New & Noteworthy

Sometimes You Need Half a Trillion

June 01, 2016

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In the Foundation series by Isaac Asimov, Hari Seldon invented a field of study called psychohistory which was able to “make general predictions” about human history. It only worked because the population of the Galactic Empire was in the quadrillions. You need huge numbers to get useful predictions.

Sometimes real life biologists need lots of individuals to see what they are looking for too. In the case of a recent paper in PNAS by Lee and Stevens, they needed to sift through half a trillion yeast to find what they were looking for. And boy was it worth it!

They saw an intron jump from one gene to another. Twice. In 500 billion tries.

This is the first example where we have caught an intron in the act of moving from one place to another. This has important implications for the study of evolution where over time introns spread or recede. It might even help us better understand cancer where intron loss can play a role.

Seeing something as rare as this means making a very specific, complicated reporter. You don’t want to manually sort those 500 billion colonies…or at least I wouldn’t want to.

Here is a “simplified” schematic of the reporter they came up with:

It is as complicated as it looks but it got the job done! Let me walk you through what everything is and how it works.

The GU and AG sequences are splice site junctions. Sequences between a GU and an AG can be spliced out.

The red gene is the S. pombe his5+ gene that has a promoter driving its expression shown with the red arrow. It is in the reverse orientation compared to the eGFP gene which is under the control of the promoter represented by the blue arrow.

The S. pombe his5+ gene works fine in S. cerevisiae his3 mutants to make up for histidine auxotrophy. But it won’t work from this construct.

See, the his5+ gene has an intron that keeps it from being translated correctly unless the intron is spliced out. But this intron can’t be spliced out when the gene is transcribed using the red promoter because the splice junctions GU2 and AG1 are in the wrong orientation. Any transcripts from the red promoter will not work because the intron cannot be spliced out.

You also can’t get His5 from the blue promoter. Even with splicing (which will work in that orientation), the gene can’t be translated because it is in the wrong orientation.

To get any his5+ transcript, the yeast needs to take the spliced RNA from between GU1 and AG2 and get it into DNA. It also needs to get rid of the intron in the middle of his5+ gene before it happens.

So what they are looking for are yeast that glow green and can survive in the absence of histidine. There are a couple of ways this can happen.

The more common way results in the his5+ gene recombining back into the plasmid such that the intron is missing from its middle. Basically the RNA between GU2 and AG1 is spliced out and the resulting RNA is reverse transcribed into DNA. This DNA then undergoes homologous recombination with the plasmid DNA resulting in a working his5+ gene. They got over 10,000 of these in their experiment.

The much less common way involves the intron being inserted into a gene within the genome. This way uses some of the same steps with one extra one—a reverse splicing event.

As I said, they got two of these with one ending up in the RPL8B gene and the other in the ADH2 gene.

Here’s how they think this happened…

The spliced out RNA from the plasmid was left for a brief time in the spliceosome (or what remained of it after the splicing reaction). During this time, a second mRNA arrived for splicing while the intron from the reporter was still there.

Then something called reverse splicing happened which basically replaced the native intron of the RPL8B or the ADH2 gene with the spliced out intron from the reporter. Next this was turned into DNA with reverse transcriptases and then this construct ended up in the genome through homologous recombination.

No wonder this was so rare! Reverse splicing is thought to be really uncommon in vivo as is reverse transcription of DNA. Add in a loitering intron on the remnants of a spliceosome and you can see why this was a 1 in 250 billion shot.

So there you have it, one way a transposon can hop. It is no transposon, but occasionally an intron can move to a new gene.

And of course we turned to the awesome power of yeast genetics to help us figure this out. Only with yeast can we sift through half a trillion cells to find the two that show us how intronogenesis, the introduction of an intron to a new site, might happen. #APOYG!

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: mobile intron, reverse splicing

Next SGD Webinar: June 1, 2016

May 26, 2016

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If you’re not already using JBrowse to view all your favorite S. cerevisiae genes…you should be! SGD’s JBrowse is a quick and easy way to browse through the information-rich yeast genome. Using JBrowse, you can visualize spatial relationships between genes, locate SGD annotations throughout the yeast genome, and align chromosomal features to hundreds of experimental data sets.

