Hey friends today. I am hanging out at Epcot's World Showcase I wanted to come out and try some more food and wine festivities mainly some adult beverages But also some snacks that I didn't get to try the last time we're here I did say before that we're gonna be coming multiple times because food and wine is massive And it's gonna take a while to get around the whole entire World Showcase at least trying one of everything But that's the plan. So let's go do this I waited till five o'clock to come out today just so I can keep cool So that's a plan or a tip for you if you want to actually avoid the high heat times come a little bit later in the evening It's gonna be nice and breezy and cool The last full video I did on food and wine it was opening day and we did a big variety of different items But like I said, there's so much that you can just keep coming back and getting more and more Today, I kind of just want to focus on some drinks and some light snacks You know, I don't want to keep on rotating, you know drink snack dessert food just a couple of things that I didn't get the last time and Continuing the food and wine series if you want to see that other video I'll put a link in the description for the opening day just in case some of the items that you were looking for We're in that video, but now I think I have a perfect spot that I want to start off at Right here at shimmering sips. They've got a couple of mimosas here And they've got a tropical mimosa a keen lime mimosa a blood orange mimosa and then a mimosa flight So we can try a couple two tree mimosas and that's a good way to start nice and light I'm not the biggest mimosa fan, but I can't turn down three mimosas especially the keen lime mimosa I find that to be the most interesting, but you know something that always catches my fancy is Shimmering sips like the name of it. I can't think of anything except for the Aladdin song I forget how it goes though I can show you the world Shining shimmering splendid A whole new world that's where I keep on thinking shimmering from because it's like that iconic line, you know, and yeah Now I got my mimosas and I'm ready to go and they look delicious. I'm probably gonna be very refreshing I am super red because I just shaved but holy moly. Look at that. I'm bald Not too sure which one I'm gonna start with maybe the Tropical mimosa which I think is that one because that one has sparkling wine passion food orange and gavi juices But I am most excited for the key line So I'm gonna wait to actually do that one. That'll be the last one. We'll start here then go here and then finish there Cheers Well, that's good. I think that is refreshing. It's a little bit heavy though Like you can see the frog that was on the top there That must be the mixture of all the different juices, but this is actually really refreshing mimosa Now for a little good old blood orange and this is just sparkling wine with blood orange juice Now I wonder if it's the same blood orange juice that I can get over in Morocco because I did get blood orange juice last time I was there just the orange juice on its own. I Don't know if these are any special kind of juices or if it's just tropical canna either way They're very delicious on a hot day at Epcot. Oh That was good. That was a lot better than the tropical one. Okay. All right. I'm gonna jump right to the key line here Key lime one. I don't know why I love key lime and key lime is very popular for a summertime No, they don't like that one enough to shock her blood orange winner winner chicken dinner The key lime has a lot of tart to it and I wouldn't expect that I thought maybe the blood orange would be very tardy, but this is my favorite one now We're gonna finish up these bad boys and get moving along Now it's time to jump from mimosas to a unique beer and you know, I love myself some beer Now this one definitely is unique by my standards and I'm very excited to try because I kind of like both things that are part of the beer Here at the noodle exchange. They've got the you pond brothers green tea pale ale a little green tea and a little pale ale Not too shabby and it's actually brewed right here in Titusville, Florida. Huh? It's like a local brew Looks like there's some lightning in the area So they closed down the outside boots and now you actually have to pay where you normally pick things up Inside the actual noodle exchange and you'll see that at a couple different boots whenever there's actually weather or lightning They usually relocate all their services indoors Here it is and I love green tea. I love Arizona green tea. You know the big 99 cent cans I went somewhere's where that's all I drank actually them and then they have that energy one the rx energy I love that too, but green tea and beer together. I don't know how that's gonna taste, but we're gonna find out That is good see I could have drank in a big one of this I got the five ounce one But I could have definitely gotten the 12 ounce and it's delicious Actually, this might be my go-to beer for the festival because this is like this is really good But this is high up there for me. I mean it doesn't have a sweet taste to it But it's got a perfect blend of you know beer without that Hoppiness, you know, I mean so if you like hoppy beers this probably isn't your way But if you like less hoppy then green tea beer all the way, huh? Who would have known I feel like the best way to get a full flavor of a beer is a big gulp So that way you can like taste everything you taste all the notes and all the hops in the Barley's You know I mean sipping it you really don't I don't I don't feel like it does it for me So usually that means the five ounce beers are kind of like a two-sip beer for me one sip and then the finisher Now we're gonna head on into the apple seed orchard and this is the only indoor boot this year They don't have a festival center, but at least we get to walk through the garden area here It's so beautiful back here, and I love licelli a it's my favorite steakhouse here on Property well in the actual Walt Disney world, and it looks like we did it just in time a port mission. It's raining Seek shelter save the queen Back to the boat oh boy it's coming up It's like really really coming hard We made it Wow that rain turned on like a light switch it is a downpour right now Well, this is really pretty in here I didn't come in here the opening night So this is my first time and this was the old Canada show and they kind of just use it as a festival center during the festival Look at that. I really really like it here, and it smells like apples so much I'm actually really happy about hanging out in here while it rains I feel like that time perfectly and I'm even always gonna rain today I don't look before I leave the house anymore I kind of just expect it to rain at some point in time for a little bit and then it gets dry again And I guess it just happened exactly at that time and here we are Here is a look at everything they have to offer on the menu They've got apple crumb tart apple chips, and then all the way at the bottom I see that they have a flight so of course if they got a flight We're gonna go with the flight because then we can try the best of all three. I Feel like the last time that I came to the apple seed orchard It was mostly ciders, and I'm not a big fan of ciders, so I skipped over it I'm opening day, but then I found out they have a whole entire beer flight here Well, look at I'm starting to fog up a little bit could see my eyes my eyes my brand And that's what we decided on getting was the beer flight They've got a big storm apple blonde ale, then they got a three daughters apple pecan brown ale And then they got a apple-y ever after sour ale. I like the name apple-y ever after so you get credits for that And then also we got the apple tart only because it looks so delicious I couldn't pass it up, and I believe it's hot because it looks like they pulled it out of a warming Container there, so that's gonna be good I think I'm gonna start off with the apple blonde ale which I would assume is the first one in this direction I feel like the apple-y ever after is gonna be the lightest beer, and I Didn't know they didn't have any like chairs to sit down in here, so I just took a knee and I'm excited to try this one That's pretty good, I mean, I don't taste much apple in there now I'm gonna hop right into the pecan and on the apple pecan. I'm excited for it Oh, yeah That's the one for me. This is it. Oh, yeah Mm-hmm And now the apple-y ever after which is definitely a very light light beer It's actually a sour, so this is gonna be I'm not a big fan of sour, so we already know this Yeah, well there is sour very sour so the apple pecan winner winner chicken dinner. This is the one I Now we're gonna dive into the tart. I grabbed a fork But I feel like forks are really ineffective when it comes to eating tarts because tarts are hard So I think I'm gonna try to just like eat it like a cookie and see how that works out for me There is a good chance. This is gonna make a big mess, so I'm prepared Mmm No mess no mess no fuss and it's very delicious. This is really really good. You can eat it like a cookie Yeah You would think like you'd get like an apple compel in the middle there But no, it's actually just like brown sugar like an actual apple like Chris so very delicious I mean, I'm shocked with this and the tart is very soft like if I knew it was this soft I probably would have eaten it with a fork, but it held together. It did its job. I Do feel like I have an apple overload though because I'm eating the tart now and then I'm washing it down with the apple sour, so Cheers I Got a little bit of this left Thank you. I have discovered though The tart will not hold up after UV in so many bites of it And there are some apple compos in there take a look at that the rest of it's all crumb, but there are apple compo in there So very good Now we're all finished up. We have to go check the situation out Sounds like it's still raining and it's probably gonna be raining for a while I don't think this is something that's gonna pass by so we might have Kind of cornered ourselves in here cornered ourselves and we got ourselves all the apple seed. Oh Yeah, this is not looking promising. Oh the lightning We might be here for a little bit That's impressive This weather makes me want to go home and curl up with the book and I think the plants are gonna love this Well, I waited out the rain for probably about a good 30 35 minutes and it's not stopping So I think we're just gonna have to go out there get a little wet and move along world showcase I wish there was somewhere I could stop and buy an umbrella because I would do that I didn't bring an umbrella with me today. I normally don't I always just forget about it But yeah, maybe I should start carrying an umbrella. It might be wise But wait, I see those water droplets are getting less and less and the sky is actually looking not too bad there So maybe we'll just give it another maybe five minutes Just a little raindrops. We're gonna be making it just fine I didn't want to ruin my new hat though because this is really snazzy. This is from Disneyland So I don't know if I'm ever gonna be able to get a chance to have another one So that's why I wanted to wait till it was like just a little drizzle right now. We're good Rain doesn't stop everybody though. A lot of people are still out and about enjoying the festivities Including me, but look at that. It definitely looks like it's definitely clearing up And a good thing about that means there would be no long lines for the boots I came out on the weekday because weekend is almost impossible lines are so long to get anything So weekday and now a rainstorm. We might be able to just walk up to some of the boots I usually have a long long line Like France that usually has a long long line Usually you'd be getting a line like right here and it looks like there is like just a very short line So now we can grab something from the France booth because normally I don't wait because if it's like that long of a line It's probably not worth it, but now we can get something. I'm I'm excited about this. I Wanted to get something new on the menu here. I've had a lot of this stuff in the past the croissant escargot Which absolutely was