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Author Aaron Ozee Secures Agreement with Stop-Motion Animation Production Studio Filming 'Regulus' Movie
eNewsChannels NEWS: -- Aaron Ozee, the author of bestselling children's book, "Regulus" (ISBN: 9781387010790), has sold the production rights from his notable creation to a prominent stop-motion animation studio that will adapt "Regulus" into an unorthodox theatrical feature.
The Hatchery: Whole Brain Consulting to speak at food industry incubator Dec. 2, 2019
eNewsChannels NEWS: -- Chicago Illinois area's William Madden, co-founder and senior partner of Whole Brain Consulting, is scheduled to present at The Hatchery from 6 to 7:30 p.m. on December 2, 2019.
Real Estate: Luxury Chicago Realtor Emily Sachs Wong Announces a Premier Home with Outstanding Lake Views
eNewsChannels NEWS: -- Luxury Chicago Agent, Emily Sachs Wong, lists a premier one-of-a-kind home offered for $2,850,000. Soaring high above the idyllic shores of Lake Michigan, mere moments from Chicago's finest beaches, biking paths, retail, and restaurant offerings, this exquisite 3,259 square foot sky-home resides in one of America's most prestigious real estate havens, the Gold Coast.
Author Aaron Ozee Navigates Bestselling Children's Book 'REGULUS' Through Chernobyl
eNewsChannels NEWS: -- Aaron Ozee, author of notable children's book, "Regulus" (ISBN: 9781387010790), has partnered with popularized international tourism organization, Young Pioneer Tours, bringing a printed copy of his charming narrative along with them during their expedition into the exclusion zone of the Chernobyl nuclear disaster.
New Book, 'Oh Da Joy' – A Conversation in the Art of Joyful Living, Resonating with Spiritual Readers Seeking to Supercharge...
eNewsChannels NEWS: -- Kharis Publishing today announces the release of "Oh Da Joy" (ISBN: 978-1946277398) by Pat Jewell, a broadly reaching new book with the spirit of "Chicken Soup for The Soul." Appealing to a wide range of readers who will find comfort and joy in this life-guide to turn the weary into the re-charged.
CHICAGO REAL ESTATE: Emily Sachs Wong offers a flawless luxury $3M residence with a rare two-tiered outdoor deck
eNewsChannels NEWS: -- Luxury Chicago Agent, Emily Sachs Wong, lists a noteworthy luxury residence offered for $3,050,000. The epitome of exceptional elegance and contemporary design awaits with this no-expense-spared custom brick and limestone masterpiece.
North Korea Ejects Aaron Ozee Children's Book 'Regulus' from the DMZ
eNewsChannels NEWS: -- Aaron Ozee, author of the notable children's book, "Regulus" (ISBN: 9781387010790), clashed with the government of North Korea when the attempt was made to bring his parable across the border at Panmunjom.
BOOK: 'Off the Hook' by Romance Author Dania Voss, Explores Challenges of Young Love, Wealth and Pro Sports
eNewsChannels NEWS: -- Chicagoland romance author Dania Voss explores the challenges of young love while dealing with the pressures of wealth and professional sports, in her latest release, new adult novella "Off the Hook" (ISBN: 978-1773399911; Ebook).
Where Did REGULUS go? Aaron Ozee Loses Bestselling Children's Book Inside Bermuda Triangle
eNewsChannels NEWS: -- Aaron Ozee, author of notable children's book, "Regulus" (ISBN: 978-1387010790), has partnered with independent aviation consultants to send a printed copy of his esteemed parable into the mythical depth of the Bermuda Triangle.
180 Pure CBD Launches Wholesale Program for Local Illinois Retailers
eNewsChannels NEWS: -- 180 Pure, a Chicago area CBD company, based in Lake Zurich, has launched a wholesale program for local retailers wishing to provide premium grade, THC-free CBD products to consumers.


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Your Atlanta BeltLine connects millions of people across Atlanta to trails and transit, better health, housing and jobs, art and education, volunteering, community and culture — and to one another
All events for November Project ATL Workout
December 2019
November Project ATL Workout
Each workout differs, but utilizes the space around them. A combination of running, body weight moves and plyometrics will keep the heart rate high. The workouts are creative and fun, and approachable to all fitness levels regardless of ability level or age. All you have to do is #justshowup.Find out more »

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Chevrolet Equinox LS w/1LS
The 2020 Chevrolet Equinox. Under the hood you'll find a 4 cylinder engine with more than 170 horsepower, providing a smooth and predictable driving experience. A turbocharger is also included as an economical means of increasing performance. It's equipped with tons of terrific amenities, but it won't break your budget. Like all wheel drive, variably intermittent wipers, air conditioning, fully automatic headlights, tilt and telescoping steering wheel, lane departure warning, power windows, and remote keyless entry. Audio features include an AM/FM radio, and 6 speakers, providing excellent sound throughout the cabin. Chevrolet ensures the safety and security of its passengers with equipment such as: dual front impact airbags, front side impact airbags, traction control, brake assist, a panic alarm, onStar, and 4 wheel disc brakes with ABS. For added security, dynamic Stability Control supplements the drivetrain. You will have a pleasant shopping experience that is fun, informative, and never high pressured. Stop by our dealership or give us a call for more information.


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Cineplex Cinemas Seton and VIP
19638 Seton Crescent SE,
19638 Seton Crescent SE,Calgary, AB T3M 2N9
No one has favorited this theater yet
As reported in the Calgary Herald, a massive fire, during construction, started by an electrical problem, swept through Cineplex Cinemas in Seton on November 4, 2017.
Scheduled to open in December the theater finally opened July 11, 2018, with seven screens and four more that followed in August.
The cinema includes an UltraAVX large format screen, D-Box motion, RealD 3D and Dolby Atmos sound, viewed on 1,900 recliners.
Contributed by Ron Pierce
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In March I joined an MLM company and I'm lost… my first reaction to network marketing was I'm not a sales person, to which I was told it's not about sales but about building relationships. The other thing that makes me crazy is I see a lot of folks who appear so fake with their bubbly "everything is coming up roses and bubble gum" that I cringe. I'm honest to a fault and happy but not giddy. I feel like I have writers block in that I'm not sure what direction to take to get my business going, the struggle is real right now… Any thoughts or suggestions I can take away.
The Federal Trade Commission issued a decision, In re Amway Corp., in 1979 in which it indicated that multi-level marketing was not illegal per se in the United States. However, Amway was found guilty of price fixing (by effectively requiring "independent" distributors to sell at the same fixed price) and making exaggerated income claims.[47][48] The FTC advises that multi-level marketing organizations with greater incentives for recruitment than product sales are to be viewed skeptically. The FTC also warns that the practice of getting commissions from recruiting new members is outlawed in most states as "pyramiding".[49]
An example of a high-profile multi-level marketing company defending its practices is Herbalife Ltd., a manufacturer and distributor of weight-loss and nutritional products with more than 500,000 distributors. Although the FTC had been investigating Herbalife, it was activist investor William Ackman who shed a national spotlight on the company by shorting $1 billion of the company's stock in 2013. Ackman accused the company of operating a pyramid scheme and backed his allegations with a bet the company's stock price would fall under the weight of the scam.
