individuals using smartpca in EIGENSOFT v6.1.4 (54, 55) without removing outliers (Fig. 3A). In addition, using VCFtools 0.1.15 (56), we estimated pairwise FST values among Chinese domestic cats, worldwide domestic cats, Chinese mountain cats, the Asiatic wildcat, and black-footed cats based on 1-Mb windows along the autosomes. Furthermore, we used autosomal SNVs to infer population genetic structure of domestic cats and Chinese mountain cats using ADMIXTURE with random seed (57). The Asiatic wildcat was excluded from the analysis because of its extremely small sample size (N = 1). The number of genetic clusters (K) was set from two to six, with five replicates for each setting and cross-validation enabled for choosing the best clustering number (fig. S2).

We applied D statistics (58) through ADMIXTOOLS (59) to detect gene flow between Chinese mountain cats F. s. bieti (bieti) and local domestic cats F. s. catus within China (X) while using worldwide domestic cats (worldwide) for comparison and black-footed cats F. nigripes (nigripes) as the outgroup and calculated all possible combinations of D (X, worldwide, bieti, and nigripes). The z scores of each F. s. catus from China were evaluated and those with the smallest z score of more than 2 were considered F. s. catus with genetic admixture from F. s. bieti (Fig. 3C and table S3). In addition, we adapted three statistical approaches to quantify the levels of genetic introgression from F. s. bieti to F. s. catus: the percentage of F. s. bieti diagnostic sites in F. s. catus genomes, f statistics (60), and the f4 ratio test (61).

To identify autosomal SNVs specific to F. s. bieti, we compared the neutral genomic regions across four F. s. bieti to 20 worldwide F. s. catus, whose chances of interbreeding with F. s. bieti were extremely low. The resultant 531,395 variants unique to F. s. bieti were then used to calculate the portion of introgression in each of the admixed Chinese domestic cats.

Using ADMIXTOOLS, we calculated the f4 ratio asf4ratio=f4(ornata,nigripes;X,worldwide)f4(ornata,nigripes;bieti,worldwide)(1)and calculated f statistics with the D statistic parameters asfstatistics=S(X,worldwide,bieti,nigripes)S(bieti1,worldwide,bieti2,nigripes)(2)where S (X, worldwide, bieti, nigripes) is the numerator of D of each domestic cat X and S (bieti1, worldwide, bieti2, nigripes) is the numerator of D with two randomly selected F. s. bieti designated as bieti1 and bieti2.

We first estimated the time of introgression from F. s. bieti to sympatric domestic cats by plotting the genome-wide distribution of F. s. bietispecific alleles found in the 10 admixed domestic cats, along the 531,395 SNVs that distinguished F. s. bieti from the domestic cat (fig. S2). Large consecutive genomic segments carrying F. s. bieti ancestry within domestic cat genomes were identified based on diagnostic variants. We used ALDER v1.03 (26) to date hybridization events based on LD decay patterns in two domestic cat populations: (i) 10 cats from F. s. bieti core range (C6 to C15, labeled hybrid1) and (ii) five individuals from the edge of that range (C1 to C5, labeled hybrid2). One group with four F. s. bieti (B1 to B4) and the other with 31 domestic cats beyond F. s. bieti distribution area (C16 to C46) represented two ancestral populations in the analysis. To find the best fitting start point (d0), we performed 11 parallel runs with d0 set from 0.5 to 5 cM, subsequently selecting 2.0 and 0.5 cM as the best parameters for hybrid1 and hybrid2 populations, respectively, according to P values and z scores (table S4).

We applied the PSMC model (62) and G-PhoCS (24) approach to infer the demographic dynamics of wildcats and domestic cats in China, including historical population sizes, divergence times, and gene flow scenarios.

The PSMC model estimated effective population size changes through time based on autosomal consensus sequences of five individuals: N2, B4, O1, C25, and W19, representing F. nigripes, F. s. bieti, F. s. ornata, F. s. catus from China, and F. s. catus worldwide, respectively. The analysis was carried out at an individual-based level with 64 atomic time intervals under the default pattern 4 + 25 2 + 4 + 6, as described by Li and Durbin (62), and with the maximum coalescent time set to 20. The estimated values were then transformed to effective population sizes and plotted with a generation time (g) of 2 years and a mutation rate () of 2.6 109 substitutions per site per generation (Fig. 5A), as calibrated in the G-PhoCS analysis. For each individual, we ran 100 bootstrap replicates to evaluate estimation robustness.