horrible. I did not like that at all I had the strawberry rose mimosa and I even had the La Passion martini slushie before I don't think that's new So I think the only new thing on the menu is the crème brûlée Grand Marnay. Oh, I like the way it sounds Crème brûlée Grand Marnay Crème brûlée Grand Marnay Not only do they got some fancy crème brûlée Grand Marnay But they also have some fancy water Evillon and I think I might be getting a little water crème brûlée mix Look at that an actual can too. You might think this was like a white clob just by the can But this is non-alcoholic and it's you know sparkling water with Cucumber and mint and also magnesium and zinc so that'll go well with my crème brûlée and Here is the crème brûlée and this looks really good now This does have alcohol on it. So if you guys plan on getting this don't give it to the kids Don't give it to them But for all the overage folk perfect. Oh Wow, you can taste the you can taste the liquor in there like you could taste it It's very good though. It's very good crème brûlée. I like it But you can definitely taste that orange liqueur in there. Oh, yeah, I Like crème brûlée. I like going to the California grill and actually getting the seasonal one I don't know if they changed up the seasonal one since they reopened But I'm not to go back and check it out, but this stuff is good. This is really really good It also reminds me of the eclair at Disney Springs or at amorettes that they have going on for the flavors of Florida because That has a grand marnée like inside of the eclair. So this is kind of just like it if you like that you'd like this Now we got a crack open the water here The fancy water it just looks so good. I was like, well, I think I might get that it's gonna be refreshing Well, here we go. We're gonna give it a go Tastes like toothpaste Like I don't know what else to say about it. Yeah, you can taste the cucumber in the mint and Yeah, I don't know if I enjoy it that much. I'm not a big sparkling water fan But it's only four dollars and I think that's the same price as a Dasani in the the store So this is a good deal and it comes in a can and everything And maybe I would get it just to get that magnesium and zinc still a little refreshing but not really like I said I'm not big into this sparkling water While I was feeding my mind because that's what it says feed your mind I was thinking about how like this is a fancy Jello shot, you know what I mean? Like so whenever they make like desserts with like alcohol on them I just feel like it's like a a fancier Jello shot, you know and Maybe I'll like finish up this and actually get moving along to my next spot Honestly, I feel like there's enough liquor in there like that it is an actual shot inside there So we're gonna take it like a Jello shot finish it up That's the way you do it That is the way I went to go throw away my items and it looks like somebody lost their sandal so sad There's one person or one sandal out there without its match The cool thing about that sandal though is that didn't fall off on top of the trash can It fell off like right here and somebody picked it up and put it on a trash can So that somebody can come over and collect it and return it to lost and found and I think that's very nice We have made our way to Japan and I wasn't able to get anything from this booth last time because there was a long line so today I think I'm gonna get the chicken bun teriyaki chicken bun and it comes with the Chicken vegetables and teriyaki sauce. It looks pretty good, but to drink I'm kind of torn because they have a Lemon drop which looks amazing because it's actually garnished with lemon jelly And then they have this platinum sake and it looks like it's a dry with the fruity flavor and then the rice logger I think I had the rice logger before so either one of these is gonna be the one and I don't know which one The rain doesn't kill this line though. Looks like it's still a long line Even though we're here later in the day and it just downpoured. I Decided on getting the lemon drop instead of the sake and boy Oh boy, let me tell you the weather has changed so quick. It was very rainy and now Golden hour. It is so beautiful here at World Showcase Look at this. I feel like I need the cheers World Showcase because it's just so beautiful out Cheers cheers friends Now we're gonna try the lemon drop now. This is a little bit bigger of a drink than I thought and I don't know if I made a mistake or if I should have just got the sake, but I'm excited. I like lemon drops That's a kick. Holy moly. Oh, these are the shivers Give you the shivers It is very very sweet and very lemony I'm gonna mix in the lemon jam there Get a little extra and then here's my teriyaki, but I like how they have it just in a paper like that Oh, this looks good. I think I might bite into this now Teriyaki buns and lemon drops in Japan at FCon Oh That's good. Oh boy That's the good stuff right there very very delicious Kind of reminds me of a hot pocket, but very soft and like a bun, you know But wow, this is really really good six dollars for this one though I feel like I should get maybe two for like eight dollars. I would do that two for eight would be good Well, the teriyaki bun was fantastic at Japan the lemon drop Not so much. I think next time I should have got the sake. So we're gonna move along. We're losing daylight here I can't even believe it says bright out and it's almost nine o'clock That's awesome. Oh It looks like they've got the American Gardens bandstand the champagne orchestra coming on at 830 Which is right now 830. Maybe we'll grab ourselves a seat and watch the show Listen we are the champagne orchestra We're gonna have some fun with you guys tonight that we're gonna do some songs that hopefully you recognize But if you don't it's probably gonna be because we did it in a different language or we made it kind of jazzy But that doesn't matter. We still want you to have fun with us. So once you clap it along Okay, don't you dance in your seats if you feel the need you cool with that? Yeah All right, so this is a song I'm guessing you've heard before but probably not like this So let's see if you notice what I Have to say the champagne orchestra was amazing Did you see how some of the performers on stage were with that? Sunset happening in the background that was beautiful and I loved all their music choices when they sang the boots are made for walking in a different language I was like, oh, I know this song and then I was like, oh wait, I love this song and I love the way they did it Very cool, and I'm happy that we have some good performances happening.  I was thinking about getting something from the Italy booth But they have one poor person out here and there was a gigantic line going almost all the way back to America I feel really bad for her. She's working really hard And I would say this is like 50 minutes probably just to get something to eat And I think they need to open up those other sides. Like that's a lot for one person. I Decided to stop in and take a look at some of the merchandise and there's a nice beauty in the beast selection here But one thing that caught my eye is chip here and at first I was like they have a new chip mug But no, this is way better. This is in measuring cups. How crazy is that look? It actually comes together as measuring cups one cup half a cup one third cup one fourth cup I think I need to get this. I mean, this is one of the coolest things. I think I've ever saw I'm sorry, but there was no way that that chip measuring cups that was not coming home with me I think that is the coolest thing ever and for 21 dollars worth my discount. You can't beat that now I'm a little worried to this glass. It's not like a plastic So like it might break, but I'll buy another one if it does And with that, I think I am done here. I'm happy. I got my chip measuring cup set I think that is the coolest thing ever and we get to try some new drinks and some new snacks There's still a lot of boots that I have not been to it. I haven't been to Italy China Mexico And all the ones that haven't opened up yet So like I said, this is gonna be a three or four part video series where I kind of just take a day and come out and Take my ways around Epcot's World Showcase enjoying all the festival Activities, I guess that's what you call them or festival boots, but overall like I said, it was an amazing day So I hope you enjoyed the video. I enjoyed making it. We'll see you next time. Bye Before we go take a look at this this is a fast-pass machine and the screen is on I wonder if we can Nope no selections. I can only wish though


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Hi, my name is Maria Doyle and I'm the application and training specialist for the Peter McCown Cancer Centre in Melbourne, Australia. Today I'm going to take you through the hands-on tutorial for CRISPR screen analysis. So in this tutorial, we're going to go through the steps for processing CRISPR screen data. We're going to identify essential genes across experimental conditions. There are some introductory slides that you can find in the link in the material here and also a bit of background in the tutorial website. Okay, so today what we're going to cover is we'll upload some data, some fast Q files of raw reads and we'll check these raw reads for quality. We'll trim adapter sequences and then we will count the guides we have and then we will test for differential abundance of guides across two conditions. So in this case a treatment and a control. Okay, so the data we're going to use in this tutorial is from a CRISPR screen from this paper here. We've got three samples we're going to use. We've got a baseline sample taken at time zero, time zero control. We've also got a sample that's been treated with drug for eight days, the APR drug, and we've got a control sample that's been treated with vehicle for eight days. That's our vehicle sample. And the aim here is to identify genes whose knockout increases the cancer cells sensitivity to the drug. So we will use fast Q files containing 1% of reads from the original data to demonstrate demonstrate the read processing steps. Okay. So what I am going to do is to go to galaxy. So I'm going to set up one window that has galaxy. And you can use here I'm going to use galaxy Australia, you could also use galaxy Europe to do this tutorial. And so I have one window with the material and one with galaxy. Okay. So go ahead and log into galaxy if you're not already logged in, I am. And then in a new history. I'm going to give it a name.  