GoPro also bundles a selfie stick/tripod combination with the camera, which is helpful. It's removable, and when it's off the Fusion can sit up on its own on a flat surface. It ships with a standard GoPro mount installed on the body. GoPro does warn that scratches can happen, and includes a protective case with a soft inner lining to use for storage and transport. A soft lens cloth does a fine job getting rid of smudges, and I reached for a disposable cleaning wipe after one of the family dogs licked the lens. That said, while I didn't try to scratch the lens, I haven't been overly careful with the camera either. The fish-eye nature doesn't lend itself to extra protection. Each of its lenses needs to see slightly behind themselves. The Fusion ($269.99 at Amazon) (Opens in a new window) doesn't have a hard glass cover over its lens like the Hero6, though. (Opens in a new window) Read Our Ricoh Theta S Review The Fusion is waterproof to 16 feet (5 meters). The outer material feels the same too, a hard rubber shell that withstands drops and the rugged treatment that action cams are often subjected to. It's finished in the same two-tone dark gray look. The Fusion takes it aesthetic cues from the Hero5 and Hero6. With a little extra software polish it would be a near shoo-in for our Editors' Choice award. GoPro promises improvement in the future, and the Fusion's video quality is better, so we're rating it higher. It delivers pro results when used with desktop editing software, but the mobile experience isn't quite as polished as what we've seen from the competing Rylo. The GoPro Fusion sets itself apart from other 360-degree cameras thanks to high-resolution capture (5.2K) and software that leverages those extra pixels to pan, zoom, and display spherical video in a traditional frame. Mobile OverCapture editing tools need a little work.Video stitching requires some serious horsepower.How to Set Up Two-Factor Authentication.How to Record the Screen on Your Windows PC or Mac.How to Convert YouTube Videos to MP3 Files.How to Save Money on Your Cell Phone Bill.How to Free Up Space on Your iPhone or iPad.How to Block Robotexts and Spam Messages.
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All 1 Cheats Unlockables Hints Easter Eggs 1 Glitches Guides Trophies. However, if you use cheats, this may make the walkthrough less interesting. Just be aware that the CT has been made on a steam game + SSE, it could not work properly for steam users or epic users. Subnautica: Below Zero Easter Eggs on Playstation 4 (PS4). Using these commands, you can easily travel around the world of Subnautica: Below Zero. If you’re ready to brave the cold, put on something warm, and. These Top 5 Tips should help you set out on the right foot and prepare you to face the icy dangers of Below Zero. This is a good alternative to teleportation. If you’re diving into Subnautica for the first time with Subnautica: Below Zero, then I have some guidance for you in this Subnatuica: Below Zero Beginner’s Guide. Type any of the following commands and press Enter. Once the box is unchecked, press Enter or to open the console. Enter any that interests you, after which the main character will be teleported there.īy the way, for the ghost mode, which allows you to fly through walls, you can use the “ghost” command (without quotes). Press F8 to open your mouse cursor, and uncheck the box. biome (biome name separated by a space)Īll biomes available are introarea, introicecave, shallowtwistybridges, twistybridges, lilypads, crystalcave, deeppads, kelp, and kelpcave.To teleport to Subnautica: Below Zero, enter the command into the console: Press F8 to move the mouse pointer, then click on the white circle next to Disable Console. The menu at the top left of the screen opens. To activate the console control, press F3 in the game. Now that the console is activated, you can open and close it using the hotkey (“~”). Like its predecessor, Subnautica: Below Zero also has its share of console controls and cheats. our hand-picked Video Game games similar to Subnautica Below Zero heres a. RELATED: How to Deter Ice Worm and Get a Thumper in Subnautica: Below Zero parks and Resorts Descenders Mods Map Codes can offer you many choices to. In the window that appears, uncheck the “Disable Console” option. Number 3: The Purple Vents How to get to the Purple Vents The aptly named Purple Vents are one of the most rewarding, yet most dangerous areas to build a base in. To find out how to enter the site, check the Finding the Koppa Mining. You may want to put large rooms on the coral itself, then bridge across each one to another with glass tubes, allowing you a great view of the depths below. These are located in the Koppa Mining Site. For this, first