5. Internet. Having a personal Web site linked to your company's Web site is becoming mandatory for the successful distributor. Your Web and e-mail addresses are the technological version of a business card and brochure. Internet recruiting still requires some high touch to entice people to view your page. Because this is of significant interest, I'll address Internet lead development techniques in detail in a future article. For now, view it as a support tool and not as an alternative to personal interaction.
The most common high-pressure tactic is the lure of getting in on the ground floor. But in direct sales, a good opportunity is a good opportunity no matter when you get in. In fact, you're safer to go with a company that has been around for more than five years (the longer the better) than a start up. Any effort a representative makes to prevent you from studying the company, talking to others, and "sleeping on it" isn't someone you want to work with.
"Joining an MLM is appealing to women who find hope in their promises of a better life: freedom, economic independence, and an endless supply of cheery trinkets. Despite professing quick-income prospects though, it's difficult for MLM consultants to earn more than pocket change. When glitzy recruitment videos yield to the reality of suburban cul-de-sacs, people selling for MLMs can be plunged into debt and psychological crisis." (Quartz)
I see Melaleuca on here. I see that as both good and bad. They are an awesome company with a great compensation plan. However, they are not an MLM. They are not even listed with the federal agency that oversees those companies. They are a Consumer Direct Marketing company. How does that differ? While I am required to purchase a certain amount each month, that's all I need to purchase. It's all products I use in my own home for myself. I don't have a monthly quota to meet. I don't have to buy product and sell it to people. The idea is that the product goes to the consumer only. In fact, it's against company policy to buy product and sell it to others. The only comparison I see are the "levels" of customerS in my group. Can you shed any light on why you think they are an MLM? Thanks, so much!


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An unbeaten half-century by Shaun Marsh put Australia in a strong position yesterday, setting India a near-record task if they are to win the third Test in Melbourne, Australia.
Marsh produced one of his finest knocks for his country with 62 to steer Australia to 261-7, giving the hosts an overall lead of 326 runs heading into the final day today.
India will have to defy history at the famous Melbourne Cricket Ground, where they have not won for 33 years, to keep the Border-Gavaskar Trophy series alive to next week's fourth and final Test in Sydney.
The highest successful run chase in the fourth innings at the MCG remains 332-7 by England against Australia in 1928.
Teams have struggled to score more than 250 in the fourth innings on the drop-in pitch in recent years, with India all out for 169 (2011) and 161 (2007) in their last two Tests at the ground, both defeats.
Australia have to decide whether to declare overnight or bat on with a total of 96 overs to be bowled on the fifth day, but opener David Warner believes more runs are needed.
"I think we need a few more runs than we've got at the moment. Shaun Marsh is batting fantastic and we saw what Ryan Harris did in the first innings [74], so hopefully he can produce something similar to that tomorrow and I think we'll assess it after we're all out," Warner said.
"It's still a very good batting wicket. The key is going to be to get that ball going a little bit, and dry up the runs and take 10 wickets," he said.
It was Marsh's fighting innings which tilted the Test Australia's way after India reduced the home side to 176-5, claiming the key wickets of Steve Smith and Chris Rogers in the process.
The tourists kept the clamps on Australia's batting in a tense final session, before Marsh clawed back the advantage.
"Any score on the final day is going to be tricky," India spinner Ravi Ashwin said.
"It's going to be hard work, but we're up for it. We're here to win games of cricket, so we'll be positive, and try and see where we can go," he said.
The tourists removed Smith for just 14 after the Aussie skipper scored 192 in the first innings, his third century of the series.
Veteran opener Rogers reached his fourth consecutive half-century of the series, but again failed to go on with it and was bowled by Ashwin for 69 nearing tea.
Rogers was dropped on 33 in the slips by Shikhar Dhawan on the second ball after a lengthy rain delay and had another big moment on 56 before his dismissal.
Umpire Richard Kettleborough called for the video umpire to check if the ball had carried for a catch claimed by Mahendra Singh Dhoni off Mohammed Shami, but replays showed the ball hitting the ground in Dhoni's glove and Rogers was given a reprieve.
India celebrated the important pyschological wicket of Smith in one of their rare successes against the prolific Aussie skipper in the series.
Smith was caught at leg slip by Ajinkya Rahane off Umesh Yadav after playing a similar shot for a boundary a few overs before.
Shane Watson again failed to nail a big score when he was caught behind off Ishant Sharma for 17.
Debutant Joe Burns was out to a poor shot and was caught behind off Sharma for 9, while Brad Haddin (13) and Mitchell Johnson (15) were also caught behind.
Australia lost the explosive Warner before lunch after he was looking threatening.


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Observations from an easily-distracted man
As I walk to class, I am easily distracted. It makes me wonder if all that Ritalin my parents gave me wasn't just because they didn't love me. Even so, I've found that a short attention span can be useful; I notice things other people don't take the time to.
For the curious, here are some things I've noticed around the University of Arizona campus:
Pigeons = Tiny, flying mobsters
Skateboarders never seem to learn that there are better things to do with one's than have your body smacked against the concrete repeatedly.
The university has a tower with a clock that doesn't work and a tower with no clock that tells the time with bells.
A menacing, barbed-wire fence. A gaping, bleak hole in the ground. A sign proclaiming that all this is part of the "New University of Arizona."
There is a moment of time in every student's life when they think "Is that person on the bike going to hit me or not?"
Smiling, enthusiastic people offering people free T-shirts, hats, or phone cards, if only you could give them your name, address, age, social security number, average household income, and your consent for them to assign you a credit card with an interest rate of 35 percent.
Just about every stranger who says "hello" to you wants money. The others are people who you don't know, but claim to know you - and become insulted because you don't say "hello" back.
If you fell from the top of the staircase inside the student union, your body might end up inside the snack machine.
A man telling people that the God will bring peace and love to all the people of the earth - except for the homosexuals, who will face eternal pain and suffering.
A $60 bike lock protecting a $20 bike.
One out of every 10 people have a metal object sticking out of their flesh - sometimes, not on purpose.
A woman walking into McClelland Hall. drinking, coffee, eating a croissant, talking on her cell-phone, and organizing her papers, all at once.
I wonder how bad the parking problem is going to get before students resort to getting an open space through heavy explosives?
There is a chance that a construction worker might be buried underneath the foundation of the new Student Union during construction.
How quickly those ad inserts in the Wildcat pile up around the Harvill building.
The love you take is equal to the love you - wait, someone else noticed that already.
An aircraft should be brought down as it flies over the U A with all the cell phones operating on campus.
Wherever there is loud, crappy music, there is a dorm room with an open window.
Some people aren't "jiggy with it."
Columns don't always have to end with something poignant.
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Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato
- Published
- Accepted
- Received
- Academic Editor
- Yuriy Orlov
- Subject Areas
- Agricultural Science, Genomics, Plant Science
- Keywords
- Single-molecular real-time sequencing, Comparative transcriptome analysis, Alternative splicing, Sweet potato
- Copyright
- © 2019 Ding et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
- Cite this article
- 2019. Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato. PeerJ 7:e7933 https://doi.org/10.7717/peerj.7933
Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in I. batatas functional genomics and molecular breeding.