And then we will load in our data. So here are our three fast Q files. So I'm going to copy this, the links for these files, go to upload data in galaxy. And I'm going to click on the rule based and upload these data, these balls as a collection. And what I'm pasting in here is actually two columns, one with the names of the samples and ones with the files names. And I'm going to create a collection to make it easier to keep the data organized in the history and to save on some file renaming. Okay, so I'm going to click build. Okay, and then a window should pop up. I need to under the rules, click add modified column definitions. I need to specify a name for each of the files. So that's the list identifier. And I'm going to say that is in column a. So these are the names for my samples. And then I need to specify the URL so where the files are located. So that's in column B. So apply that. The final thing I need to do is give this collection a name. I'm going to call it fast cues. And I'm going to upload that. Okay, and you should see it appearing here in your history. So at the moment it's great means it's a job waiting to run waiting to upload. And when it starts uploading, it'll turn yellow. And then when it's finished uploading, it'll be green. So now it's turned yellow. So you can see, so it's uploading there and it's a list. So this is what a collection is. It's like a folder. So a list and containing multiple files. Okay, and then in the tutorial website, you can see the steps we have gone through here. That should upload shortly. Okay, great. It's great and see we've got a list. So our collection with three items by click on the name. There's our three samples there. You can click on the name of each of the files. And see it's fast cue format and get a little peek into it. So these are our fast cue reads. So if we click on the eyeball, I can have a look at the file in the main pane. And I can close off this panel on the left. Okay, just get a bit more space. So these are our reads. So each of these four lines is one read. So you've got the first line is the read name. And we've got a sequence for the read. So in this case, there's 75 bases, but space surplus. And then we've got the quality score for each of the bases. Okay. So I'll go back because what we'll do now is we'll use a program called fast QC to have a look at the quality of these reads. So I'm typing fast QC in the tool search bar here. And then clicking on fast QC to open up that tool. Okay. So because we are using this collection list, I need, we need to click on the data set collection icon. And then select our collection here. Fast cues. And we also need, we don't need it, but we're going to upload a file containing the, some adapter sequences, including our CRISPR adapter sequences. So we can have a look at these in the raw data. Okay, so we're going to upload this file here. So I'm going to copy that. Go to the upload data. So this time, I'm not making a collection out of that. This fall is just a single file. So I'm going to use pace fetch and paste in the link to this adapters file here and click start. Close and that should appear here in green when it's uploaded. So to explain here with CRISPR screen data, we're interested in the single guides. So these are these sequences in blue. See a minute of it. So sequences in blue. And this, the read the spaces surrounding them in red are the adapter sequences that we don't care about and want to remove. We're going to upload this adapter list file so we can have a look at these adapter sequences in our data. Make that a bit smaller. Okay, so we've selected our fast cues. Now we are going to select our adapters file. So the, as it says here fast you see will explicitly look for these adapters. And if we want to peek in this file, we can see what it is. So the first four, one, two, three, four, the first four are sequences fast you see will look for by default. So we'll just include those and then we've added in two more that are CRISPR sequences. So these regions in red around the guide. Okay, so I'm going to run that. And what we'll do is so fast you see will output a report for each of our three samples. So we can use a tool called multi you see search for in the toolbar to aggregate these multiple reports into a single one to make it easier to view the results. You've got many samples. So we tell multi qc what type of what tool reviews to generate the reports we want to summarize. So in that case it's fast you see it's raw data. It's in a, it's in a collection. So this raw data here. And we click execute. Okay, and that's waiting to run there and it's running now. And this will give us one. And then on the web page that will summarize the fast you see results for our three samples. It's just finishing now. And I'm actually I'm going to add a tag to make it easier to see what this is in the history. So there's a button here for creating tags. So if I type a hash, and then I can say call it fast qc on trend. That just makes it easier to for me to know in the history what multi qc is summarizing there. Click on the eyeball we can have a look at our fast qc report. We can see how many sequences we have for each of our samples. Just got a small amount here for this data set for the tutorial. There is a galaxy tutorial on quality control that goes through these back to see plot and in detail. We're here. We just are going to have a look at the base quality. So we can see that along our read length. So we can see our readers 75 bases. We've got good quality for our basis along the rates. So the sequencing has been has been good. The other thing we also really want to have a look at is the adapter content. So if we go down to our adapter content section. So what we can see is the doctors present and this is using the file of the adapter list file that we supplied for to fast qc. So we can see that at the beginning. So again, this is the length of our reads. And at the beginning of the reads, we can see some of the five prime CRISPR adapter has been detected at different positions in the reads. That's because of the staggered and the stagger sequence that's present in. And it's using this protocol explained in the tutorial. And we can see at the ends of our reads from about 40 onwards we're starting to see the CRISPR three prime adapter. Slightly in less amount than the five prime, because the three prime and basically tends to be a bit lower towards the ends of reads in general. Okay, but that's picking up the adapters that we expect. So we've got some examples of CRISPR adapters and we can also see the small little bit of a lumina adapter present there which means you've got some short sequences or a small amount of primer timer there. Okay. Okay, so now we'll trim the adapters from our reads and we'll use cut adapt tool for that. So I'll type that in the toolbar. So our data is single and again we need to say it's a collection. I'm going to input our fast cues. And then we're going to say, tell kit cut it out we want to trim adapters from the front. So we are going to trim. This is the adapter sequence here. Enter that sequence. And I will select to output the report. And explain. Okay, so, so here. So as I said our guides are these regions in blue. We are telling cut adapter we want to trim this front part to the bit in front of the blue by putting in the five prime adapter sequence. So if we could, then we could also trim off the, the three prime adapters or shorten the reads to just have the guides, what magic, the tool we're going to use for the analysis will take just the guide sequence from the front of the adapters from the front of the adapters. So we really only need to trim these by prime adapters. Okay, so cut adapters finished running as before, we can summarize the cut adapt report using multi QC. So we'll do that. Okay, need to say the input is cut adapt. And it is cut adapt report we're going to summarize. This is just so we can have a look at the trimming that's been done. That one's waiting to run. And while it's waiting to run, I can add a tag. So I'm going to add a tag cut adapt report. Okay, and that's there and that's like just to make it easier to see which, again, what the multi QC is summarizing. That's wrong. So we can have a look at the multi QC report. So the trimming report from cut adapt. So here we can see for each of our samples we've had a similar amount trimmed by 34%. That's been trimmed from the front from the firecrime man of each read. And see that we've got no none of our reads have been dropped to about 100% because we just told cut it off to trim reads from the front. And then in this trim sequence lens we can see how many reads with the different lengths of drafters have been trimmed. Okay, so if I click, you click on these buttons can get the plots to show whoops. Okay, so we can have a look at say the counts, the numbers of reads that have been trimmed for at each of these lens so we can see we've got many 20 21 etc basis trim from the front of the reads up to 31 basis. And that's actually what we expect for this. So this is a sequencing protocol because of the use of these stagger sequences, and that's explained in more in the link here, you can see what those stagger sequences are. And so this is what we expect to see for the trimming for this data set. We can also see that the samples are fairly similar. It's maybe a bit more trimming and performed for the control the T zero control sample. So that looks pretty good. And if you want, then you can go on and run fast QC and multi QC again on the trimmed data set. So on the output of could adapt the fast Q's that could adapt has produced just to see how the trimming affects the adapters. And we can see that there's now no adapter, no CRISPR adapter present in the first 20 basis of the reads. It's been removed. Okay. So now we can go and use magic. So that's a toolkit for CRISPR screen analysis. And we will use magic count first. So that will count how many reads we have for each guide. And we will do that. So first we need to import our library file. So magic count will count how many reads we have for each of these guides in the library file we supply. So this tutorial this data set we're using the Brunello library which is a human genome wide and knockout library. So I'm going to upload the Brunello library file. So I use paste fetch here. Okay, and that should appear in history. So I'm going to upload in the meantime. I will search for magic count. So this is it. So what we're going to input here is we are going to input again our collection of our trend reads. So that's the output of could adapt. So we can select our library file need to wait for it to finish uploading. And this is the Brunello file this has just over 77,000 guides in this library. So there's four guides for every targeting every human gene. And then there's a thousand non targeting controls. This is what the fall looks like. The first column is an easy ID identifier for each of the guide RNAs. The second column contains the sequence of each of the guides. And the third column has what gene it's targeting. So as you can see, the first four rows are for guides that are targeting the a one bg gene. Okay, I've got 77,000 of them. So I can now select Brunello as as it's uploaded. And in the output options, I'm going to select output the summary statistics, and also plots. Okay, and execute that. Okay, so and that's going to output three files. So counts for every guide, the summary statistics and then the plots. However, this is using 1% of the original data so these aren't as maybe useful for you to see as what are compared to what a real data set looks like. So we're going to, at this point now import these three files for the full samples. So that's these links here. So you can see what that looks like so I'm going to copy those links. So data. Paste in the links here. Start that. Okay, so these three files are the same as these three, except in this case this is 1% of the data and this is the full files full fast key files. And the tutorial website goes through the different columns that are in each of these output files and what they are for so I'll just point out some of the key things. Okay, great. Okay, so first we'll have a look at the count summary file. So it's that one here. I'll click on the eyeball to view it. Okay, great. So we'll name the label of our sample. And this tells us how many reads we had in the files we inputted. So in this case it's eight new 20 million. And then how many then mapped and what percentage mapped. So we are looking for at least 60%. But we get 80%. So that's very good. It also tells us how many guides we have, and how many have zero counts how many have no no reads mapping to them at all. So we hope that to be no more than 1%. So that's okay for this data set. And then we also get this genie index, which is important metric to have a look at. So this is a measure of how even the distribution of guides are. So if we are doing a positive selection screen. It would be okay to have high values here because we just expect only a few cells view guides to be present. We're doing a negative selection screen. So we want to see small genie index values here. And that's what we say so that's good. Okay, there's a bit more on the genie index explained here. Okay, so these metrics for these samples are pretty good. And we can also have a look at the plots that magic count produces here. So if we click on the eyeball. Okay, we get some box plots that shows us that the distribution is pretty pretty similar for our samples. There's a little bit more variability at the day eight time points compared to time zero and distribution is pretty similar. Again in this histogram type plot. The control is a bit lower because there's more more points plotted. And we also get a PCA plot. This would be useful if we had. So we can see if our replicates are grouping together. And if we have a good separation between our different samples are different groups. We also get a little heat map showing the hierarchical