Our upcoming webinar on June 1st will provide a short 15-minute tutorial on the basics of JBrowse. We will demonstrate how to navigate the genome with JBrowse, locate your favorite genes or chromosomal features, and visualize experimental data with data tracks. Whether you’re an experienced GBrowse user looking to try JBrowse for the first time, or someone new to genome browsing as a whole, this webinar is sure to help you get started.

If you are interested in attending this event, please register using this online form: http://bit.ly/SGDwebinar3

This is the third episode of the SGD Webinar Series. For more information on the SGD Webinar Series, please visit our wiki page: SGD Webinar Series.

Categories: Announcements, Tutorial

Control (Apple) F for Genetics

May 18, 2016

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A long time ago, through the mists of time, finding a phrase or a passage in a document was hard. If you didn’t have the foresight to highlight it, you were stuck.

You could reread the whole document but that might take way too much time. Or you could narrow down where to look by remembering the chapter it was in and rereading just that.

Nowadays of course, finding that phrase or passage is trivial in a Google or Word doc. You just use the find function!

Finding the genetic variant responsible for a given trait is still, in many ways, in the age of the typewriter. A genetic association study can find a part of the DNA responsible for that trait but homing in on the exact variant is terribly time consuming and labor-intensive.

Enter that wonder of a gene editing tool, CRISPR.

In a new study in Science, Sadhu and coworkers have essentially turned CRISPR into a find function for genetic association studies. They use CRISPR’s double stranded DNA cutting ability and mitotic recombination in a heterozygote yeast strain to blanket a region of DNA with crossover events.

Sifting through the results let them find the actual amino acid change responsible for manganese sensitivity with much less effort than would have been required with the old method. They only needed to use 358 lines instead of the 7,500 or so they predict they would have had to go through without good old CRISPR.

And think how much time might be saved with a more complicated beast! Given its short generation time and relatively small genome, yeast is like a magazine article. A human is more like War and Peace. This new system might make linking human traits/diseases to the causative SNP much simpler.

The usual way to link a trait to the DNA that causes it is to see what known traits tend to get passed down with it. If you know trait A is at position X on chromosome Y and the trait you are interested in, trait B, is usually passed down with A, then trait A and trait B are close to each other (this is called linkage mapping). This is the easy part.

The hard part is getting from A to B. To get finer mapping, you need to rely on the recombination that happens during meiosis. This is the step where this new study can help.

Instead of relying on the slow, natural process of meiotic recombination, these authors use the CRISPR-Cas9 system to jump start mitotic recombination.

They start off with a heterozygote in which one of the parent strains has a trait and the second does not. They chose the lab strain, BY, and the vineyard strain, RM, as the parents.

Next they designed 95 guide RNAs that would direct Cas9 to 95 different spots along the left arm of chromosome 7. Sadhu and coworkers targeted sites that were heterozygous in the two strains.

Once Cas9 cuts the DNA, the cell uses homologous recombination to repair the cut using the DNA on the other chromosome in the pair as the template. This results in a loss of heterozygosity (LOH) which can reveal recessive traits.

They picked 384 lines, approximately 4 from each cutting event and found that 95% of these had undergone LOH with hardly any off-target effects.

They next measured the growth of these strains in 12 different conditions and found that one trait, growth on 10 mM manganese sulfate could work for their purposes. Using the more traditional approach with 768 segregants obtained through meiotic recombination, they were able to narrow it down to an overlapping piece of DNA of 3,900 bases. However, using CRISPR-Cas9, they were able to narrow down the location of the variant to a 2,900 base pair stretch of DNA. Score one for the new method!

Next they did finer mapping by targeting the 2,900 bases they had identified with CRISPR-Cas9. Even though they only used three guide RNAs that targeted three locations, they were able to look at many more places on the DNA than this because the length of DNA repaired around the cut varies a bit between strains.