press the F3 key, to control the cursor press F8. To begin, you will need to find four Prawn Suit Fragments. If this does not bother you, then first you need to activate the console. Teleportation in Subnautica: Below Zero is a cheating feature, not a common game feature. Subnautica: Below Zero Teleportation Guide Therefore, in this guide, we will tell you how to teleport in this game. And Subnautica: Below Zero is no exception. The desire to move quickly in open-world games is quite popular. I think if Heather was holding the camera here, it would be very shaky, and she certainly wouldn't be strolling into the basement, I would've thought she'd be running to catch up with Mike. But, personally, I don't think Heather is carrying her camera at this point, when the POV enters the basement and focuses on Mike just prior to it being dropped, ending the film.Īs mentioned in my previous post, who or whatever is carrying the camera at this point, it certainly isn't Heather, as the camera is being held very steady, and the movement into the basement is very slow. Ok, it could be because she is using Josh's large camera, and her mouth is further away from the mic. Heather's screams sound very distant, compared to how loud her screams would sound if she was holding the camera at this point. When we cut back to Heather's B&W camera POV after Mike drops his camera in the basement - something doesn't seem right. But I think there's more to it then that. Josh was tortured and killed by the Blair Witch, she makes Mike stand in the basement corner, while she kills Heather as she enters the basement, then she kills Mike. It's the sequence afterwards that raises the most questions, the sequence where we are witnessing Heather's last POV before her death as she enters the basement, or so we're led to believe it's Heather's last POV. Whatever the case, everything leading up to the point where Mike enters the basement and drops his camera, is pretty straight forward. Of course, this theory is only possible if there is NO witch. Which leaves us with the possibility that there was a conspiracy between Josh and Mike to kill Heather, and Mike was indeed leading Heather into the house, lead her to the top floor which is the furthest point from the basement, run downstairs as if responding to Josh's screams, he enters basement, drops camera and reunites with Josh, Mike takes his place in the basement corner, Heather enters basement, Josh attacks and kills Heather from behind, her camera drops, end of film. But if it wasn't Mike in the corner, who was it? Someone else dressed in Mike clothes? No, it is unlikely that Mike worked alone as the killer. I also mentioned that Mike could be the killer, but that seems unlikely, because then who was it that attacked Heather from behind while Mike was standing in the corner? That is, if it even was Mike that was standing in the corner, which leaves Mike free to attack Heather. I mentioned in my previous post, that there is a possibility that Josh could be the killer, whether it be under the Witch's spell or not. Carbonite has\will be acquiring Crash Plan, and that explains the unequal "Partnership"? Someone else mentioned that Venture Capitalists are to blame for this nonsense. Maybe switching to Pro for home might work but again, I am not sure if I would stay with you now. Moving to Carbonite doesn't sound like an option based on how much storage I would need. I have somewhere between 5 and 6 TB of data backed up from my home machine. What incentive do I have and the business I work for have for staying with you? Your product has worked so well for us but now you are asking us to leave. Talk about a double whammy! Crash Plan, you have basically cut my business and myself off at the knees here. Earlier this year, they announce that they will no longer support running their product on a Server OS, and now they are telling me I cant use their product at home. I'm sure I am not the only one in this boat but my company has been using Crash Plan Pro for backing up our file servers and I have been using Crash Plan Home for my computer at home. Plain duplication covers none of these issues, and while it is handy if someone steals my PC, or for me to look at my cat videos on multiple devices, it's not a real business solution. A dead disk or blown server or RAID array means loading all the good stuff into new equipment. One needs to be able to restore to other than the original location.Backups are typically periodic snapshots, as opposed to realtime backup so that if things like ransomware do come along, it's possible to stop backups before uploading infected files, or versioning is in place, or some vendors claim to be able to spot infected files and block their uploading.Files deleted on the host PC are never deleted in the cloud backup system (or onsite backup, for that matter).The ability to carry some number of versions of a file to be able to restore to an earlier point.Infected and encrypted ransomware files are copied into the cloud as well, leaving me with no clean files to get back.