clustering of our samples. And this helps us to see that are in this data set are again our day eight time points are a bit more similar than our time zero baseline sample. Okay. Okay, so now that we've generated our counts. So just have a look this is actual counts follow we're going to go on and use. So where we've got a row for every guide, same what gene, and it's targeting, and then also how many counts that guide has for each of our, in this case three samples. So now we're going to input that into magic test to be able to compare our conditions of interest. So, like a magic test. Okay, so here. So from magic test what we want to do is input the guidance. And we will specify that our treated sample is in our first column. So it's actually caught it in the first column if that's column zero. And we want to compare that to our controls up to our vehicle sample. So drug to vehicle, then column one then. And we want to output what we want to put we want to put a normalize cancel because that's useful if you want to plot the normalize can values for different genes and guides for those genes. And we'll also choose to output the plot. Okay, so execute that. Just to say that there is a magic test is using an algorithm called robust ranking aggregation so the RA. And then this is a visual of what it's doing. And in essence, it's helping us to identify essential essential genes by identifying guides that are highly ranked, either highly enriched or highly depleted in our treatment versus our control. Okay, and so note on if you have biological or technical replicates. You can input them into magic and can separate the names by comma. There are biological replicates for the samples for the experiments used here but we just for the sake of time and in the tutorial you're using just the single samples. Okay, and what we're going to see is that magic test outputs a gene summary file and guide summary file. So those output also the normalize cancel and we get a PDF report with some plots. Okay. And again, the columns in the different files are explained here in the website. And we'll have a look at the gene summary file. Okay, so here we get a row for each gene so it's 20,000. Okay, so in this, you can see every row is a gene. And we get the number of guides that are targeting that gene as well as metrics. So, our score p value FDR and ranking off that gene, both for negative selection and for positive selection. And this is a negative selection screen we're looking for genes and that are where the guides have decreased and with the treatment compared to control because we're interested in and drug sensitivity. Okay. And we also get a CSG RNA summary form. So use the peep. We have a view here. In this case we have a row for every guide so go over 77,000. And this gives us the counts for the guide in the treated and the control condition. So if we want to have a look at the individual guides and how they're performing. Okay, and then the PDF report we get output. This gives us plots of the top 10. And then we have a list of the top 10 genes. So these are ranked by the by magics or a score or p value. You can think of the RRA score is similar to p value. And that it's showing us the most significantly ranked genes. Okay. So here we get our top 10 genes. And then we get the results whether we look at the RRA score or the p value. And then we get plots for these top 10 genes. Or their guides. So every gene has four guides. And so we see how the abundance of those guides is changing in the control compared to the treatment of our drug and our vehicle. As I said, we're interested in guides that are dropping out that are increasing the cell sensitivity to the drug for this particular experiment. So we would hope to see that these four guides for vehicle are all higher than what they are in the drug. So if we look though, we can see that this may not be the case for every gene in our in our top 10 samples. So if we look at this FL1 gene, this is the fourth top negatively selected gene. But if we look at the actual guides, we can say that one guide is decreased. It's very decreased in drug compared to vehicle control. But one of the other guys is slightly increased and two of the others aren't changing that much. So by looking at these counts for this, for the individual guides, we can see maybe we wouldn't have as much confidence in that gene being one of our key genes, essential genes here compared to some of the others. So it's worth having a look at the individual guide values. Okay. Okay. And something that's quite commonly done is to create volcano plots for results. So we can do that with the volcano plot tool in Galaxy. And we first need to prepare reformat the gene summary file so we can create the volcano plot. Because as we saw in the gene summary file, we've got p values for both negative and for positive and logful change values as well. So we really just want four columns like this for the volcano plot gene, one p value, one FDF, one adjusted p value and a logful change column. So we can use. So, so what we're going to do is basically described here. So we are going to select if the negative selected p value is smaller than the positively selected p value. That means the gene is negative selected. So we use that p value. And if the negative p selected p value is larger than the positive p value, the gene is actually positively selected. So we are going to use all to select to create these p value and logful change columns. So I'm going to copy that. And go to the octal. Okay, so it's the gene summary file we want to reformat. Okay, and that's going to convert our gene summary file into this. Then we can use. We have a look here. We can see we've got our four columns now. And we just need to specify what our false discovery rate column is. Our p value. And logful change. And our gene labels. And then we can ask it to label our, for example, our top 10 most significant genes and execute that. Okay, we have a look at the results. Okay, we get a volcano part produced. We can see significance on the y axis. And logful change on the X. And we've got our positively selected genes to the right of the zero. And our negatively selected genes to the left. So in this case, these are negative selective genes of interest. And if we want to identify pathways, central pathways between our conditions between our drug treatment and vehicle control. And then we can use. Many different tools we could use, but we use one called FG SCA, which stands for fast gene set enrichment analysis. What we need for that though is a follow up pathways. So there are. For human there are hallmark pathways available from the Broad MCDB. Website. So there's one of these hallmark fall we have here. So we'll import that. And actually, I should have set the type to tabular, but if you do what I did, you can actually change the data type then and set it to tabular here through the pencil. And because it needs to be tabular format for the FG SCA till. So it should be in between for the tool to stop running to upload. Okay. So there we go. Okay. And now we are going to actually, just before we use FG SCA, we need to prepare our gene summary for us. So we need just the gene symbols and the negative score. So from the gene summary for so from here. Because we're most interested in the negatively selected genes for this experiment, we're just going to chop out this, the gene and that negative score column. So I'm going to use the cut columns of a table to and use that to select first and third columns from our gene summary file. Okay, we have a look. You can see, we've just got those two columns there. And now, if we look for FG SCA to. Okay, we are going to input this. So these are the genes, their scores to rank by. We are then going to input our pathways or our follow with our gene sets, and just to show you what that looks like here. So everywhere was a pathway. The name of the pathway is a link to more information on the pathway from the GMC DP website. The genes that are members of that pathway are in the subsequent columns. Okay. We'll say we're interested in gene set of size 15 and above. So we're not too small. And we will have lots. Okay. We get two outputs here. So then we get a ranked list of pathways and also PDF file containing some plots. So if we look at the ranked category list, we can see we've got pathways with their p values, just a p values, and we can see top pathway is oxidative phosphorylation. And we also get some plots then that show us. So how these genes in the gene sets. They are in our ranked list of, of genes, sort of genes being ranked from left to right, and shows us how these gene sets, for example, from the oxidative phosphorylation are ranked in our data. Okay. And there's another tutorial in galaxy called genes to pathways that gives a bit more information on this FG SCA tool. So if you have, if you want to use multiple conditions. So there's another module for magic called magic Emily, which you can choose to run. So the one we've been using magic test only allows you to compare two conditions. So for example what we were doing our drug versus our vehicle at time point eight. So if you had more than that more complex designs, you could use this magic Emily algorithm. And it is slower to run so it takes about 30 minutes or so on this data set so we won't run it here you can run that yourself if you like. So I want to note that instead of outputting scores for both positive and negative selection, it outputs a single value, a beta score, and a negative beta score indicates negative selection and positive score indicates positive selection. Okay, and for that, the Emily, you create sign matrix as one example there, and more information on the magic website. Okay. And again, you can visualize your results from the Emily using volcano pots. Okay, so hopefully this tutorial has helped you see how you can analyze CRISPR screen data. So using standard sequencing tools such as fast QC multi QC adapter trimming tools I could adapt. And then how you can use the magic toolkit to count and counter guides, and to test for differences in guide abundance and across different areas. And then also how you can create some downstream visualization such as volcano plots and perform some pathway analysis with tools like FGSA. Okay. And there's some links here if you want to get help with this. If you have any feedback, we, there's a little form here would love it if you would complete that. And thank you very much for listening and taking part in this tutorial today.