In this study, we sequenced full-length transcriptomes in I. batatas and its diploid ancestor I. trifida by single-molecule real-time sequencing and Illumina second-generation sequencing technologies. With the generated datasets, we conducted comprehensive intraspecific and interspecific sequence analyses and experimental characterization.
A total of 53,861/51,184 high-quality long-read transcripts were obtained, which covered about 10,439/10,452 loci in the I. batatas/I. trifida genome. These datasets enabled us to predict open reading frames successfully in 96.83%/96.82% of transcripts and identify 34,963/33,637 full-length cDNA sequences, 1,401/1,457 transcription factors, 25,315/27,090 simple sequence repeats, 1,656/1,389 long non-coding RNAs, and 5,251/8,901 alternative splicing events. Approximately, 32.34%/38.54% of transcripts and 46.22%/51.18% multi-exon transcripts underwent alternative splicing in I. batatas/I. trifida. Moreover, we validated one alternative splicing event in each of 10 genes and identified tuberous-root-specific expressed isoforms from a starch-branching enzyme, an alpha-glucan phosphorylase, a neutral invertase, and several ABC transporters. Overall, the collection and analysis of large-scale long-read transcripts generated in this study will serve as a valuable resource for the I. batatas research community, which may accelerate the progress in its structural, functional, and comparative genomics studies.
Sweet potato (Ipomoea batatas (L.) Lam.) is the seventh most important crop in the world and it ensures food supply and safety in many developing countries. I. batatas is a hexaploid plant with a complex and heterozygous genome (2n = 6 × = 90, 3–4 gigabase pairs in genome size (Magoon, Krishnan & Vijaya, 1970; Ozias-Akins & Jarret, 1994)). A preliminary genome estimate has revealed two genome polyploidization events occurring about 0.8 and 0.5 million years ago (Yang et al., 2017). Nevertheless, the complete reference genome of I. batatas remains lacking, which hinders the progress in molecular dissections of its evolutionary scenario and agronomically important traits. Moreover, I. batatas is a self-incompatible and thus obligate, outcrossing species (Martin, 1965). It is almost impossible to develop typical mapping populations such as F2 and recombinant inbred lines for constructing high-density linkage maps and classical genetic analyses. To date, no successful investigation in forward genetics (i.e., quantitative trait locus mapping and subsequently map-based cloning) of I. batatas has been reported. Therefore, RNA sequencing (i.e., RNA-seq, whole transcriptome shotgun sequencing (Wang, Gerstein & Snyder, 2009)) has been widely used as an attractive alternative to whole genome sequencing for gene mining in I. batatas (Schafleitner et al., 2010; Wang et al., 2010; Nurit et al., 2013). However, all reported transcriptomes in I. batatas were derived from second-generation sequencing platforms, which generate relatively short reads (i.e., hundreds of base pairs per read) and are disadvantageous in obtaining full-length transcripts (Koren et al., 2012). To date, the collection and analysis of large-scale full-length cDNA sequences have not been done in I. batatas, which is fundamental to its structural and functional genomics studies.
Ipomoea trifida (H.B.K.) G. Don has been considered as the diploid ancestor of I. batatas and accumulative evidence supports this hypothesis (Srisuwan, Sihachakr & Siljak-Yakovlev, 2006; Wu et al., 2018). Nevertheless, the evolutionary scenario underlying the origin and domestication of I. batatas remains unclear. Unlike I. batatas, I. trifida does not form tuberous roots, and thus comparative analysis of I. batatas and I. trifida may provide insights into the evolution and domestication of I. batatas. Although the reference genome of I. trifida becomes available recently (Wu et al., 2018) and short-read transcriptomes of I. trifida have been analyzed in a few projects (Cao et al., 2016; Ponniah et al., 2017), no study involving the large-scale collection and analysis of full-length cDNA sequences in I. trifida has been reported.
Long-read or full-length cDNA sequences are fundamental to structural and functional genomics studies. First, they provide complete information of transcribed sequences, which are required to gene function analyses. Second, they facilitate accurate predictions of gene models (i.e., to define proper orientation, order, and boundary of exons). Third, they may be utilized in validating or correcting the scaffold assembly in genome sequencing projects. Fourth, they are particularly useful to analyze alternative splicing of transcript isoforms, which is important to increase transcriptome diversity and adaptation potential of an organism. In the past, collecting full-length cDNA sequences was expensive, labor intensive, and time consuming (Seki et al., 2002; Shoshi et al., 2003). The advent of a third-generation sequencing platform (i.e., single-molecule real-time (SMRT) sequencing) has revolutionized DNA sequencing and thus genome/transcriptome studies (Eid et al., 2010). Long reads of up to 20-kb in size, albeit with a relatively high error rate, can be produced by SMRT sequencing (Roberts, Carneiro & Schatz, 2013; Au et al., 2013). Today, high-throughput sequencing combining second-generation sequencing (to generate short reads with high base quality) and SMRT sequencing (to produce long reads with a relatively high error rate) has become an attractive option in genome and transcriptome studies (Au et al., 2013; Sharon et al., 2013; Xu et al., 2015). In the present study, we performed SMRT sequencing to generate large-scale full-length or long-read transcripts from I. batatas and I. trifida, respectively. Comprehensive intraspecific and interspecific sequence analyses were conducted, which has provided a valuable resource for the research community to exploit the origin of I. batatas.
Materials & Methods
Plant material and RNA preparation
Xushu18, one of the most widely cultivated I. batatas varieties in China, was selected for transcriptome sequencing in this study. Eight tissues of young leaves, mature leaves, apical shoots, mature stems, fibrous roots, initiating tuberous roots, expanding tuberous roots, and mature tuberous roots from one individual were collected and pooled together in approximately equivalent weights (Figs. 1A–1H). Similarly, tissues of young leaves, mature leaves, shoots, stems, and roots of a diploid I. trifida plant were collected and pooled. Collected samples were frozen in liquid nitrogen immediately after collection and stored at −80 °C until use.
Total RNAs were extracted using Tiangen RNA preparation kits (Tiangen Biotech, Beijing, China) following the provided protocol. RNA quality and quantity were determined using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Qualified RNA samples were subsequently used in constructing PacBio cDNA or RNA-seq libraries.
PacBio cDNA library construction and SMRT sequencing
cDNA was synthesized using a SMARTer PCR cDNA Synthesis Kit, optimized for preparing full-length cDNA (Takara Clontech Biotech, Dalian, China). Size fractionation and selection (1–2 kb, 2–3 kb, and >3 kb) were performed using the BluePippin™ Size Selection System (Sage Science, Beverly, MA, USA). The SMRT bell libraries were constructed with the Pacific Biosciences DNA Template Prep Kit 2.0. SMRT sequencing was then performed on the Pacific Bioscience RS II platform using the provided protocol.