They isolated 358 lines of which 46 or 13.1% had a recombination event in the 2,900 bases they were interested in. Only 0.7% of segregants obtained the old way were in the right place. Score another one for the new method!

After measuring how well these 46 lines grew in manganese sulfate, the researchers were able to narrow the search to a single polymorphism that resulted in an amino acid change in the PMR1 gene. A perfectly reasonable result, given that Pmr1p is a manganese transporter. They then showed that this variant, pmr1-F548L, did indeed make a strain sensitive to manganese all on its own.

By targeting recombination events to regions of interest, these authors have provided proof of principle for a technique that should make connecting traits (phenotype) with DNA variants (genotype) much easier than it is currently. Behold yet again the awesome power of yeast genetics (#APOYG)!

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: CRISPR/Cas9, genotype, mapping, phenotype

New SGD Help: Downloading and Uploading JBrowse Data Tracks

May 10, 2016

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SGD’s new JBrowse genome browser allows quick and easy browsing of the information-rich yeast genome.

Take a look at our newest video tutorial to learn how to download or upload JBrowse data tracks. Let us know if you have any questions or suggestions.

For more SGD Help Videos, visit our YouTube channel, and be sure to subscribe so you don’t miss anything!

Categories: Tutorial

Can’t Get There Like That

May 04, 2016

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As HBO’s Silicon Valley scathingly relates, the mapping app from Apple was truly terrible when it was first launched. There are all kinds of funny (scary?) stories in which people following the directions ended up in the wrong place. (Click here for a few more of the epic fails.)

And sometimes it would show impossible ways to get from one location to the other. For example, to get to a certain place, my iPhone would recommend two different routes. After choosing the seemingly easiest route I quickly realized that it would take me through a building. Sometimes, even though it seems there are a couple of different ways to get somewhere, there is actually only one.

It turns out that this can be true in gene expression too. While you might increase expression by either mutating the promoter or duplicating the whole gene, sometimes only duplication is enough. There is just one route from here to there.

This point is driven home in a new study out in GENETICS by Rich and coworkers. Here they show that, under sulfate-limiting conditions, the only way that yeast can boost the expression of the SUL1 high affinity sulfate transporter enough to thrive is by duplicating it.

In many previous experiments, whenever yeast is starved for sulfate, after 100 generations or so the population almost always ends up selecting for a SUL1 duplication rather than increasing expression with a promoter mutation. This duplication results in a fitness advantage of 35% or more, which makes sense – when there isn’t much sulfate available, those cells that can get more of what’s there will outcompete their neighbors.

There are a couple of possible reasons things go down like this. It could be that the duplication is simply the most likely way to increase expression enough to survive, meaning that promoter mutations are possible but rare. Alternatively there may be no way to mutate the promoter enough to adequately increase the activity in such a short window of time. You simply can’t get to enough increased fitness by this route.

The first step in figuring out how to get somewhere is to map the roads in the area. This is also what Rich and coworkers needed to do – they needed to figure out how SUL1 is regulated. They did this in the standard way by nibbling away bits of the sequences upstream of the gene until there was a significant impact of gene expression. This is how they identified the 493 base pairs upstream of SUL1 as its promoter.

In the meantime, they also managed to develop a broadly applicable methodology for investigating any promoter – saturation mutagenesis, chemostat selection, and DNA sequencing to track variants.

The next step was to generate a library of mostly single point mutations in this promoter using error-prone PCR. As might be expected, most of the mutants had no effect or decreased gene expression but a few did increase activity. However, none of these last set increased expression as much as duplicating the gene.

They found 8 mutants that gave a 5% or better increase in fitness with the best being a 9.4% increase. Even when they combined these point mutations they could not increase the fitness much beyond 11%, not even half of the increase in fitness that an extra SUL1 gene gives. It seems that to get the most bang for its buck, yeast needs to duplicate SUL1. At least in the time frame of the experiment, that is.

Using what is essentially the scanning mutagenesis of this promoter, they were able to identify three sites that were important for SUL1 regulation. One site at -465 to -448 corresponded to a Cbf1-Met28-Met4 regulatory site, the second site at around -407 was most similar to either a Met31 or Met32 site and the third site at around -350 matched a Met32 site.