So, um, yeah, like Coby said earlier, uh, Tamron and Randall were meant to be here giving a presentation, um, building modern service-oriented architectures. I can't even get through that without trying to falsely. So yeah, so they asked me to give it on their behalf. So my name's Tamron. I'm Randall Thomas. And so we were too busy enjoying our whiskey last night to really put this presentation together for you.  So that just sounded great to me. So yeah, woo-hoo. You can all thank me, Randall, later. Except for the fact that exactly none of that's happening. So, um, Rain, where are you? Steven Baker, where are you? Get up here. And Eric. So, if you've been to RubyConf and you've seen our, uh, so you think you can code panel, where we basically just get up here and argue with each other, that's pretty much what we're gonna do. So, hope you enjoy it. Oh, we're gonna take a, we're, we'd like to take questions. So, if there's anything that you would like to know how not to do, feel free to just toss a hand up and we'll tell you how not to do it, because that's what we're really, really good at. How do you not write Ben String? I'm actually gonna give this one to Rain. I can't think of a case where we'd ever not want to write Ben String. So I can't answer that. Literally, you literally always want Ben String. All the time. And I want whiskey, all right. So, have you guys, uh, have you guys seen the recent blog post about Unclerc and if Rails is possibly failing? No. Anyone, have anyone seen that? No. Don't be silly.  It was never successful in others' place. Yeah. It never scaled. Right, exactly, it never scaled. It wasn't web-scaled. It was just, it was just a thing that tied us over between Koval and Nub. No. Right, yeah. It was on Rails. Like what? We're about to intercal. Intercal, right? Intercal on stilts. What was that? What did that mean? Intercal on stilts. Yes. So this is only going to work if you guys ask questions. So, start doing that. We're happy. We're going to do a drink. This is, uh, this is something, I don't actually know exactly what it is, but it's made by experiments. Hogshead. You mean, it's Hogshead? Yeah. Yeah, you can get it in back. We have a question here. Yeah. This is semi-serious, so you can put it up. We have another question in the back. Do it. Go ahead. Okay. Let me, let me just bring my mic around. I can yell. Okay. Make Kolton do it. Yeah, where's Kolton? Do all of them get to work here? Yeah, where's Kolton? Kolton. Where are you at? Kolton to charge. I didn't even have a question. But I have a mic. I haven't asked yet. Yeah. Okay, so I wanted to know, uh, Twitter switched off of, uh, Rails mostly. Go ahead. Um, go ahead. I want to know, is it, is it possible to make a scale at that level? Yeah. Or do we have to go on to something else? Serious questions or no, or serious answers or no? Oh, I've got a, we've got a work, it's an autograph at work that does 3 billion requests a week. All. All of it. That's it. I mean, I don't know how many Twitter does a week, so. Audience participation. I process at least 15% of Twitter every day. Every day. All day long. 24 hours a day. Good job. How much staff? In Ruby. What? Staff? Yeah. Uh, we're up to, well we weren't two for a while. We're up to seven. Oh my God. Yeah, that's amazing. How many in your team? How many in your process? When are you moving to Skalton? Oh, actually our intern took over for two Rails developers that left. So. Require. Require. Yeah, Colton's here. So if anybody needs a drink. Require relative versus require with load path, mainly. If you make a load path, then you're doing a good job. Great job. Do it. Every gem. Make sure, make sure there's like other gems too. Yeah, other gems too. Our spec did that once, it was great. Yeah, and personally, I would really, I would like to request that everyone use Unship to do that. Oh yeah, I forgot about that. Here are you guys? Find other things that you don't want there and then remove them. Don't ever forget that you have access to Rails. You can change the state of anything. Hold on, hold on, hold on, hold on. Here are you guys? I just said that, I'm sorry. That's fine, we're just some people, it's not a big deal. Just some people. And if you want to go for bonus points, delete stuff out of loaded features. So what questions should we answer? Sorry, seriously though. I'm Ben, I've been doing Ruby for a few years. Ben is blithing not blaving.  My name is Ben Blithing. What blithing? Say it slow. Blivee. All right, the other thing that Ben does in the community, Ben and a guy named Shane Becker actually created the first conference to run in Seattle. Actually, second because RubyConf was held there in 2002. So anyway, so Ben and Shane Becker put together a Cascadia Ruby conference and they announced it recently. It's going to be coming up the first weekend in August in Seattle. So Ben has been an active part of the community. Thank you for visiting my conference, Kevin. You're welcome. I wasn't going to do it myself. I'm trying to decide if I want to do the rest of these. Everyone does the person just next to him. Okay. This is Stephen Becker. He's Canadian. Ask him to say out. That was good. Stephen, let's see. Most recently, Stephen was working. I don't actually have any idea. I think he was a G5, whatever. It doesn't really matter. I was a G5 and it's so bad. So conference sponsors G5, apparently. Awesome, awesome. Thanks. Do I get to talk about Eric? No. I'm going to take organizer prerogative here. The other thing that Becker has done in spite of generally not wanting to claim it, he wrote the first version of R-Spec before turning it over to Dave Estells.  I'm sorry. Let me set the room and say it here. Shalinsky. Sorry, not Estells. So now I'm going to give the mic back to Becker and let him tell you about Eric. Okay. Eric's from Seattle and he wrote everything you use or something that is required by everything you use. Yes. Yes. How many people love Ho? Everybody. Yes, you do. If you don't love Ho. Get out. Is there anyone who likes Ho that's not from Seattle besides Mike? No. It's not from Seattle. It's from Seattle. Seattle would just tend to say cooler than me in Canada. This is... No. Oh, Kobe. More organizer prerogative. So, yeah, Eric, if you didn't catch the references, he goes by Dr. Brandon on Twitter. I'm right. All right. Well, I don't know what I think. And he also, he maintains Ruby Jams. He's a Ruby core committer. And I hired him once. Once. He's thinking about hiring me again. So... Well, he's... Maybe. So this is Rain and Rain is getting married next week. Let's see. Rain, let's see. Rain spoke last year and then got harassed by a man where he killed two as, say, rather inappropriate things. Unfortunately, he's not here again. And Rain is much happier. I don't know what else to say about Rain. Oh, no. Kobe, let's go first. The purple pimp hat does really well with the orange hair, though. Yes, it would. Let's see. Rain. It took me about a year before I pronounced his name, right? I kept wanting to call him Rain. Rain is currently at, is currently at Living Social. He's done lots of interesting things with Cloud at PHP Fog, where they did a lot of Ruby, at least on the underside of things. Ironically, no. Ironically, no. He's also presented and been active in the community for, I don't know how many years, longer than I have been. And I started in those six. Apparently, apparently someone would know that it's seven years. And he generally hosts the, the last two years as hosted the Think You Can Code presentations at Ruby Company. If you haven't seen those, now I'm going to fit my own company. You can go to confricks.com and look at the talks that were there from previous years. They generally go better than this one is right now. I guarantee they're a lot funnier than this one. This one is not going up there. No. Thank God. There's not even, this isn't even being recorded. Thank God. I'm also not a Ruby core committer. And I don't maintain Ruby chips. And this is Renee, who I've never met before in my life.  Before the conference. So I can't really say anything, but if someone else would like to say something, that would be really awesome. Renee lives in Seattle, and she was formerly a Blue Box group. And now she works for herself, which she says is much, much better. The best thing about Renee is if you can get her to start laughing even a little, she's not going to stop until tomorrow. Talking borough. The other thing that Renee has done a tremendous amount of working in the community is an organization called Railsbridge. So she was actually down here the last two days in a brand new workshop yesterday. And for those of you who don't know, Railsbridge is an effort, a group that's designed to help get more women involved with software development, specifically the Ruby and Rails community. So the workshop had one requirement. You either had to be female, or you had to accompany one to the workshop that we have. But have great attendance with that yesterday. So that gives you a brief overview of who they all are. They're also helping the time slot right now that snow has caused to be an issue. To be completely clear, none of us knew about this before last night when we were already drunk. So I apologize on behalf of all of my family and six upper rain. This is completely horrible. You're the one that told me not to do my slides. Yeah, that's true. I have a question. Unless I wasn't paying attention. So the question was, where the hell are Tamara and Randall? And as far as I know, they're being slaggers billing in San Francisco right now. Oh, damn. Precisely the question I asked Tamara last night to be clear. Randall bought me a lot of booze over the years. A lot of booze. I have nothing bad to say about Randall. I have nothing sober around that guy very often. I have a question for you. Whiskey, rocks, or lead? Whatever you want. I like this. Neat personally, but it's up to you. I'm not a big fan of ice. It's straight out the bottle. And it depends on if it's Scotcher Irish Whiskey, really. What are you going to say about Irish Whiskey? This is one of the things America has done better than Canadians have done. Whatever. Are we asking the audience? No, but I think it just provided the segment as cheap. What do you guys think about the new Rails 4?  Anybody out there want to jump in on this? I hear it's coming up around the time the Techsmate 3 comes out. It's coming up around the time the Techsmate 3 comes out. Forever. So I've worked with Aaron Patterson for many years. For about the last two years, he's been working on Rails. And when he started, he had fewer gray hairs. But I think that is for your benefit, because Rails 4 will be amazing. He's also older now. I don't know about that, but if you get older, you get older. Can you stop living facts? Get in the way of the story. So working with Aaron and watching him do Rails, there's a lot of face poems. And then he looks at his sausage. And then he goes and plays with his cat. So that's what Aaron does, too. Does it do any order? Sometimes it goes Rails, sausage, cat, sausage. I don't know what he does.  I haven't seen him actually play with the sausage yet. No, he's got that. Not in person. He's got a case. I'm talking about matching the sausage with the record. What's the case with sausage? That's a little off the panel. Can you guys open up with this? His voice is pretty whiskey. That's more questions than Aaron. I like the Florida recognize Brian Day. Fine, Brian. What do you guys want to see in Rails Fall?  No. What do you want to see in Rails Fall to come? I want to see people using it. No, that's kidding. The world right now is all between, like, let's upgrade this Rails 2 app to a Rails 3 app.   And that's what I would like to see for Rails 4. I know that's a pipe dreamer. Sorry, I killed that. Okay, we'll answer the question. Yeah, you. Can you guys use Ruby for anything? Do you have another one? Eric, actually. Actually, I was about to say in the last question, I haven't ridden any Rails since, like, 2006. I've been just pure Ruby back in stuff for the most part since then. Occasionally, we'll maintain a Rails app wherever I'm working, but most of the time, yeah, I'm just writing Ruby. You're right. Right now, I work for Living Social. And, yeah, we're building, the funny thing to bring it back to Tamer shit is we're actually moving towards a service-oriented architecture using Ruby for the back-end services, some of them are HGDP, some of them aren't. The HGDP ones for the most part aren't Rails. So, anyway, serious answer. Sorry. I haven't done much Rails either in the last two years, but I'm mostly committing on RDoC, RubyGems, Rake, Ruby, whatever. All the important stuff. All the stuff, all the back-end ecosystem stuff that everything else depends on. But also, like, I use Ruby to do all the random, fun programming stuff I want to do as well. Like, I've got RubyScript to control my fireplace. That's pretty sweet. I use Ruby to work on a little project called Puppet, which is used by a lot of organizations that you've probably heard of, like New York Stock Exchange. And for the record, when their service went down, that was not Puppet's fault. So Puppet is a configuration management tool that's managing possibly on the order of 100,000 or more machines around the world in hundreds or thousands of organizations, a lot of universities. It's as old as Rails, it's as big as Rails, and it's used in far more real-world situations. It's all, it's about, I don't want to get into an argument about that. It's almost as old as Rails. It's also almost as good as the adventure. And it's almost as good as Chef. In the same way that it's almost as old as Rails. Well, so I was going to say, Chef Scripts have been using a lot to set up servers in contrast to Puppet, but like a little better. They are both Ruby, so it's all Ruby with not Rails. Also I was going to say, when we played at the Ed, we know chips that you can program on Ruby. And they're really cool. So, we're the one, they're awesome. There are a lot of things you can't do, Ruby. Yeah. Yeah, it's trying to complete it. Exactly.  