Illumina RNA-Seq library construction and sequencing
The RNA-Seq libraries were constructed using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Beverly, MA, USA), following the manufacturer's protocol. Qualified libraries were applied to transcriptome sequencing using an Illumina Hiseq 2500 (Illumina, San Diego, CA, USA) to generate 150-bp paired-end sequence reads (2 × 150 bp). High-throughput sequencing reported in this study was performed in the Biomarker Technology Co. (Beijing, China).
Quality filtering and error correction of SMRT long reads
The SMRT subreads were filtered using the standard protocols in the SMRT Analysis software suite (http://www.pacificbiosciences.com), and reads of insert (ROIs) were obtained using the standard protocols in the SMRT Analysis software suite (parameters: minFullPass=0, minPredictedAccuracy=75). After examining for poly(A)signals and 5′ and 3′ adaptors, full-length and non-full-length cDNA reads were recognized. Consensus isoforms were identified using the algorithm of iterative clustering for error correction and further polished to obtain high-quality consensus isoforms. The raw Illumina reads were filtered to remove adaptor sequences, ambiguous reads with 'N' bases, and low-quality reads. Afterward, error correction of low-quality isoforms was conducted using the Illumina reads with the software proovread 2.13.841 (parameters: –coverage=50 –overwrite, –no-sampling) (Hackl et al., 2014). Redundant isoforms were then removed to generate a high-quality transcript dataset for each species (i.e., Ib53861 for I. batatas and It51184 for I. trifida, respectively) using the program CD-HIT 4.6.142 (parameters: -c 0.99 -T 6 -G 0 -aL 0.90 -AL 100 -aS 0.99 -AS 30 -o) (Li & Godzik, 2006).
Functional assignment of transcripts
Functional annotations were conducted by using BLASTX (cutoff E-value ≤ 1e−5) against different protein and nucleotide databases of COG (clusters of orthologous Groups; https://www.ncbi.nlm.nih.gov/COG/), GO (gene ontology; http://geneontology.org/), KEGG (kyoto encyclopedia of genes and genomes; https://www.kegg.jp/), Pfam (a database of conserved protein families or domains; http://pfam.xfam.org/), Swiss-prot (a manually annotated, non-redundant protein database; https://www.uniprot.org/), TrEMBL (an automatically annotated protein database; https://www.uniprot.org/), and NR (NCBI non-redundant proteins; https://www.ncbi.nlm.nih.gov/). For each transcript in each database searching, the functional information of the best matched sequence was assigned to the query transcript.
Predictions of open reading frames and simple sequence repeats
To predict putative open reading frames (ORFs) in transcripts, we used the package TransDecoder v2.0.1 (https://transdecoder.github.io/) to define coding sequences (CDS). The predicted CDS were searched and confirmed by BLASTX (E-value ≤1e−5) against the protein databases of NR, SWISS-PROT, and KEGG. Those transcripts containing complete ORFs as well as 5′- and 3′-UTR (untranslated regions) were designated as full-length transcripts. To identify putative simple sequence repeats (SSRs) in our sequences, the tool MISA (MIcroSAtellite identification tool; http://pgrc.ipk-gatersleben.de/misa) was employed. Only transcripts that were ≥500 bp in size were included in SSR detection.
Identification of transcription factor gene families
This was done according to our previous publication (Ding et al., 2017). Briefly, for each transcription factor gene family, the Hidden Markov Model (HMM) profile of the Pfam domain (when available) was downloaded from the Pfam database (http://pfam.xfam.org) and used as a query to survey all predicted proteins out of our transcript datasets using HMMER (http://www.hmmer.org). When no HMM profile was available for a gene family, all protein sequences belonging to the gene family in A. thaliana were downloaded (http://www.arabidopsis.org) and used as query sequences to search for our predicted protein datasets using BLASTP (E-value ≤1e−10). One redundant sequence was removed if two proteins shared the identity of amino acids equal to or larger than 97%. All identified non-redundant proteins were confirmed the existence of featured domains by searching the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The confirmed protein sequences as well as their corresponding transcripts were compiled (Only those gene families containing more than 10 members in at least one of our transcriptomes were presented).
Prediction of long non-coding RNAs
To sort non-coding RNAs from putative protein-coding ones, we employed each of four computational approaches including CPC (Kong et al., 2007), CNCI (Liang et al., 2013), Pfam (Finn et al., 2016), and CPAT (Wang et al., 2013). Putative protein-coding RNAs were filtered out using a minimum length and exon number threshold according the instructions of programs. For each species, the intersection of the four resulting lists were obtained as final lncRNA candidates.
Identification and validation of alternative splicing
To identify alternative splicing (AS) events, all transcripts of Ib53861 and It51184 were mapped to the the genomic contigs in I. batatas (Yang et al., 2017) and I. trifida (Wu et al., 2018), respectively, by using the program GMAP (Wu & Watanabe, 2005). The tool AStalavista v3.2 was employed to identify putative AS events (Foissac & Sammeth, 2007). Subsequently, 16 of AS events were selected and 10 of them were successfully confirmed by RT-PCR. Total RNA was isolated from the eight tissues in a I. batatas cultivar (Xushu22) as described above. The cDNA was synthesized using a cDNA Synthesis Kit (ProbeGene, China) and used as the template for PCR amplification. Afterward, PCR products were visualized in agarose gel.
SMRT sequencing and generation of full-length transcriptomes
To obtain large-scale long-read transcripts for I. batatas and I. trifida, respectively, SMRT sequencing was performed using a Pacific RSII sequencing platform. Eight different tissues collected from a single plant of each species were pooled and used in mRNA extraction. Three size-fractionated, full-length cDNA libraries were constructed and subsequently sequenced in four SMRT cells (Figs. 1I and 1J; 1–2 kb for one cell, 2–3 kb for two cells, and >3 kb for one cell). In I. batatas, we obtained 220,035 reads of the insert (total bases: 701,923,565), which included 49.9% of full-length non-chimeric and 46.6% of non-full-length reads (Table 1, Fig. 1K), whereas in I. trifida, 195,188 reads of the insert (total bases: 527,497,043) were generated, of which 52.1% and 43.9% were full-length non-chimeric and non-full-length reads, respectively (Table 1, Fig. 1L).
|I. batatas
|I. trifida
|Reads of insert of PacBio sequencing
|Bases of insert of PacBio sequencing (bp)
|Reads of Illumina sequencing for correction
|Bases of Illumina sequencing for correction (bp)
|Number of non-full-length PacBio reads
|Number of full-length non-chimeric PacBio reads
|Average length of full-length non-chimeric PacBio reads (bp)
|Number of non-redundant transcripts after correction
|N50 of non-redundant transcripts after correction (bp)
|Mean of non-redundant transcripts after correction (bp)
|Number of non-redundant full-length transcripts after correction
Given that SMRT sequencing generates a high error rate, it is necessary to perform error correction, which includes self-correction by iterative clustering of circular-consensus reads and correction with high-quality Illumina short reads. To this end, cDNA libraries were prepared from the same samples that were used for SMRT sequencing, and deep RNA sequencing was conducted using an Illumina Hiseq2500 platform. A total of 71,360,785 and 39,372,131 clean reads (total bases: 17,972,706,252 and 11,772,267,169, respectively) were obtained and used to correct the SMRT reads in I. batatas and I. trifida, respectively (Table 1). After error correction, redundant transcripts were removed. Finally, we obtained 53,861 transcripts for I. batatas (named as Ib53861; N50: 2,933 bp; mean: 2,421 bp) and 51,184 for I. trifida (named as It51184; N50: 2,642 bp; mean: 2,190 bp). Those transcripts containing complete coding sequences (CDSs) as well as 5′- and 3′-UTR (untranslated regions) were defined as full-length transcripts. Approximately 34,963 and 33,637 full-length transcripts were identified for I. batatas (named as Ib34963) and I. trifida (named as It33637), respectively (File S1).