Mutations that resulted in increased activity tended to bring one of these three sites closer to the consensus transcription factor binding site. For example, the strongest point mutation, -353T>G, did this with the Met32 site at -350.

Even with these stronger consensus sequences for sulfate-regulatory transcription factors, none of these point mutations could get the yeast to where it needed to be in the fitness landscape in order to be able to thrive under sulfur limitation. Point mutations just can’t hold a candle to simply duplicating the SUL1 gene. Sometimes there really is just one way to get from point A to point B.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: adaptation, chemostat selection, fitness, gene duplication, saturation mutagenesis

Sign Up Now for the Next SGD Webinar: May 4th, 2016

April 26, 2016

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If you’re not already using YeastMine to answer all your questions about S. cerevisiae genes and gene products…you should be! SGD’s YeastMine is a powerful search tool that can retrieve, compare, and analyze data on thousands of genes at a time, greatly reducing the time needed to answer real, practical research questions. Through YeastMine, questions such as “What proportion of plasma membrane proteins are essential?” or “How many different gene products physically interact with the mitochondrial ribosome?” can be answered within minutes.

Next week on May 4th, 2016 at 9:30 AM PDT, we will provide a brief webinar tutorial on how to run queries and create gene lists in SGD’s YeastMine. As a practical demonstration of YeastMine, we will also showcase a research scenario in which yeast-human homology data is used to predict potential chemotherapy targets for human cancers.

Space for this webinar is limited. To reserve your spot, please register for the event using this online form: http://bit.ly/registerFor2ndSGDwebinar

This is the second episode of the SGD Webinar Series. If you missed the first one, or if you’d like to find out more about upcoming webinars, please visit our wiki page: SGD Webinar Series.

Categories: Announcements, Yeast and Human Disease

Chocolate and Coffee Too?

April 20, 2016

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Most of us know about yeast’s big part in making bread and booze. But those aren’t yeast’s only wonderful gifts. It also plays a big role in chocolate and coffee too. Is there anything this marvelous microorganism can’t do?

A new study by Ludlow and coworkers in Current Biology set out to look at the strains involved in cacao and coffee fermentation. Unlike the extensively studied wine strains, these have mostly been ignored up until now.

These researchers found that cacao, coffee and wine strains were very much different from one another. And they also found that unlike wine yeasts, which are pretty much the same most everywhere in the world, coffee and cacao strains are different depending on where they come from.

But the cacao and coffee strains did have one thing in common. Each had a lot in common with the other strains in its country and even on its continent.

So, for example, coffee strains from all over South America are very similar to each other but different from the coffee strains from Africa. And the opposite is true as well. African coffee strains are all pretty similar to each other but different from the South American strains. The same sort of thing is true for cacao strains as well.

You can think of coffee and cacao strains as the large, flightless birds of the yeast universe. Like a rhea, emu, or ostrich, they stay on their own continent. Wine yeasts are more like those chickens that are basically the same worldwide because humans have taken them along with them in their migrations.

The first step in their analysis was for Ludlow and coworkers was to get a hold of a bunch of different samples of cacao and coffee yeast strains from all over the world. They managed to get 78 cacao strains from 13 different countries and 67 coffee strains from 14 countries. The countries were from Central and South America, Africa, Indonesia and the Middle East.

The next step was to compare the genomes of these strains with each other and with the wine strains. They decided to use a technique called restriction site-associated DNA sequencing, or RAD-seq, that would give them an in depth look at around 3% of the yeast genome. As there was already a database with 35 wine strains that used the same method, Ludlow and coworkers only needed to generate data for their newly isolated strains.

These data revealed that coffee and cacao yeast strains were very different from one another. It also showed that the closer two coffee or cacao strains were to each other geographically, the closer they were together genetically. Using just these strains they could accurately predict the country of origin for a coffee yeast strain 79% of the time and 86% of the time for those associated with cacao.

The researchers next expanded the number of species they compared by using two additional databases. One included RAD-seq data from 262 strains from a wide variety of different places while the other contained another 57 strains.