I wonder if the other one is trying to complete it. New question, right? So, what are the things you want to be, that you should know? The panel is talking about where I heard him. What are the things in Ruby that people should know that they don't know? Ben String. Obviously. Call CC. Oh, for Christ's sake. Go watch the second so you think you can code. Ben String is not, it's not worth spending any mental energy on. I mean, for you. I don't mind, but for you it's not worth it. Oh, sure. Okay, fine. Serious answers. I wish more people understood the difference and extend and include. Oh, I'm totally with you. I would like to see less abuse of inheritance. We've only got a few minutes. I would like for people to use better names for classes and modules. Base, instance methods, and class methods. Yes, it's a module.  Name it something smart. So, I want people to use fewer complicated mocking frameworks and just use DEF. Zero is good. Yes. DEF. You just use DEF. Maybe class.new. We don't want to throw that in there, but DEF. Yeah. Shit, anonymous classes. I wish more people did that. I did that recently, so there you go. What are we on? What are we on this time? I'm going. I need to answer. Crap. What's your programming? Pretty cool. Yeah, more people did that. Tell your friends. Yeah, tell your friends. Yes, over here. The question is, if you could rename the splat operator, what would you rename it to? Astros. What's wrong with splat? Nothing. Blah. What would you call it? I would call it the thing that sometimes turns a raise into not a raise, and sometimes turns not a raise into a raise. Operator. Base argument. Don't let it decor. Back to ho is unsure we all have some time. Never mind. So, I wrote a blog post about this, so you can get a lengthy argument, but for everyone who has not read it, there was a lovely person who decided that Ruby should have man pages shipped with him. I think this is a fantastic idea. More documentation is great. However, the way that he did it to package it with your gem was you would require this file in the library in your gem spec, and then it would go in monkey patch, Ruby gems, and throw files in there, kind of like, well, no, the dependency being like, oh, rake can do for you, and then you just have a rake task, and you say, oh, well, this depends on the package task, depends on this, put the stuff in the right spot, done. And then you can use it with whatever you want. So, you know, negative points for simplifying things, we all use rails. Yeah, but you're going to protect people just from what you say they should. You heard it, come on down. Yeah, sorry, when we do so you think you can code at RubyConf, we make it really clear up front that you should not listen to anything we say. And I guess we failed to do that today, so sorry. You don't know, we have an empty chair if you want it. Yeah!  So, don't do that. Don't read it? Don't read the documentation. Got you. You already won. Because more documentation is good, so don't read it. So, I have a question, it's a follow up to the, what do we wish people used more in Ruby, which is, what do we wish people used less in Ruby? Okay. Starting with you then. Did I give you some time? Yes. Nope. Ruby 1A, Ruby Enterprise Edition. Thank you, Kevin. Less? Less. Oh, meta programming. Yeah. Class arrow arrow cell.  That drives me crazy. Rescue from the inception. Yes. Rescue nail. Yeah, yeah. Trailing rescue nails. That's gross. So, like, you know, when you call something and you don't want it to blow up, so you put rescue nail at the end, so it just returns nail instead of blowing up.  There's this law called the meter. I would like people to do less of that. Less to meter? No, less of the... Less throwing away exceptions that are being called. The problem with that case is that often you may have another exception that gets thrown for some other reason that you are swallowing all exceptions. If you just rescued, like, IO error or whatever the error you are trying to rescue is, now you do not swallow exceptions. The big problem with that is that it breaks the chain of evidence.  And you have no way to figure out where to go next if you know what's wrong. And the only way to figure it out is to remove the rescue nail. But if you need to do that, why is it there in the first place? Maybe you should just not cause errors that you expect to happen. Absolutely. Just don't ever do it. Sorry. Sorry, we got into teaching there again. Anybody have a question that doesn't have a real answer? Yes. I think the documentation is doing it more as better. I have many, many invariant opinions on this. So I have a question just sort of to frame this a little bit. Why do you want know our doctor and know our eye? Why do you not want documentation? It's like three minutes to it. Okay. So should the documentation be generated ahead of time and shipped with the gem that way? No. Oh, that's awesome if you have an internet connection, which I almost never do. Well, how do you use all the gems without the gem? Level install local? I'm not kidding. The problem that I have know our doctor and know our eye is that people are installing stuff with them documentation. Okay. And I don't have a problem with this on a per project on a local basis. For this project, I don't want, I just said project, you are welcome. On this project, I don't want documentation because I have it elsewhere at the system level. And that's okay. There's nothing wrong with that. That should be explicit. When you're in the default in RubyGems should be always to install documentation because I want you to have the assembly who writes documentation from time to time. I want you to read it. Gem help and I think has how to go into it off by default. How many of you have ever actually use the documentation that is generated when you install a gem? So I have used it and it's still the wrong default. Right. If you need it, go generate. It's fine. The problem is, the problem is that we are basically making everyone pay a tax or something that I use 0.5% of the time. But everyone has to pay the tax all the time and nobody understands why. Most people have never used that documentation. The French case though. You're the one percent that uses that documentation that's installed. I'm the one percent that uses that documentation. I didn't count one percent at hand. If I'm the one percent that uses that documentation, I'm talking about the documentation in general. I'm talking about the documentation that's installed. But he just has to have the hands on it. So you can always generate that later. So the question is, when a new user does gem install rails, somebody who never used rails before and he types in pseudo gem install rails because that's what someone told him to do. Now they have to wait five minutes to get everything up and running. We should try to make that amount of time be left. If the solution to that is to make our doc factory, that's great. But the fact of the matter is that today, we are making people wait an extremely long amount of time to get up and running. We should not. Yeah, that's good. So comment says, successfully install n gems. You can use those gems. You need to control Z and Z down.  But I'm not going to change the default because I don't want to. Brian Davis, tell you not to change the default or is that your personal feelings? Okay, we can do that. Brian Davis has kittens. Eric doesn't have any kittens. I'm not real puppy. I'm not a puppy. That's a dog. You're not worthy of sympathy. It's a dog, dude. I think the bottom line is that documentation is an advanced feature. And advanced people know how to install it. And the people who aren't the advanced users aren't going to use it anyway. Let's talk about other things now. Not documentation but using the documentation that Ruby gem is installed in your system. Why don't we now talk about this anymore? We have a repeat of what we know about documentation. How do you guys... Alright, fine. Never mind. I'm patched in. Looking at it right now. The parser is amazing. We'll get it. Cool story, bro. How do you guys feel about turning the standard library into gems? And also, Steve, can you pronounce JavaScript? There you go. Java script. For you people because you put Ws in it. Java script. Where did all these extra Ws come from? Where did the story go, bro? So, okay, what was the question again? Sorry. No, we said Mapa. This one's going to blow your mind. I'm going to Vivachi in Seattle to hang out with these pundits. You know what? That's a good question, actually. I just want you to be consistent. I'm all about consistency. You can be wrong, bro. I learned that word by hearing it so that you are absolutely correct. When I go to Vivachi, I order a latte. And some pasta. No, I say a big one. I think the technical phrase is a big one, eh? Steve, what was your other question? Standard library is a gem. Great, great. Yes, and I need to go if I'm not here about that. So, the thing that's hard about that is that the standard library right now is actually pretty hard to pin the specific versions of Ruby. So, we could fix that but today the way that the standard library happens to be developed is that every time a feature changes in Ruby that necessitates changes in the standard library, they just go change it. And then that thing that has been changed no longer works on older versions, and it works. So, what you would have to do is you would have to have... People would have to treat the standard library like they treat any other gem which is... No. Backwards. Sometimes you get backward things. Okay, there we go. Things like encodings probably shouldn't be backwarded. When it's dead, it's fine. We've got another couple of months on the clock, but... Whatever feature is going to add in 2.0 may not be backwardable. The point is that up until now the latest standard library has been developed is very locked to specific versions and that would have to change. Question. I got one. It's for Mr. Baker. Who do you prefer, Terrence or Phillip?  Am I correct? No, they are not from Canada. Yeah, they are from Canada. I never giggle like that. Yes. When you open photos, do you probably clap your head? I don't think so. Are you sad at the news? I do my best. She's really not going to stop. Alright, I've got a... I was just looking at the schedule. And we've gotten Yehuda scheduled at 3.30 for the closing keynote for today. We've actually scheduled a fairly long break after this talk. Would that be a Yehuda and Steve game match? Well, I wanted to take this back for contrary to Ben, who is all about not educating. I've got a couple of questions that I want to ask. All I've got these guys up here. For the people in the audience, how many of you have actually contributed to an open source project? How many of you maintain an open source project? Because one of the dynamics that I've seen and had some interaction with between Yehuda and Eric and Ryan and watching things come together between Rails and Merb and some of the things that happened there. There's a lot of passion that's required to generate software and a lot of effort and hours and people's lives that go into creating the open source work that we all participate in and benefit from. There are consistently, and you get this with any time you take a fairly smart set of people and say work together under your own cooperative format. So you get lots of clashing. But the point that Eric was making earlier about Aaron Patterson developing gray hairs is so Aaron came to his boss at the time while we were all working at AT&T Interactive and basically put together a proposal that said it had a pie graph that said I spend eight hours a day working for AT&T Interactive. I spend four or five hours a day working on open source software and I think there's two more slots in there. I don't remember exactly what they were. One had to do with sleep and the other one was life or something. And then he proposed another graph that said what I would like this to be is 12 hours a day of open source. And that's effectively what he got. That's why his hair is going gray. And that's exactly it. So one of the things is there's a ton of effort that goes into this stuff. I generally, for those of you who don't know me, I