Basic sequence analysis of the full-length transcriptomes
The transcripts of Ib53861 and It51184 were functionally assigned and classified according to sequence similarities using BLASTx or tBLASTx (E-value ≤1e−5) against different protein and nucleotide databases. Overall, we successfully identified homologous sequences for 97.25% of Ib53861 and 97.34% of It51184 in the public databases, and the rates of successful validation in a single database ranged from 41.67% to 96.46% (File S2). These results indicate that most of the genes in our datasets are truly transcribed sequences in I. batatas and/or I. trifida. Furthermore, from the datasets of Ib53861 and It51184, 104,540/94,174 open reading frames (File S1), 25,315/27,090 simple sequence repeats (Files S3–S5), 1,401/1,457 transcription factors (Files S6–S8), 1,656/1,389 long non-coding RNAs (Files S9 and S10), and 5,251/8,901 alternative splicing events (Files S11–S14) were identified. These data provide fundamental information for functional genomics study and molecular breeding in I. batatas and comparative biology study between I. batatas and I. trifida.
Analysis of long non-coding RNA
Recent studies have shown that lncRNAs act as key regulators in a wide range of biological processes. In the present study, we in silico identified 1,656 and 1,389 candidate lncRNAs out of Ib53861 and It51184, respectively (Figs. 2A and 2B; Files S9 and S10). Amongst, 421 I. batatas and 355 I. trifida transcripts could be recognized as sense, intergenic, intronic, or antisense lncRNAs (Fig. 2C). Notably, there were only 344 common candidate lncRNAs (i.e., homologs in sequences; cutoff: identity >200 bp & >90%) between the identified 1,656 I. batatas and 1,389 I. trifida transcripts, suggesting remarkable divergence in lncRNA biogenesis and thus their regulatory mechanisms between two species (Fig. 2D). These data suggest that different lncRNA members may be involved in different tissue/organ developmental processes in I. batatas.
Analysis of Alternative splicing
Alternative splicing (AS) is a posttranscriptional regulatory mechanism to increase transcriptome diversity, yet little is known about its roles in the development of tuberous root and the evolution of I. batatas. In the present study, we identified 5,251 and 8,901 AS events out of 10,562 and 17,826 transcript isoforms in I. batatas and I. trifida, respectively (Table 2; Files S11–S14). The AS events were divided into five major types: intron retention (IR), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), exon skipping (ES), and mutually exclusive exon (MEX; Fig. 3A). The proportion of each AS type was comparable between I. batatas and I. trifida and the majority of AS events were IR in either species (Fig. 3B). Overall, the alternatively spliced isoforms accounted for 32.34% or 38.54% of all isoforms successfully mapped to I. batatas scaffolds or I. trifida genome (Table 2), which should have largely increased the complexity of transcriptomes in either species. Notably, 37% of the alternatively spliced isoforms in I. batatas were not alternatively spliced or not detected in I. trifida and so were 63% of the alternatively spliced isoforms in I. trifida, suggesting substantial divergence in AS biogenesis and thus their regulatory mechanisms between two species (Fig. 3C). The isoform number per AS event ranged from 2 to 35 (mean, 4.98) in I. batatas and from 2 to 46 (mean, 4.55) in I. trifida (Table 2; Fig. 3D). In total, 2,074 loci in I. batatas and 3,640 in I. trifida were involved in the detected AS events (Table 2). The maximal number of AS events per locus was 45 (mean, 2.57) in I. batatas and 38 (mean, 2.45) in I. trifida (Table 2; Fig. 3E).
|I. batatas
|I. trfida
|Number of isoforms of the datasets
|Number of isoforms mapped to genome sequences
|Number of isoforms (with multiple involvements) in AS events
|Number of isoforms (with one involvement) in AS events
|Number of detected AS events
|Maximal number of isoforms in a single AS event
|Mean number of isoforms per AS event
|Number of loci occuring AS events
|Maximal number of AS events in a single locus
|Mean number of AS events per locus
|Mean number of isoforms (with one involvement) per locus
|Proportion of isofroms undergone AS
|Number of estimated loci in the datasets
To assess our large-scale predictions of AS events, we manually examined 40 genes that were predicted as containing AS events and found 8 of them were likely false candidates. We then designed primers to examine 16 AS events, each of which located in one gene, by RT-PCR across eight tissues of an I. batatas variety (Xushu22), and successfully confirmed 10 of them (Figs. 3F and 3G). According these results, we concluded that at least 50% of our AS predictions were valid. Given that we only examined one of multiple AS events in each gene and only in one I. batatas variety, our data should be underestimated. Therefore, our large-scale AS analysis has provided a useful resource for studying biological functions of transcript isoforms and the regulatory mechanism of alternative splicing during the evolution of I. batatas.
For example, starch-branching enzymes (EC 126.96.36.199) are one of key enzymes involved in plant starch biosynthesis and sugar metabolism (Zeeman, Kossmann & Smith, 2010). In our analysis, we detected multiple AS events (i.e., one ES and one IR events) in a putative I. batatas starch-branching enzyme I and verified two AS isoforms, whose expression changed over different tissues (Fig. 3G, Gene01). In aboveground tissues (i.e., T01 to T04) and fibrous roots (i.e., T05), the two isoforms were expressed at a similar level; whereas in tuberous roots (i.e., T06 to T08), the smaller isoform were specifically expressed (Fig. 3G, Gene01). Plant alpha-glucan phosphorylases, also named as starch phosphorylase (EC 188.8.131.52), are another important family of enzymes involved in carbohydrate metabolism (Rathore et al., 2009). Our results revealed distinct splicing mechanisms existed between aboveground and belowground tissues in the examined I. batatas alpha-glucan phosphorylase (Fig. 3G, Gene02). In addition, divergent gene-expression and splicing patterns were also observed in other investigated genes including a neutral invertase, an E3 ubiquitin-protein ligase, a pentatricopeptide repeat-containing protein, and a few ABC transporters (Fig. 3G, Gene03–10). These data revealed that alternative splicing and thus transcriptome regulation might play important roles during the development of tuberous roots in I. batatas.
Understanding the genetic basis and evolutionary scenario underlying agronomically important traits is one of central research themes in the hexaploid crop I. batatas. However, achieving this goal is doomed to be challenging because of the complexity of its genome structure (Isobe, Shirasawa & Hirakawa, 2017). In the present study, we applied a hybrid sequencing approach to generate and analyze large-scale full-length or long-read transcripts and their expression profiles in I. batatas. Our study would be beneficial to the I. batatas research community at least in the following aspects: gene cloning, gene family analysis, development of cDNA-derived marker for breeding, gene model prediction, genome assembly, and study of genetic variation within or among species. For example, we have demonstrated an example of fast gene cloning and gene family analysis basing on our transcriptome datasets (Ding et al., 2017). Overall, our study has provided a fundamental resource for functional genomics study in I. batatas, which would certainly facilitate genetic dissections of the origin of tuberous root as well as other traits.