Ī true backup system has to have the following characteristics:.A file deleted inadvertently on the PC is also deleted in the cloud with no way to get it back.There is no versioning of files to allow me to go back and get an earlier version if a user overwrites a file.But, it, like other file duplication products, leaves me with several serious deficiencies. OneDrive is indeed a copy of the same file in another location. I've used the free version for years, and considered paid versions, but really, just have not needed the additional features.īackup is not quite that simple. Someone else mentioned versioning, which is a feature of backup, obviously good, and supported by Syncback paid versions.īTW, I have no financial ties to Syncback. It may not be offsite/cloud backup by itself, but it's still backup. Backup, in its simplest form, is as a copy of the same file in a different location. Which backup service are you moving to now that CrashPlan is going away, and why? Share with the class in the comments.Regarding SyncBack, one said it wasn't backup. Hopefully, these services will offer easy, automated methods for moving your data before your subscription runs out. The only way this makes sense is if you only have a small amount of space and want all your files and data to live exclusively in Google’s cloud (and even then, I’d recommend that you consider other options). Google Drive now offers a Backup and Sync tool, but it’s expensive at $100 a year per TB of storage. The only drawbacks are that it doesn’t yet offer a dedicated Linux client (you can use B2, but it could be pricier depending on your needs) and that it doesn’t allow for unlimited file versioning. At $50 a year, it’s 10 bucks cheaper than the other two. If you choose to move, you’ll be charged $60 a year for unlimited storage with a single computer, and you won’t be charged for the first 60 days.Īnother formidable alternative is Backblaze. However, this service doesn’t include support for computer-to-computer backups, meaning you can’t copy your files over to a second system.ĬrashPlan has also partnered with its competitor Carbonite to help users make the switch. You’ll get a 75 percent discount on this plan for 12 months after you’ve migrated. Reality#6: Anne is a legal liability to the establishment.įantasy#1: Owners just want to make money and the problem goes away on its own.įantasy#2: Let’s not intervene when spa employees have issues with the spa director and not investigate what is happening in the spa.įantasy#3: Shoot first and ask questions later type of managing style will do the trick!įINAL: GET INVOLVED BEFORE IT IS TOO LATE. Reality #5: Erin Battle and Legedu Naanee are the owners of Sente Bella Med Spa and will not do anything about the “problem”. Can anyone explain why? As it is explained above………and then some. I am so glad I came in, because Evan is fantastic. I suffer from chronic neck pain and found Massage Heights on Yelp. Reality#4: High turnover since Anne Benedito has been the spa director. 55 Off 60-Minute Massage with Add-On at Massage Heights. Of course, she told everyone in the spa about it. Here’s another one, Anne was told in confidence about an employee’s health issues and she promised not to disclose it to anyone. This is very false and I mean room for slander. She even went out of her way to insinuate that a massage therapist was having an affair with her client. Reality#3: Anne would spread malicious gossip to the point to make herself feel better. She thinks that employees are out to get her, so then her vindictive nature comes out. Why you must ask? Well Anne feels threatened when someone knows how to approach things the right way. She relishes every moment having employees wrongfully terminated from Sente Bella Med Spa. She never harshly disciplines male staffers-just only females. ed un punto di riferimento per il cibo italiano nella bella Dublino. She has tarnished employees reputations, fabricated information about the spa operations, and harasses specifically female employees. We have 704 reviews of Pinocchio Italian Restaurant - Temple Bar - Restaurant. At Bella Sant we’re here to treat you to that time. Time to remember why we work so hard, say yes too often, and rarely take time out for ourselves. Time to zone out, stretch, breathe, relax, and maybe even nap. Reality#2: Spa Director Anne Bandito is a callous individual who habitually manipulates staff around