don't write a lot of code and that's because I've written enough code in Ryan and Eric's presence to know that I'm better off hiring other people to get my code written. So the big piece and one of the things that I've tried to do and one of the reasons why I'm going from G5 which is a smaller vocally ran startup to AT&T which is where I'm unemployed at the moment, but I will more than likely be inside of AT&T in the not too distant future. One of the reasons for that is because they represent a very large pool of resources a.k.a. a very large pool of money and a very large pool of technical resources. And you've now got me on my diatribe. So if you want me to stop raise your hand. No, I like to speak your hand. Walk on. Alright, so one of the things that I believe in and one of the things that I'm trying to do by going back into AT&T is open source software is not free. And for every one of you who have released a project, you've paid part of that cost. And part of what we're trying to do and part of what I'm trying to do at AT&T and what we've accomplished at AT&T Interactive where I believe this is a true statement and Eric can correct me but Eric and Ryan now at this point are pretty much doing close to 100% of their time is also out of open source software. So we've now got Interactive, basically employees on that team, three engineers working on open source projects full time. They do not have other delivery responsibilities. When Salesforce.com bought Paroku, they then went out and hired Maps and they got other contributors. No boo. Who are also in hiring them their job descriptions and what they were doing did not change. Actually I guess job Maps has a new title but his effective role did not change. So we were making inroads with a lot of corporations or some corporations to help fund open source software. So instead of paying hundreds of thousands of dollars a year to license B.E.A. or WebLogic or one of the other large job frameworks we're shifting and trying to shift the corporate mentality to the point where open source isn't free. For small companies you end up letting your guys work on or your people work on open source software with some portion of their time and release things that they create to benefit your company that don't necessarily have intellectual property value to your company but do facilitate software development in general and let them release that stuff because it benefits the community at large and the only way that the open source community is going to continue to grow and continue to get healthy and I think this applies to software development in general is by spreading that attitude and part of it to Eric and I think Ray said this also is Aaron is older now. Time does flow, we all get older. Eventually you may be an intern now you may be an associate software engineer you may be a senior engineer down the road you're going to end up running your own company owning your own company being a director in a larger company being a principal architect in a larger company keep this in mind keep the fact that open source software is not free in mind because otherwise there is a chance that the open source environment collapses but it does, we go back to a proprietary world which in my opinion is not a very pretty choice so part of the software craftsmanship part of this movement is to continue to push that whether it's your own personal contribution you do through your open source projects at home or it's something you can do as part of your workforce continue to push to make that happen and then the part that I started on is there are going to be disagreements there was the Ruby gems and what I read is slime gems which was actually a slim gem issue there's going to be some degree of drama in the community around some of these things think about what you're saying think about how you're saying it remember that some of the people that you were arguing with do put a lot of time into this you put a lot of time into this a lot of the times we have to figure out how to just get past those pieces some of them is accepting some of them you don't want to accept because it's wrong and that's why we work things you create an additional version and fragmentation in my opinion is not horribly bad because generally what happens is in the case of Rails and Mirv they eventually came back together but they did that because there was some actual hard long thought that went into what happens if we continue down the major fragmented road with those two frameworks so I'm going to give the mic to Yehuda here for a minute but anyway the big piece is continue doing what you're doing with open source continue trying to make yourself better continue trying to make those around you better it's not utopian it serves your own best interest I'm not a big fan of work doing everything for others I do a lot of this stuff for my own benefit so was that all good? I just kind of want to reiterate what you were saying in general open source software is harder than it looks and in general there are actual design differences that cause the disagreements and I think in general what I usually try to do is try to understand what there's either assumptions or design differences that exist and try to understand those it's very easy to get up and say like this is crazy these people are doing retired things that's easy and I think in general people spend some thousands of hours of time writing software are usually not doing retired things they may have different values than you for instance I have very different values from the node community but I understand what their values are and identifying why my values and their values are different allows me to more easily either decide when to argue or when to just be like I think in general when there are fights like that's basically why Mervin Rails worked is that like there was a fight we sat down and said ok what are the actual differences it turned out that the differences and values were mostly like prioritization questions and adding a bunch more resources basically solve the prioritization problem that's basically the core thing to happen there so I think it's better to figure out what the values the axioms that people have are than to just say like this person The thing about MIRB is that we had very low resources. We had specific goals that we wanted to achieve and I think Matt's point about it was about performance. There have definitely been performance regressions, especially around active record. I think people don't usually do benchmarks of just action back, which actually has pretty good performance characteristics. But when you throw an active record, there were like pretty crazy regressions. But in the larger overall question of like, people who write plugins should have a more solid grant system. I think that for me that was the big thing. People who are doing advanced stuff, they should have a more solid grant stand on. They shouldn't be on quicksand, which was the case in Rails 2. I think we got to the point where the API that plugins use is actually decently well defined now for most cases. It's obviously not perfect, but the fact that MIRB, when people upgrade to Rails 3.2, usually active record plugins are not great. Usually, action controller plugins are not great. Dan? I think so. You can tell me if I'm correct or if I'm wrong, but my feeling at this point is that active record has gotten most of the interesting features that were in that mapper that I cared about. Yeah, it has gotten a lot of the features. I don't know if we're necessarily going to benefit from our merger right now. We're actually looking at doing some stuff, actually a true data mapper, where the objects are actually just normal objects and your mapper is separate, so you can test it separately. Oh, yes. Yeah, thank you. Nice. So the way to emerge in general is if the disagreements are resource constraints, then you can fix it with the merge, right? That basically, what happened with Rails is that Rails was like, we would love to make to add more modularity, but we're basically, we're all a bunch of volunteers and we have no full-time. An engineer was like, seems good, we'll put two full-time people on it. Suddenly, there was no more resource constraint problem about being to be more modular, right? And that meant that the actual points of contention didn't really exist anymore. One needs a higher dam. This is a little bit... Hold on, hold on. I'm going to buy Dan a beer for that. Colton, can you get Dan a beer? That's a nice thing. Do you get yourself a beer? I'm going to buy Dan a beer. Get Colton a beer, if you wouldn't mind. No, that's good. All right. Thank you. Oh, if you have something unrelated, do you mind if I just pop in real quick first? No. So how many people actually get paid, say, to do open source maybe more than 20% of the time? How many would like to? Why haven't you asked your boss if you can? Why haven't you just done it? Yeah, seriously, just do it. What's stopping you? Yeah.  A lot of employees that are working on projects are stuck in a corporate environment that don't have the luxury of contributing what they're producing to open source. Like for me, for instance, I can contribute to stuff that I produce at work, get paid for it, and then make it open source. And not a lot of people have the luxury of... A lot of employers want to lock down everything that their employees produce. I've worked in that environment and I've asked my boss and I've done permission. Anything I create is the intellectual property of my employer. Yeah, but... So is mine. So is mine. So depending on your actual work agreement with your employer. Yeah, but I mean, the point I'm making is that there's no reason why that can't also be open source. I release a ton of code that's copyright-living social. It's really not that easy. A lot of the people who are contributing open source are getting paid to do so. The question is the legal environment, right? I think so. Are you guys using open source projects in your... Not especially, but when I write stuff, usually it depends. Well, if you're using an open source product, at least because you go back to that. I mean... I'm waiting for a lot of us who are writing applications. A lot of us are working in real. A lot of the code is not so interesting. For some of the code that is interesting, at least for me lately, it's pretty damn proprietary. I think why I'm clear, really, is not the one I'm giving out. So then why is this stuff that's not interesting, not interesting? It's kind of boilerplate. Can't quite be automated? There's almost both three clubs. I mean, there's an area... You have to do some serious... I heard it goes... I appreciate it. But it's not always... Yes? No, I agree. So I got to where I am today by doing a lot of open source stuff. And sometimes it was just like, oh, we're working on this thing. It's kind of interesting. So I'm just... It's generic enough that everybody else should be able to do this. And how many people have heard of AR Mailer? So I wrote that, and I extracted it out of 43 Things Codebase because I was like, well, this is something that we needed because Rails didn't provide a good enough way to send emails. So I was like, well, we'll just put it in the database and then we'll have another thing that sends it from the database and then it can be completely decoupled. Page won't slow down. And I didn't ask any permission. I just did it. Nobody cared. But I was working with a company that was small and didn't really care what I did. But I think there's a lot of it where you can just be like, this is a small thing, especially tiny things. You just do it. And don't ask permission.  If they fire you for a reason like that, you probably don't want to work there. Yeah, yeah, exactly. If you get fired, there's probably a good sign that that's not a good place to work for. That family. Well, yeah. A lot of places are hiring. Yes.  So it usually has nothing to do with that, right? It usually has to do with the fact that the stuff you work on at work time is owned by your employer. And so there's a bureaucracy by which you have to find someone who will give you permission to do it. I didn't ask. Yeah. So in a small company, you can get away with it.  