This analysis generated 12 distinct groups of yeast, many of which had been identified before. Their new strains formed four new groups which they called South America Cacao, Africa Cacao, South America Coffee and Africa Coffee.

By comparing the four groups to the eight older ones, they were able to see that the new groups were not novel but instead were made up of mixtures of some of the other known groups. And who they shared alleles with depended on where the yeast strain was located. So, for example, the two South America groups shared alleles with the North American Oak group while the two African groups shared alleles with the Asian and European groups.

It looks like that, unlike with wine where there are a limited set of best yeast strains that most people use, the yeast strains involved with making chocolate and coffee are continent and even country specific. This suggests that people either took their wine yeast with them when they moved or that cross contamination throughout the world resulted in homogenization of wine yeasts. Sort of like the chickens Pacific Islanders brought with them whenever they settled on a new island.

It is a very different story for cacao and coffee yeast strains. While there was cross contamination within countries and even continents, there was no worldwide contamination. The rhea stayed in South America and the ostrich in Africa.

Wherever these strains come from, they work hard to make our chocolate, coffee, bread, wine, and beer. What an amazing bounty! Make some room, Dog, good ol’ Saccharomyces cerevisiae is joining you as man’s best friend.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: admixture, migration, population diversity

Lessons from Yeast: Poisoning Cancer

April 06, 2016

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In the book Dune, the mentat Thufir Hawat is captured by the evil Harkonnens and given a residual poison. He can only stay alive by getting a constant dose of the antidote. Once it is withdrawn, he will die.

A new study in the journal GENETICS by Dodgson and coworkers shows that the same sort of thing can happen to yeast that carry an extra chromosome. In this case, certain genes on the extra chromosome turn out to be like the residual poison. And a second gene turns out to be the antidote.

Once that second gene is deleted, the yeast cell dies. It has been deprived of its antidote.

This synthetic lethal phenotype isn’t just a cool finding in yeast either. Cancer cells invariably have extra and missing chromosomes. If scientists could find similar “antidote genes” in specific types of cancers and target them, then the cancer cell would die. And this would happen without damaging the other cells of the body that have a typical number of chromosomes.

The first thing these researchers did was to make separate yeast strains each with an extra chromosome I, V, VIII, IX, XI, XII, or XVI. The next step was to see what happens when every gene was deleted individually, one at a time, from each strain.

As expected, these yeast did pretty well when a gene on the extra chromosome was deleted. So, for example, a strain with an extra chromosome I tolerated a gene deleted from chromosome I. This makes sense as this just brings that gene back to its normal copy number.

But this was not the case with chromosomes VIII and XI. Here deleting genes on the extra chromosome often had a negative effect. This suggested that the screen probably had a high number of false positives and these researchers later confirmed this.

Likely reasons for the high number of false positives include the strain with the extra chromosome being W303 and the deletion strain being S288C, errors in the deletion collection itself, and what they refer to as neighboring gene effects. Basically this last one is the effect that deleting a gene has on nearby genes.

Once Dodgson and coworkers corrected for these problems, they found two broad sets of phenotypes – general and chromosome specific.

The general ones were the ones shared by most or all of the strains. These were deletions that affected the yeast no matter which chromosome they had an extra copy of.

For the most part, these genes were enriched for the Gene Ontology (GO) term vesicle-mediated transport, indicating that they have something to do with the transportation of substances in membrane-bounded vesicles. For example, deletion of MNN10, HOC1, and MNN11, genes all involved in protein transport and membrane-related processes, had a negative effect on many of the yeast strains with an extra chromosome. Consistent with this, brefeldin A, a drug that targets protein trafficking, negatively affected most of the strains.

Another gene that affected many of these strains when deleted was TPS1. This gene encodes a subunit of trehalose-6-phosphate synthase, a key enzyme for making trehalose, a molecule that helps yeast deal with stress. Perhaps not surprisingly, having an extra chromosome is stressful!