her including the owners. Bella Sant, the best luxury day spa in Boston, was founded on the idea that we could all use a little more me time. Reality#1: Working with a pit of Vipers brings nothing but employees loathing their work environment. Her debut collection, Ayiti, explores the Haitian diaspora through fiction, nonfiction, and poetry. And their upcoming novel, The Death of Vivek Oji, comes out later this year. Pet explores the realities of a society striving for peace to the point of willfully ignoring the monsters in their midst, all from the perspective of a young trans girl. Freshwater is the surreal story of a Nigerian woman living with fractured selves and was nominated for the Lambda Literary Award. Their books have haunted and amazed me, and I’m sure they will you, too. Akwaeke Emeziįrom fabulist literary fiction to young adult fantasy, Akwaeke Emezi has a voice like no other writer today. Several of the books and novellas in the series feature queer characters and stories, including Once Ghosted, Twice Shy (a prince’s assistant and a jewelry maker get a second chance) A Prince on Paper (the underestimated cousin of the new Princess of Thesolo and the bisexual step-prince of Liechtienbourg go from side eye to heart eyes), and Can’t Escape Love (a nerdy bi businesswoman and puzzle-obsessed designer make a deal and wind up falling for each other in the process). Alyssa ColeĪlyssa Cole is the author who got me into romance with her incredible Reluctant Royals series, about a group of friends and the royals they become inexplicably mixed up with. Whether exploring adolescence-as in Another Brooklyn-memory and generational identity-as in Red at the Bone-or recounting her own childhood-as in Brown Girl Dreaming-Woodson’s remarkably insightful and tactile writing brings her stories to life in a way unlike any other. Queer identity is intrinsic to Woodson’s work, as is the beautiful, lyrical prose she’s well known for. Jemisin’s excellent short story collection How Long Til Black Future Month? and fantasy series The Fifth Season also feature incredible characters, queer and otherwise. Her most recent hit The City We Became, about the living avatars of New York City and its boroughs, includes multiple queer characters fighting hard to save the city they love. Jemisin for her incredible work in SFF (unless you either live under a rock or eschew sci-fi and fantasy altogether) but her writing also often includes powerful queer story lines. Most of their books explore queer identity in some way- Hurricane Child, This is Kind of an Epic Love Story, King and the Dragonflies-and explore heartfelt stories of identity and belonging. Their newest book, Felix Ever After, is a story about a queer, black, trans teen looking for his own happily ever. Kacen Callender is a powerhouse of today’s queer fiction, writing books spanning from middle grade and YA to adult. From joyful stories about queer Black girls to memoirs of what it means to be Black and gay, these authors present incredible and affirming stories to read now or anytime. They include some of my personal favorites, some classics, and some on my TBR that I still really need to get around to, because I have over 1,000 books on my Goodreads Want-To-Read shelf (bet you can relate). And these 20 Black authors of LGBTQ books are a great place to start, exploring experiences, both fictional and non, of what it means to be queer and Black. It widens your worldview, helps you reevaluate ingrained biases and beliefs, and, hopefully, exposes you to incredible new authors and books you might not have discovered if you only ever explored stodgy old lists of classics featuring solely straight cis white authors. Diversifying your bookshelf is incredibly important. With Pride Month in the rearview mirror, it’s also a great time to reexamine your TBR and your bookshelves-make sure the books you’re reading don’t solely reflect one identity, whether one portion of the LGBTQ community or mostly white authors. What about nonbinary authors? Black queer authors? Disabled authors? Neurodivergent authors? Queer experiences, for example, don’t necessarily look the same for any two people-especially if one of them is white and another Black. But is it really? Because it’s easy to miss the intersection of identities-or miss certain identities entirely-if you accept that as enough. Beyond that, though, I think it’s easy to fall into the trap of thinking you’re doing enough, or reading widely enough, if you’re reading books that are non-white or non-straight or non-male. What it means is simple: evaluate the kinds of books you’re reading and make sure they aren’t all reflecting a white, western-AKA colonizer-point of view. The phrase “decolonize your bookshelf” has