We used the G-PhoCS to estimate the demographic parameters such as historical population size, divergence time, and migration rate based on coalescent-based Markov chain Monte Carlo and a given topology (24, 63, 64). To identify neutral loci for the analysis, the autosomal sequences of the hard-masked domestic cat genome assembly (felCat8) were further masked to remove CpG islands and exons with 1-kb flanking regions based on UCSC genome annotations. Following established procedures (24, 64), we recognized 34,418 unlinked loci, each 1-kb long with a minimum interlocus distance of 50 kb and containing less than 10% masked sites.

We performed G-PhoCS analysis based on a given topology of the four Felis lineages and its estimated parameters (fig. S3A) and with 10 representative individuals, including four F. s. bieti (B1, B2, B3, and B4), three F. s. catus (C20, C25, and W19), one F. s. ornata (O1), and one F. nigripes (N2). To avoid possible interference between migration bands and the time cost correlated with the demography models complexity, we performed a prior analysis to identify significant migration bands with C25, B2, O1, and N2. All 18 possible migration bands were considered in the model, two parallel runs were conducted with 500,000 generations sampled every 100 generations, and all results were cross-checked to ensure convergence. Four significant migration bands were detected in this preliminary run, with a total migration rate (mtot = m ) around or more than 0.1 (fig. S3B).

Then, we ran 12 independent analyses with four individuals from each of the four lineages and the four migration bands detected in the prior analysis while considering all combinations of domestic cats and Chinese mountain cats. Each analysis was performed with two parallel runs of 1,000,000 generations each, sampled every 100 generations, and the trace files were combined to obtain final demographic parameter estimations, with the first 30% of the generations as burn-in (fig. S3C and table S5). The estimated parameters ( and ) were converted to the effective population size (Ne), divergence time (T), and coalescent time (Tdiv) according to Gronaus formulas (24): = 4 Ne , = T /g, and _div = + 0.5 . The mutation rate = 2.6 109 was calibrated according to the divergence time between the black-footed cat and the domestic cat lineages (T_Felis_div), 3 Ma ago (8).

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Genomic evidence for the Chinese mountain cat as a wildcat conspecific (Felis silvestris bieti) and its introgression to domestic cats - Science...

Express News Service

BENGALURU:While there has been a dip in new cases, the emergence of the Delta-plus variant has become a cause for worry. To address the issue, experts and members of the Covid Sequencing Task Force have suggested intensifying genome sequencing to larger populations.

According to the Health Department records, in the last six months, 2,300 genome samples have been sequenced by NIMHANS in coordination with the NCBS. As per the information from NIMHANS, the test results of 918 are still under way.

To increase sequencing, Bruhat Bengaluru Mahanagara Palike has also partnered with Molecular Solutions Care Health LLP. Over the last two days, five samples of the seven who tested positive for the variant have been taken for sequencing.

As per the present guidelines, 5% of samples of those who tested positive are sent for genome sequencing. These are chosen based on the characteristics of the virus during RTP-CR tests. Talks are on to increase this percentage as the number of Delta-plus variant cases are rising, a BBMP official said.

A member of the task force explained that samples for genome sequencing are chosen based on the area where the cases are reported, the clusters and the regions. The results of the sequencing will be effective only when it is on a continuous basis and on a larger scale. At present, it is based on statistical models.

Discussions on intensifying the scale of testing are also being held with the National Centre for Disease Control and the ministrys coordination committee to have a larger web for genome sequencing. Talks are also on to have a separate strategy for Karnataka, compared to what India is following, because of its cosmopolitan nature and floating population, an expert said.

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Genome sequencing to be intensified in Bengaluru - The New Indian Express

A new first of its kind institution aimed at enhancing local genomic research as well as diagnosing and treating patients with genetic disorders has been unveiled in the United Arab Emirates (UAE).

The Center for Genomic Discovery is a joint venture between the Mohammed Bin Rashid University of Medicine and Health Sciences (MBRU) and the Al Jalila Genomics Center of the Al Jalila Childrens Specialty Hospital (AJCH).