In addition to the genes that affect many strains with an extra chromosome, there were also genes that were chromosome specific. The best characterized of these was the EDE1 gene in the strain with an extra chromosome IX. Deleting EDE1 in this strain increased its doubling time by more than 80 minutes while only causing an increase of 5 minutes in the doubling time of wild type yeast. This was a severe phenotype in their assay.

They next tried to find which gene on chromosome IX might be responsible for the severe effect of deleting EDE1. Since EDE1 is known to be involved in endocytosis, they looked for genes involved in the same process. And they found one – PRK1.

The strain with a deleted EDE1 gene and an extra chromosome IX was rescued by deleting one copy of the PRK1 gene. The extra PRK1 gene was the poison and the EDE1 gene was the antidote.

If a similar pair could be found in cancers that often have the same set of extra chromosomes, then perhaps scientists could develop drugs that target an antidote gene. Now the cancer cells would die and the “normal” cells would be fine. Thanks again, yeast, for pointing us toward new ways to treat human disease.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight, Yeast and Human Disease

Tags: aneuploidy, cancer, synthetic lethal

Apply Now for the 2016 Yeast Genetics & Genomics Course

March 30, 2016

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For almost 50 years, the legendary Yeast Genetics & Genomics course has been taught each summer at Cold Spring Harbor Laboratory. (OK, the name didn’t include “Genomics” in the beginning…). The list of people who have taken the course reads like a Who’s Who of yeast research, including Nobel laureates and many of today’s leading scientists.

The application deadline is April 15th, so don’t miss your chance! Find all the details and application form here.

This year’s instructors – Grant Brown, Maitreya Dunham, and Marc Gartenberg – have designed a course (July 26 – August 15) that provides a comprehensive education in all things yeast, from classical genetics through up-to-the-minute genomics. Students will perform and interpret experiments, learning about things like:

• How to Find and Analyze Yeast Information Using SGD
• Isolation and Characterization of Mutants
• Transformation of Plasmids & Integrating DNAs
• Meiosis & Tetrad Dissection as well as mitotic recombination
• Synthetic Genetic Array Analysis
• Next-Gen. whole-genome and multiplexed DNA barcode sequencing
• Genome-based methods of analysis
• Visualization of yeast using light and fluorescence microscopy
• Exploring synthetic biology with CRISPR/CAS9-directed engineering of biosynthetic pathways

Techniques have been summarized in a completely updated course manual, which was recently published by CSHL Press.

Scientists who aren’t part of large, well-known yeast labs are especially encouraged to apply – for example, professors and instructors who want to incorporate yeast into their undergraduate genetics classrooms; scientists who want to transition from mathematical, computational, or engineering disciplines into bench science; and researchers from small labs or institutions where it would otherwise be difficult to learn the fundamentals of yeast genetics and genomics. Significant stipends (in the 30-50% range of total fees) are available to individuals expressing a need for financial support and who are selected into the course.

Besides its scientific content, the fun and camaraderie at the course is also legendary. In between all the hard work there are late-night chats at the bar and swimming at the beach. There’s a fierce competition between students at the various CSHL courses in the Plate Race, which is a relay in which teams have to carry stacks of 40 Petri dishes (used, of course). There’s also a sailboat trip, a microscopy contest, and a mysterious “Dr. Evil” lab!

Last year’s Yeast Genetics & Genomics Course was loads of fun – don’t miss out!

Categories: Announcements

Updated Genome Browser

March 27, 2016

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In an effort to provide a comprehensive view of sequence-based functional elements in Saccharomyces cerevisiae, we have upgraded our genome browser, and added new data tracks, to allow users to quickly and easily browse the information-rich yeast genome. We invite authors to work with us to integrate published data into our new JBrowse genome viewer pre- and/or post-publication. Please contact us if you are interested in participating or have questions and comments. Watch for the regular addition of new tracks to SGD’s JBrowse in the future!

Take a look at our newest video tutorial to get acquainted with JBrowse, and let us know if you have any questions or suggestions.

For more SGD Help Videos, visit our YouTube channel, and be sure to subscribe so you don’t miss anything!

Categories: Data updates, Website changes

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