come into common reader vernacular in recent years. Many viewers have given reviews on this new season and have enjoyed watching all the episodes of The Man Who Fell to Earth season 1. The last season of The Man Who Fell to Earth is trending, and the audience who have watched season 1 has given positive responses. The Man Who Fell to Earth Season 2 Popularity And just like people’s demand, the storyline of the series has followed the novel accurately, and the series also has excellent performance characteristics. The new Voot series, The Man Who Fell to Earth, is based on the famous sci-fi novel, and people who have read the novel expect the production studio to follow the accurate story of the novel and build the character as well as the novel. What We Can Expect From The Man Who Fell to Earth Season 2 Please stay in touch with us because as soon as we receive any new information on this subject, we will post it here. As a result, it is no longer necessary to assume the spoiler at this time. The Man Who Fell to Earth Season 2 Spoilerĭespite the fact that the 2nd Season of The Man Who Fell to Earth has not been officially announced. A brilliant continuation of the novel written by Walter Tevis and the legendary film starring David Bowie that was released in 1976. Together, they realize that they must first save our world by restoring his world. Even though he is working hard to adjust to our culture and become more “human,” her faith in people is low. What Is The Storyline Of The Man Who Fell to EarthĪn extraterrestrial being played by Chiwetel Ejiofor crashes his spacecraft deep within the oilfields of New Mexico with a mission: he must locate the brilliant scientist Justin Falls, played by Naomie Harris, who is the only person on earth who can assist in the survival of his species. Cast: Chiwetel Ejiofor, Naomie Harris, Clarke Peters.© 2022 Hookshot Media, partner of ReedPop. Join 397,256 people following Push Square: Soapbox: I'm Sorry, But Your Cute Cat Game Isn't That Great PS4 to PS5: All Games with Confirmed Free Upgrades New PS5, PS4 Games This Week (25th July to 31st July) PS5 Stock: Where to Buy PlayStation 5 and When in July 2022 FIFA 22 Guide: Tips, Tricks, and How to Win More Matches.Is there anything else you want to know about all Squad Battles rewards, release dates, and times in FIFA 22? Have a browse of our FIFA 22 guide, and let us know in the comments section below. This page also includes a list of all Squad Battles rewards above. Asia/Oceania: 4:05pm JST / 3:05pm AWST / 5:05pm AESTĭo keep in mind that you'll need to be at least Bronze Rank Three in Squad Battles to be eligible for rewards.We've included a number of popular time zones below, so you know when to expect your rewards to be available: Depending on where you live, all Squad Battles rewards will be available at a different time, determined by your time zone. You can check your Rank at any time by selecting Squad Battles > Your Rank from the FIFA Ultimate Team menu.Īll Squad Battles rewards are listed below: Bronze 3įIFA 22: All Squad Battles Rewards Release Dates and TimesĪll Squad Battles rewards have a release date and time of Sunday at 7:05AM UTC. However, once you have played 40 matches you can't earn any more Battle Points for that week, so try to win as many games as possible on the highest possible difficulty tier you're comfortable with. Keep in mind that your Rank can change depending upon what the rest of the game's community does. FIFA 22: All Squad Battles Rewards Release Dates and TimesįIFA 22: Squad Battles - All Rewards, Release Dates, and Times.FIFA 22: Squad Battles - All Rewards, Release Dates, and Times. You can also find Division Rivals - All Rewards, Release Dates, and Times, and FUT Champions - All Rewards, Release Dates, and Times. As part of our FIFA 22 guide, we're going to share all Squad Battles rewards, release dates, and times.įor more information on FIFA Ultimate Team, including Best Formations and Custom Tactics for FUT, How to Make Coins in FUT, All FUT Packs Explained, When to Buy and Sell Players in FUT, and All New TOTW Players in FUT, then click the links. This mode allows you to test out different teams and tactics, while also earning some pretty major rewards in the form of Packs and Coins. What are all Squad Battles rewards, release dates, and times in FIFA 22? If you're playing FIFA Ultimate Team, also known as FUT 22, then there's a good chance you'll be spending a large amount of time up against the CPU in Squad Battles. VeryPDF Released an Online PDF Editor to allow you to edit, comment and annotate PDF files in web browser directly. 