AS commonly occurs in eukaryotes. In humans, more than 90% of genes were found to be alternatively spliced and the predominant AS type was exon-skipping (Wang et al., 2008). In higher plants, the AS frequency in intron-containing genes approximately ranged from 33% to 60% with intron retention as the major type (Filichkin et al., 2010; Zhang et al., 2010; Shen et al., 2014; Thatcher & Li, 2014). In our study, we observed an overall AS frequency of 32.34% in I. batatas isoforms (Table 2). Considering about 30.03% of isoforms contained a single exon in our dataset, the AS frequency in intron-containing isoforms in I. batatas was approximately 46.22%. The estimated AS frequency in intron-containing isoforms in I. trifida was 51.18%, a little bit higher than that of I. batatas. The major AS type was intron retention in either I. batatas or I. trifida, similar as observed in other plants. These data highlighted the prevalence of AS in both I. batatas and I. trifida, which would certainly increase the complexity of their transcriptomes. In addition, we also examined the AS pattern across eight tissues in 10 I. batatas genes and found that many isoforms exhibited a tissue-specific expression pattern (Fig. 3G). These results imply that the generation of AS isoforms in a tissue-dependent manner have contributed substantially to organ/tissue development and species evolution in I. batatas.
AS and gene/genome duplication are two fundamental biological processes contributing to transcriptome and proteome diversity. The relationship between these two evolutionary mechanisms remains debatable. Some studies have reported that the AS frequency decreased after gene duplication and genome polyploidization (Kopelman, Lancet & Yanai, 2005; Su et al., 2006). In contrast, some other reports argued that the evolutionary relationship between AS and gene/genome duplication was more complex and must be cautiously anticipated (Lin et al., 2008; Roux & Robinsonrechavi, 2011; Iñiguez & Hernández, 2017). In this study, our transcriptome-wide AS analysis revealed comparable AS patterns between I. batatas and I. trifida, in terms of mean number of isoforms per AS event or per locus, mean number of AS events per locus, and proportion of isoforms undergone AS (Table 2; Fig. 3). These data showed that the overall AS frequency (not between specific duplicated gene pairs) was not evidently decreased after the genome hexaploidization in I. batatas.
Although I. batatas is a global crop of great agronomic importance, advances in its functional genomics study and molecular breeding remain limited because of the complexity of its genome. Here we report the first collections and analyses of large-scale full-length or long-read transcripts in I. batatas and its putative diploid ancestor I. trifida using single-molecule real-time sequencing. By performing comprehensive intraspecific and interspecific sequence analyses, we provide a valuable resource for genetic marker development, gene discovery, and gene function study in I. batatas, as well as comparative biology study between I. batatas and I. trifida. Furthermore, we analyzed transcriptome-wide long non-coding RNA and alternative splicing, which revealed tissue-specific-expressed transcript isoforms and the importance of transcriptome regulation during the speciation and domestication of I. batatas.


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TheSixthAxis talks No Man's Sky and how the games pre-launch pitch may have mislead many coming into this space-faring survival sim:
"From the feel and functionality of space travel down to the game's gorgeous sci-fi stylings, there's an awful lot Hello Games has managed to nail with No Man's Sky. Despite never stepping foot on its intergalactic hype train, I could still see from a distance why so many people were losing their minds."
It says something about today's gaming world when a developer says clearly "Don't buy this expecting a multiplayer game" and then there's an uproar about the lack of multiplayer.
I swear the only hate on this game continues to come from people who wanted the game to be something it was never said to be. They wanted AssassinsCreed/FarCry/Fallout/ GTA in space - otherwise known as a game that holds your hand and everybody has the same story.
I agree that a lot of heat is coming from the idea of the game being something else. So far I am loving the game for what it is
concerning the multiplayer he (Sean mrrey) has stated multiple times that you are very unlikely to bump into someone but he did State you can play with them if you do. There are multiple articles interviews videos of him in it saying that the game has multiplayer.
That's the issue. PC Gamers have already data mind the game to show that there's not even an indication that the game even had any kind of multiplayer. Apparently there's a sticker on the back of the retail box that covers up the logo for multiplayer. Sean keeps avoiding the question if there's multiplayer and if you can actually see people . People are upset because they lied about it, and yet people are quick to defend it yet if it were any other game you would be chewed up to death.
Many people talked about the game they wanted NMS to be. The problem is the guys at Hello did nothing to clear up what people thought the game was. If anything Sean was misleading with some of his comments. Very unlikely sounds like the possibility exists. He should have been clear and stated it won't happen since there is no possible way to see someone else online. I honestly don't even know how you can call a game "online single player" unless it's a flash game you play in your browser. Even the box art was wrong on early copies. https://geeksnack.com/no-ma...
I think the bigger question is why didn't the developers add multi-player to the game? I think it would be a much more interesting game if you could play with friends. It's not like there is a heavy single player campaign to play through that would be impacted by making it multi-player.
The game could have been like WoW in space. Where you can do your own thing or join up with others and all traipse around the galaxy together. That would have been pretty cool and the adventures of others throughout the universe could / could not influence ones own ability to explore and be part of the action or fly solo.
It wasnt so much about action and blowing stuff up as much as it would have been fun to explore and mine with others. And who knows... it may be added on after the fact. Lots of games initially start out as single player with multiplayer elements added on later.
" And who knows... it may be added on after the fact. Lots of games initially start out as single player with multiplayer elements added on later."
He said it was something they were looking at and would come later...later. You know, as in later. Then again pointing this out prevents people from downplaying the game in the name of traditional multiplayer.
if they just came out and said that there is no way to interact with anyone or see them online that would have been fine. but that isn't what he said or implied.
You see the problem with that entire "oh he didn't clear it up" argument is that he did on twitter and the NMS website.
Aug 8th
"To be super clear - No Man's Sky is not a multiplayer game. Please don't go in looking for that experience."
Then you'd have to understand how procedural generation works. It would have been impossible to have multiplayer in such a game since each game is generated randomly. It would be like asking two people, each with a set of 4 dice, to roll the same number at EXACTLY the same moment in time.
Yes that's impossible. You'll have better chances to get struck by lightning.
@Hugs: Yeah, sure buddy. Spending years of hyping the game as a multiplayer game should be forgotten because the DAY BEFORE the game is released the creator tweets that it's not a multiplayer game. Yeah, sure..
Let's just forgive him for straight up lying to everybody.
"Sean Murray: The only way to know what you look like is for somebody else to, y'know, see you
Colbert: Can you run into other people, players, in the game?
Sean Murray: Yes"
Sean is a Peter Molyneux wannabe, and too many people let's him get away with it. Sheeps.
@death- "Many people talked about the game they wanted NMS to be. "
That's irrelevant to hello games so they talked about something that the team never stated or even hinted at ever being the game?