The necessary interdisciplinary activities including patient recruitment, genomic data analysis, and functional characterisation cannot be undertaken without the Center, its founding institutions, and the interdisciplinary ecosystem they have created, said Fahad Ali, Assistant Professor of Molecular Biology at MBRU. At the Center, functional experiments will be designed to characterise any novel candidate genes or mutations, to establish new gene-disease associations, and to explore potential therapeutic targets.

And work has already started with one family, offering promising progress, said Stefan Du Plessis, founding Dean of Research and Graduate Studies at MBRU and a member of the Centers Steering Committee.

At least one novel gene has been identified by whole-exome sequencing a complex genomic test which surveys all 20,000+ human genes in search for tiny changes which might be disease causing, he said. Functional analysis is still ongoing but preliminary data strongly suggests a role in disease. We have also identified patients outside the UAE, in the Gulf region, with the same clinical conditions, mutations and gene.

We are now establishing collaborations with researchers from those sites to characterise this gene. The fact that patients from different backgrounds with similar clinical features have mutations in the same gene further establishes a potential new gene-disease discovery.

WHY IT MATTERS

Speaking at Arab Health 2021, Amer Sharif, Vice Chancellor of MBRU, described the formation of the centre as a major milestone for healthcare research in the UAE.

These are life-changing outcomes that underline the power of research and the role of MBRU and our academic health system partners as research-intensive institutes, he said. The establishment of the Center of Genomic Discovery through an integrated academic health system will allow us to innovate in genomics application and gene discovery. This will also enable us to realise our vision of advancing health through cutting-edge academic research and nurturing future scientists serving individuals and communities in the UAE and the region.

The Center for Genomic Discovery will reportedly seek to engage undiagnosed pediatric patients with suspected hereditary disorders whose clinical genomic testing at Al Jalila Childrens such as whole-exome sequencing and chromosomal microarrays failed to identify any definitive genetic causes.

THE LARGER CONTEXT

The launch of the centre coincides with the formation of the board of the Emirati Genome Program, whose mission is to provide preventive and personalised healthcare for the Emirati population.

Headed by His Highness Sheikh Khalid bin Mohamed bin Zayed Al Nahyan, Member of the Abu Dhabi Executive Council and Chairman of the Abu Dhabi Executive Office, the Emirati Genome Program is reportedly being designed to enable precise and customised medical treatment that will support a more robust healthcare system in the UAE.

MBRUs Sharif is a member of the board.

Meanwhile, AJCSH has also announced it is to formulate a steering group to study the workflow of genome sequencing in an intensive care setting. The hospital has partnered with Illumina Netherlands BV to develop this practical knowledge improving the use of testing, and fostering greater understanding of best-use cases, clinical indicators and the health economics of genome sequencing in this specialised setting.

There is increasing evidence for rapid, efficient and cost-efficient genome sequencing in newborns and babies that will save lives, said Mohamed Al Awadhi, COO of AJCH. The application of next-generation sequencing has revolutionised the process of making complex diagnoses in paediatric medicine, significantly shortening the time for accurate diagnosis and optimal clinical management in critically ill children.

Thanks to the support of our partners and stakeholders, this working group will once again push the boundaries in the quest to save young lives.

ON THE RECORD

This is a major development for Dubai and the UAE, said Ahmad Abou Tayoun, Director of Al Jalila Childrens Genomics Center and Associate Professor of Genetics at MBRU. The new Center leverages the clinical, genomics, and functional research infrastructure and human resources at Al Jalila Childrens and MBRU to propel interdisciplinary activities.

Our clinicians and researchers are experts in their respective fields who can help resolve undiagnosed patients with highly suspected inherited disorders. Furthermore, the Center will create a training and research site for Masters and Ph.D. students, as well as post-doctoral fellows at MBRU.

He added: Ultimately, the Centers main goal is to make novel genetic discoveries in the pediatric patient population in the UAE and the region, and leverage these discoveries to develop new diagnostic tools and uncover personalised pathways to restore normal phenotypes in affected patients.

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Center for Genomic Discovery launched in the United Arab Emirates - Mobihealth News