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Step 3: You will see VeryPDF Digital Signature Tool dialog, Step 2: Open a PDF file, click “ Advanced”->“ Digital Signature” menus, Step 1: Download and install latest version of PDF Editor v3.0 from following URL, PDF Digital Signature (PDF Signer) Screenshots: Permission options are include following: Allow Assembly, Allow Copy, Allow Degraded Printing, Allow Form Filling, Allow Modify Annotations, Allow Modify Contents, Allow Printing, Allow ScreenReaders. PDF Encryption – Encrypt your PDF file by User Password, Owner Password and permission options. Including a timestamp helps to prove that the document wasn’t changed after you had signed it and it reduces the chances of an invalid signature. Timestamping – Like signatures, timestamps are easier to verify when they’re associated with a timestamp authority’s trusted certificate. Validation – Signatures produced with PDF Digital Signature can be verified using standard Adobe Reader 7.0+ or Adobe Acrobat product. 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Options like auto background refresh, flash reflow, and scene tree make it easy to trim, move, highlight, or reduce the background elements in your photos or video to create the exact graphic. With these stunning effects, you can create an overall visual effect for any clip. In addition to all these benefits, Adobe Premiere can also import and edit other files such as AVI files, HTML, QuickTime movies and so forth.įeatures like auto focus, multi-angle backgrounds, and chroma key effects are available to make your photos, video, and images pop. Openshot is really awesome and a professional video editing software. It also supports the latest video editing technologies, including Avid Media Access, Digital Video Posting, and High Efficiency Video Exchange. These benefits also include the fact that Premiere can edit footage in real time, improving the speed and accuracy of your final results. Video editing features include its intuitive interface, superior graphics, powerful processors, and a wide array of motion graphics options. Kdenlive 15.04.0 It becomes part of the KDE official project. What Video Editing Software do YouTubers Use? - 3D Insider, advantages of Adobe Premiere include its extensive features and ease of use. Take a look of three of its most remarkable released: Kdenlive 0.7 Rewrite version from the K Desktop Environment 3 version to KDE Platform 4.1 users For creators of all skill levels and across all devices. The 7 best Instagram photo editors to edit like an influencer, . Open-source video editing tool for Windows, Mac and Linux users which helps with 3D animated titles, audio mixing/editing, frame accuracy, clip resizing, desktop integration and more., Best photo and video editing apps - Microsoft Store., Top 10 YouTube Video Editing Apps for iPhone and Andr….VideoPad is one of the best tools for YouTube video editing. This is one of the free video editors for YouTube that is perfect for vloggers for many reasons. Along with Apple’s Final Cut Pro,it is the accepted standard in movie and TV production.īesides, What are some good YouTube video editing apps? With OpenShot, you can create animated 3D titles, enhance the sound, change clip size, and more. Adobe Premiere Pro CC Premiere Pro CC is a professional-grade video editing software. OpenShot is another open-source video editing program for Windows, Mac OS, and Linux that will be a perfect replacement for Cyberlink PowerDirector 12.iMovie While the best-known YouTubers have moved on to software with more in-depth editing options,iMovie remains a mainstay of many content creators.In this manner, What do most people use for editing YouTube videos? Final Cut is one of the most popular editing tools available in 2020.…īeside this, What editing apps do influencers use? - VSCO - Snapseed - Prisma - Afterlight - Adobe Lightroom - Huji Cam - Foodie iMovie comes free with all Apple devices,so it has long been the first port-of-call for those new to video editing,and many well-known YouTubers still use the program. The video editor is also available for Android and iOS. You can also get discounts on special offers. The annual plan is better and available for 69.99. You can buy the Windows and Mac monthly subscription for 19.99. What apps do YouTubers use to edit their videos?