"The problem is the guys at Hello did nothing to clear up what people thought the game was."
So if 100 people have a 100 different ideas of what they want no man Sky to be why on Earth should Hello games go out of their way in explaining to hundreds of different people what the game isn't ,when it's easier to just State what it is?
I'm sorry but ignorance is not a virtue and it's a completely stupid excuse you're basically saying for the dumb slow dim-witted people we should have someone go out and continue to repeat themselves as opposed to just ignore them.
it is not any developer's fault that a gamer decides to make up what they think the game is when the information has been provided to the public and all the damn places that it legally needs to be on their website on their Facebook page on their YouTube page and all over the public.
Being stupid is not an excuse.
If you think multiplayer makes a game interesting why don't you actually go play a multiplayer game? In fact if the damn developers telling you that this is what it's not, wouldn't that already stop you from purchasing the game in the first place?
I'm sorry but it's not the developer's fault that stupid people exist and decide that they don't want to listen to facts regarding a game.
If you honestly believe that the developer withheld information or misguided the public or advertised the game falsely take your claims to court.
Whenever I hear Gamers try to bring up such grievances against developers are publishers usually what holds them in their tracks is telling them if their grievances are real they should have no problem taking it to court.
The very thing you're talking about is illegal!!! if what you're saying is absolutely 100% true then it technically means you could take it to a court of law and sue them for false advertising.
So let's sue Activision because Call of Duty is not an MMORPG kart racing game?
still implying that if a miracle happened and you did bump into someone, that would be the only way you could see your character.. 😶
I'm pretty sure this is what most people are upset about.
Exactly, not only that, that was said over a year before release but people still insist it all changed 3 days before.
What the hell is wrong with you people!?
He clearly said there was multiplayer & clearly said that it was possible to meet other people. In fact claiming it would be the only possible way you would ever find out what your character model actually looked like. People have been uploading the very video where he makes this claim & you people are still childishly defending him.
It's ironic that while trying to make the point of others being stupid you come out the worst in your own argument without anyone else having to even get involved.
He never said there was multiplayer, that entails playing a game 'with' others rather than just existing in the same server/shard. He said the only way you would be able to know what you look like is if someone else saw you. I'm not certain if that last item has been fixed or not, but the statement of there being "multiplayer" is false, as that entails a system design of playing with others and that is not what he has ever said. Journalists have said it, but he has not.
"The only answer that I can think of for this is a really technical one. If we were to make a game where we synchronized every player, what they were doing with every other player, then that would be impossible and no one has ever done that." Sean Murray 2014.
"To be super clear — No Man's Sky is not a multiplayer game. Please don't go in looking for that experience," Sean Murray 2016
What is confusing to you?
It seems a lot more ridiculous to start screaming about lies when it's obvious the game has server problems. I'm still having to upload discoveries more than once, for example.
Expecting the game to have every player in the same instance is equally idiotic, and the different times of day in the infamous "proof of lies" video should strongly imply to any rational person that those two players weren't in the same instance.
I watched that video and thought "technical issues"
You watched it and grabbed your digital torch and pitchfork.
I know which seems more rational to me.
The uproar is because literally up until 3 days before release he have been told countless times "Yes you CAN find other players, you CAN see them, however the chance of that is extremely low" So obviously people are going in expecting to find someone only to find out we've been lied too for 5 years with a tweet saying vaguely that there isn't multiplayer which the common public won't see but have read about the low chance of meeting players on multiple occasions.
I do question how easy it was for people to find one another in the game when he did say it would be almost impossible. Makes me wonder if the world is "that big" or is it a simulated size while only certain portions are being utilized to start with while others aren't?
He said it was impossible in 2014. Then, in 2016 he said, it isn't a multiplayer game.
Internet search engines are your friend.
The uproar is about an imagined proof of lies.
Articles about games releasing with technical issues, using NMS as an example, would make perfect sense. Articles about "they lied!" make no sense. The game is obviously having server issues.
That's what I can't figure out. I totally understand how games like this can be polarizing...but the devs have said, for months and over many vids and interviews, that the odds of you running into another player are very very low. I just don't understand why this is suddenly so surprising.
Saying the odds are very low isn't the same as saying it's not possible. From the sounds of it, the game is an offline game that uploads information like names and discoveries made by other people. That would explain why you can't see other players. It's misleading at best and at worst simply a lie. It feels like a bait and switch tactic which is why some people are upset.
I'm sorry but the game was promote with Multiplayer in mind until recently.
The video about it is pretty clear when Sean's asked about did he created a big massive Multi player world, to witch he respond a big yes.
Also all the time he's been asked about it he mentioned that yes it was possible to meet players but hint that it would be nearly impossible due to the size of the universe and not because of server room where basically you have to be in the same 16 to 20 person room to see the other player.
And to conclude, NMS did cover up old boxes that had the online logo and change the rating accordingly, saying it's printing error would been a major major problems since it would mean that all committees that approve, art, legal mumbo jumbo, quality std would have fail.
I'm sorry but no it wasn't. In 2014 Sean Murray, in an interview with Game Informer stated:
"The only answer that I can think of for this is a really technical one. If we were to make a game where we synchronized every player, what they were doing with every other player, then that would be impossible and no one has ever done that."
And just in case you were confused by the word IMPOSSIBLE. He said this in 2016:
"To be super clear — No Man's Sky is not a multiplayer game. Please don't go in looking for that experience," Murray
Here! How about a simple answer! no the game is a single player experience instead here are the answer!
But I know the voice were probably change and it wasn't him!
Says alot about people who are one track minded and failed to notice he states many times about meeting others until that tweet.
A game that large is a total waste to one person in my opinion
So, stating that it would be impossible for the 2 years leading up to launch means he said you could meet up? That isn't even one track mind. That is off the rails delusional.
Don't bother with this one, thorstein. He's having a lot of fun with his virtual torch and pitchfork.
I see server issues or other technical problems.
He sees "Ermagerd they LIED!"
Another example of self entitled millennials giving false information and delusional thoughts.
It's well documented you could see another player if it's possible, by luck you have had people attempting it and failed, you've had stickers placed on the back of a game replacing multiplayer features to single player and only one tweet just before launch he asked people to into the mind set of being a single player.
Now you are blaming servers which is false hope when some people have checked packets, codes and made it clear there is no multiplayer feature to meet anyone.
He should of made it clear it's a single player experience well before coming out. The more you support nonsense like this the more companies will take advantage.
@thorstein: You are incredible do you know that? He says one time in two years that all players synchronizing would be impossible and one time the days before release that the game isn't a multiplayer game and because of that you just swat away every other time he flat out lied?
How can there be any server issues when the game don't even send any information out?
Are you a couple of shills payed to defend the game or do you just enjoy Sean sitting on your face and shitting down your throats?
If anyone is coming off like an entitled millenial it's you and the other crybabies.
If I thought it would make a bit of difference I'd find every Murray quote through the years that should have clued you in to the game not being multiplayer oriented, but I know it would be a waste of my time.
Have fun with your delusional crusade.
Do yourself a favour Pongwater and keep up, old article and a large list of missing features now on reddit.