It's even less. I didn't ask. I haven't asked. I just asked to work on open source at AT&T. So there's a problem. So now what we're basically doing is we're introducing open source into the wild that's owned by people who have not given permission to license it. I agree with you completely that that's not a problem. No, so even if you don't care about whether it's theft or not, what you're doing is you are actually, you are making the choice to license something on it. You are basically giving up the copyright of your employer. And I personally am very fearful of the day where that nuclear bomb explodes. I feel like that open source is full of that and I'm scared of it. Well, that's a good CEO problem. I agree completely that that's not okay, but that should not stop anybody from asking permission. Ask your manager. Have your manager just push on it until you get permission, and you will. If you can't sell it to your management, then it's not valuable to the company to do so. But most of the time for this generic stuff that costs a lot of money to maintain that other people can help you maintain, that's beneficial to the company anyway. I think to be able to sell it to the CEO or it's not valuable to the company. I suspect that we're probably on a good falling ground that we would like to be able to release things into the open source that we do at work that are not proprietary. And you guys are right that there are policies in place that often prevent you from doing that at work in those environments. But these guys are right that we as people in this market in the Ruby job market as sellers of our services in a very good place, because it's such a hot market, because it's such a seller's market, and we're in a stronger negotiating position. We can make changes to those policies far more effectively today than we could, say, five or ten years ago. And those policies are not going to change unless we work towards changing them. I don't think subverting them is correct, but if we're not trying to change them, you can't complain that they're not changing. So we're going to company in, basically Vancouver called First Bay, probably I haven't heard of because we don't like Canadians. And so we move money between, we do a lot of the services that pay to help us, but between businesses. And so one of the things that happened recently, a year ago, I guess, we realized, or before I joined the team, the team realized that a lot of what we do is crud. It's just the basic, let's list some data, let's edit the data, so on and so forth. And so they created a project called Active Admin. The entire web interface of our application is Active Admin. That gives us, the rest of us, the ability to focus on what we do, which is our core competency, which is actually moving large sums of money around every single night.  If you have a small piece of your code and you have 100 users or even 1,000 users, and you have a piece of code and 1,000 users at most are using that, if you have 1,000 users. But if you release it as open source, unlimited number of users can use that piece of code. It can exercise it. They can find bugs, they can add improvements, they can help you. I don't think there's any reason why you can't sell this to a CEO. There should be no reason why. Yeah. In a company, there's hiring reputation. Absolutely. When we are looking at people, we look at new folks based on their open source contributions as well. Oh, sorry. The question, the seed was that people are valued, but hiring new people, they're valuing the contributions to the community. So, if people don't even work there, they're not allowed to work with that person? Yeah. Right. So, that's the other side of it. If somebody is active in the open source community, people don't want to work there. It's a recruiting budget. I worked at ThoughtWorks for a little while. That sucked. But Martin Fowler is made out of the recruiting budget. That's what I'm paid out of. Exactly. So, Ryan. And Aaron. And Aaron. In the back. So, the one breakdown that I see with this, though, is that you're assuming that your CEO, or in my case, people hire up, even understand what open source is. It's like Congress voting on SOPA. I mean, they have no idea what you're saying. You should be able to explain this to them. Open source is not rocket surgery. So, it is to it. If you have to talk to a lawyer, whose head is stuck up a certain orifice, it makes it difficult. This is the country that invented the assembly line and the reasonable part. Here's the thing. If they are recently intelligent people, you can get through to them. It may take some time. You may have to find new ways to frame it. But it's possible. I've seen it done a million times. There's a book all about this. I don't get serious. I don't get serious. So, I've gone through this a couple of times now. When I started at AT&T Interactive, which is where Ryan and Aaron work, is a wholly owned subsidiary of AT&T. And as such, there is a hand-waving agreement between a guy who runs the division that Interactive is part of. So, AT&T Interactive is the only company where the CEO reports to the CEO, who reports to the CEO, who reports to the CEO. So, there's literally four layers of chief executive officers. So, I know what you're talking about in the bureaucracy, but Interactive had this hand-waving agreement which was basically the guy at the head of the division had the agreement with the CTO at AT&T that said, Interactive can break a lot of rules and because you're not on the trusted telecom network, we're going to let you do that. Now, when, because I left Interactive and came to work for G5, and Chris Kragels over here in the corner, who is a CTO of G5, one of the things that was a real pitch for me to go to G5 was when they'd gone and did the funding and brought in the venture money, Chris went through and looked at the entire code base and basically said, all of the code that's required to run our application, 40% of it's coming from open source. The other 60%, or I could be wrong on the numbers, the other 60% is stuff that we've developed in-house. Because of that, if you want to attract the right talent in the company, we have to have some commitment to supporting the open source effort. Now, if you're in an organization where the CEO barely knows that your department exists or if you're in an entertainment company in LA where IT is a cost center, it's not intellectual property rights, then the battle is going to be hard. The battle is going to be, may very well be impossible. And to your point, yeah, we all have families. Most of us work and we do this because we're passionate about it. The reason you're here, I don't know, how many people are here paid for this out of their own pocket?  How many people here are on vacation right now? So you've got a little bit of cooperation from your employers. So it is a struggle. The other piece I was talking about though, I left AT&T Interactive because my boss went from being the CTO of Interactive to taking a VP position at AT&T Corporate. And I was supposed to move with him and my teams were supposed to move with us. And if that transition had gone through, what Ryan and Eric and Aaron do as part of their daily job was a terminatable offense inside of AT&T Corporate because AT&T had policies within the, this is what they call the CTO organization. They had policies that said you can use open source software if it's on this white list. And you can fill out exception paperwork to get onto the white list. And we figured out how to manipulate the system so that you quit putting down version numbers and you only put major version numbers so you didn't have to go back through the process every time. But that was their idea of open source. You could contribute a patch back to an open source project after legal review of the code. Legal review of the code. There was no concept within that part of the organization of releasing an open source project. It hadn't occurred to them and it wasn't in their mindset to take a project and set it loose in a lot to give up an intellectual property grant. This is the other fun part. If you don't know, Graphiz is released by AT&T. Graphiz is released by AT&T Labs. So there are lots of segments within the larger AT&T organization who managed to get exceptions in how this behavior happens. And the team that I'm going to be rejoining, if you didn't see the announcement, AT&T officially joined the OpenStack community and is now a partner as part of it. I'm not sure what the terminology is, I don't research it, but they have actually put the AT&T logo, which the folks in the branding department literally look at. If our logo shows up on your website, it's worth this much money to you. Do they call it the Death Star? They do not call it the Death Star. We have graphics of the Death Star, superimposed on the logo and it's awesome. Anyway, so there are absolute issues with doing this within large companies, within small companies where the value of what you're doing is not always there or is not always perceived. Part of it, and part of it is if you can't, it's that maximum, if you can't change your job, change your job, that's not an easy answer and it's not always an easy answer and sometimes it's not the right answer. I mean, Wozniak did it with the original Apple One when he went to HP with his design and basically said, do you guys want this? If not, can I have permission? If you're not going to do anything with it, can I have the permission to release it or go do my own thing with it? And I think that's the main thing is if you've got something that you think is going to be valuable to the community, at least have that discussion. Try to have that discussion if you can, which is this represents no intellectual property value to the company. If you want to do something more with it and you want to help put resources behind it, great, it's valuable. It's valuable to me, I'm passionate about it, I care about it. And if you don't want to own it and help beat it and nourish it, then let me let it go. Let me put it into the community.  Some people aren't going to get that, they aren't going to care, they're going to tell you to go back to your cube and leave them alone. And part of that comes back to that's when you should really be looking, whether it's Tim and Crawford's idea of just going to work for yourself or looking for another employer. There are a lot of people hiring, there are a lot of caveats on hiring. I had the discussion with, when Eric said we're trying to try to hire him again, we had a discussion the other day about whether or not there was any value in moving them from interactive to AT&T. We're not done with that discussion, but right now they're in a better place where they're being paid out of an engineering, marketing, recruiting budget. If they move to my team, I need them to deliver stuff that is proprietary and can't be open source. So there would be some loss in the amount of time they could spend on open source. That's more or less on the table at the moment. But there's a lot to this process and I'm probably running on that, huh? It's 3.15. Okay, so we've got, let's take a 15-minute break. I can have one other. Sure. There are some companies who absolutely will refuse to allow you to, so an alternative that could be to allowing their employers to have five hours a week. Yeah, I mean the point, the thing is, you just have to ask, and it's important to you ask, and if they say no, find a new job. It's not like quit immediately, but start looking for a job they can fulfill. Start attending conferences, start talking to our user groups, local groups, talk to other people.  I don't care about it. You'll be in point in 15 minutes. Yes. Alright, let's keep your hands up for just a minute. The question was, who's in companies that are hiring? Oh, yeah. Okay, now for the next question is, if you are a hiring manager who is hiring, keep your hands up. Okay, look around the room if you are not happy with where you're at. There are faces here who are responsible for bringing new people onto teams who probably have a better understanding of open source than your current employer. If you're in that room. Alright, well, thank you all for your participation. We're going to take the 15 minute break, and then we're going to come back, and you're just going to give us our cue.