Īlso, What do famous youtubers use to edit their videos? There are two plans for PowerDirector Video Editor. Magisto Video Editor&Maker - iOS/Android.KineMaster is a video editor for Android that lets you use the latest hardware to create professional films. Similarly, people ask, What apps can I use to make YouTube videos? Final Cut Pro is one of the most popular editing tools available in 2020. iMovie is free and works across all Apple devices. If you're looking for the absolute best,industry-standard,video-editing software,then look no further than Adobe Premier Pro. Secondly, What programs do YouTubers use to edit their videos? Faced with a long video conversion: a 7:30 minute video (a part of which was accelerated at 800) was exported in 20 minutes. Then, What are the best YouTube video editing apps? Best YouTube video editor software and apps, 1. VSDC Free Video Editor exceeds the standard array of functions required for modest editing goals. A professional video editor, including editing, color correction, audio production, video effects and motion graphics four high-end video production. Classic free YouTube video editor that lets you create Hollywood-style trailers and amazing 4K resolution video clips. Keeping this in consideration, What apps do YouTubers use to edit their videos? What Apps Do YouTubers Use to Edit Their Videos? iMovie. An Ultimate Comparison of Desktop Software and Apps Best YouTube Video Editor for Beginner and Semi-pro - Highly Recommended - Part 3. 10 Best YouTube Editing Apps for iOS and Android - Part 2. What apps do YouTubers use to edit their videos?. Therefore, paired-end sequencing leads to fewer false positives. To be declared a duplicate, both ends of one fragment need to match both ends of another fragment, which is far less likely to occur by chance. On the other hand, after paired-end sequencing, both ends of the original DNA fragments are defined. Thus, many false positives may result, and perfectly good reads are removed from further analysis. With single-end reads, there is only one position to compare, and so any reads whose 5' ends match are considered duplicates. Genrich) typically identify duplicates based on comparing ends of aligned reads. However, computational programs that remove PCR duplicates (e.g. As noted above, PCR duplicates are artifacts of the procedure, and they should be removed as part of the analysis pipeline (see below for more details). PCR duplicates are identified more accurately. A single-end read defines only one end of the fragment, and, although computational methods to infer fragment lengths from single-end reads exist, they tend to be inaccurate. This can be done only with paired-end reads. With ATAC-seq, we are interested in knowing both ends of the DNA fragments generated by the assay, since the ends indicate where the transposase inserted. Additional sequence data, such as with paired-end sequencing, helps to reduce these alignment ambiguities. Many genomes contain numerous repetitive elements, and failing to align reads to certain genomic regions unambiguously renders those regions less accessible to the assay. More sequence data leads to better alignment results. Sequencing modeįor ATAC-seq, we recommend paired-end sequencing, for several reasons. (2015) recommend more than 50 million mapped reads per sample. For studies of human samples, Buenrostro et al. The optimal sequencing depth varies based on the size of the reference genome and the degree of open chromatin expected. This helps to reduce PCR duplicates, which are exact copies of DNA fragments that can interfere with the biological signal of interest. In preparing libraries for sequencing, the samples should be amplified using as few PCR cycles as possible. Such a control could be useful to help define regions of the genome that are more challenging to sequence or to align reads unambiguously. by sonication), has adapters ligated, and is sequenced along with the ATAC sample. A control for a given sample would be genomic DNA from the sample that, instead of transposase treatment, is fragmented (e.g. With ATAC-seq, control groups are not typically run, presumably due to the expense and the limited value obtained. To begin with, two replicates per experimental group are sufficient. This ensures that any signals observed are due to biological effects and not idiosyncracies of one particular sample or its processing. Like most high-throughput sequencing applications, ATAC-seq requires that biological replicates be run. Here are a few additional things to consider when planning an ATAC-seq experiment: 1. The developers of the ATAC-seq method have published a detailed protocol for the laboratory procedure (Buenrostro et al., 2015). ATAC-seq overview ( Buenrostro et al., 2015). High-throughput sequencing then yields reads that indicate these regions of increased accessibility. It utilizes a hyperactive Tn5 transposase to insert sequencing adapters into open chromatin regions (Fig. The original version can be found here.ĪTAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) is a method for determining chromatin accessibility across the genome. NOTE: this is an updated version of the ATAC-seq Guidelines. |
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