I'd hate to be someone like yourself defending pure nonsense and waffling on and on in denial.
https://youtu.be/ZqeN6hj4dZ... this is why people expected multiplayer.
There is nothing wrong with gamers. It is the gaming media that is largely the problem.
I'd like this game a whole lot more if I knew where to get an atlas pass 😭
Go to any beacon and select the outpost option. Build a bunch of bypass chips until you get 2 or 3 command centers from the beacon. Go to them and interact with the console in the middle of the room and you'll get a puzzle. If you get it right, you'll get a chance to get v2 or v3. So far I've only gotten v2. I'm not sure if you can get them a manufacturing facilities that also pop up with that option on the beacon. So far, every confirmed Atlas Pass has been from command centers.
Follow the anamoly path that will lead you to the Atlas, it will give you the option from the Galactic map.
They will give you the Atlas pass v1.
Perhaps what @xphear said works, but I got mine from Space Anomalies popping up randomly in any solar system while your exploring. I haven't gotten any passes from command centers, but I've gotten V1 and V2 from Space Anomalies.
People making excuses for this game not having multiplayer. Shit even Mind craft has CO OP. lol To miss such a vital component is mind boggling.
Before any goes about how they enjoyed playing SOLO in souls games... every souls game had multiplayer.
I disagree with the fact that every game needs MP just as I do that every game needs SP (though I would say that my preference is for there to always be SP, but I'm not going to tell the developer it 'should' be there). It's up to the developer. If you don't like games w/o MP, then don't buy them. That's the best way to tell a developer what you want in the end and if they listen they listen, if they don't, they don't.
I like single player games with a good story as much as multi-player focused games. The thing with NMS is it doesn't have a deep story to drive it. It's simply creating random worlds where you do the same thing over and over until you get bored of doing it. For a game to focus on grinding so much, there needs to be a loot system or other type of reward system for doing it. You can upgrade your ship/suit in NMS, but there isn't much incentive to do it since you aren't leveling or even working towards a goal set by the stories narrative.
This is a game that screams for multiplayer since it relies so much on the players to create a story/reason to play.
ye but then again not every game needs a SP either... Can you really not see the benefits of NMS having a MP?
1000% agreed, I'm in the same boat.
I'm tired of people begging for single player games to have multiplayer component just as much as I'm tired of people begging for multiplayer games that have single player component if the creator of the game created a concept that specifically has to do with single player let them focus on the concept of the game's creation if a game was made specifically to be a multiplayer only title I want to see what this team is trying to convey.
It's actually why if I even get Titanfall 2 I'm only getting it for its multiplayer simply because the team has already expressed that they had no interest in even creating a single player for the original Titanfall , why should I suddenly care about its sequel's knowing that they don't even care about such a portion of their game?
Dice will not make a single player game that comes near Uncharted, naughty dog will not create a multiplayer portion that touches a Battlefield game.
Let's respect Developers for what they bring to the table and stop trying to ask for this jack-of-all-trades game that is like 15 genres. Lol
@wellyuk- the concept of No Man's sky was never created to be a multiplayer title.
I could see the benefits of No Man's Sky being a huge Avast MMO just like I could see the benefits of Dead Space being an amazing cover based multiplayer competitive shooter like Gears of War but the reality is it's just what you believe the game was going to be as a multiplayer game or simply Fanboy wishful thinking?
There's what you want and there is reality this is no different than the people that bag for single-player portions in games that are primarily multiplayer expecting some Bioshock like single player but this is also no different than games that have multiplayer portions that were attacked on in Gamers expecting it to be this amazing competitive Battlefield or Counter-Strike like experience.
What has Halo games done to have ever shown they could create such a complex Network MMORPG?
Did you seemingly forget that the game was made by about 5 people?
Again there is what you fabricated in your mind and actually reality I could assure you Battlefield 1 single player is not going to be some Half-Life or BioShock 2 defining single player, just like Titanfall 2 single player is not going to be some Game of the Year contender, it's a great example to show that people cried about Titanfall one not having some deep single player , well do you really believe with its sequel you're going to get that?
People were talking about RPG's they were even talking about Mass Effect and other type games in regards to what Titanfall one single player could have been completely, disregarding that the team has zero history of ever producing such a title, they've basically made up a fantasy in their mind of what they think it could be, absolutely disregarding all reality....
So when you say that no man's Sky multiplayer could be a fascinating thing are you saying this based on absolutely unrealistic wild dream? What has this team done that could ever support what you're actually saying?
Buddy this is no different than the people that were making it sound like Titanfall 1 single player could have been some Milestone story and experience....
I never believed that even for a slight moment based on the history of the team and we now finally get to see once and for all when Titanfall 2 story is shown to Gamers this is what you guys were going to get, please stop making up and fabricating with your imagination what you think the outcome will be based on nothing but fantasy.
No man's sky was made from a team of 5 people how many MMOs do you know where made in such a short period in time from a team of this little?
What you think would have been a great thing I can assure you was never going to be reality , consider Star Citizen is still being developed and its base concept is no man sky's BUT an MMO!
***ye but then again not every game needs a SP either... Can you really not see the benefits of NMS having a MP?***
Did you not read what I wrote? First sentence even addresses SP. And, regardless of the "benefits" there are also negatives to it having MP, such as creating another game that is online required when the focus becomes MP focused as well as griefing by other players or the like (depends on the design, but just look at games like Rust and State of Decay for how other players actually drive people away from the game rather than to the game).
I like to leave those decisions up to the developers. If I ever made a game, it would be SP only for the sole fact that it makes balancing the game design 1000x easier to do. I'd like to think that the people would respect my decision to do that rather than argue it should be there.
I wasn't going to mention souls games but I do recall playing The Witcher 3 and Fallout 4 solo..
Aye, but FO and Witcher was rich with story. In the main storyline and those side quests. Something NMS does not have. Much of the boredom many are complaining about would have been mute if Co op was added.
How do you make excuses when the developer has been very open and up front about multiplayer for 2 years. He stated that it would be impossible to sync up players...in 2014. "The only answer that I can think of for this is a really technical one. If we were to make a game where we synchronized every player, what they were doing with every other player, then that would be impossible and no one has ever done that."
Then, right up to launch he said: "To be super clear — No Man's Sky is not a multiplayer game. Please don't go in looking for that experience," Murray
Thinking this game was multiplayer is mindboggling.
Read my comment. I say that it is stupid that they didn't ADD it. So much space wasted. It sitting at the score that its sitting on metacritic for a reason. Much of the complaints of boredom could have been elevated with co op. Not talking pvp.
Sorry, I didn't catch his tweet 3 days before the game came out. On top of that I had already preordered it.
However he has stated (in another tweet mind you) that it is possible and they are having server issues, so people should wait until they get their bearings before crucifying them. Now if the pseudo multiplayer comes out at the same time a patch is released, that would be laughable.
What excuses? The game was never marketed to actually be a multiplayer game, the developer came out several times and stated it's not a multiplayer game.
How could that be a vital component when the concept of the game was never even created with the idea of multiple people essentially playing with each other?
I'd actually rather the sequel have such a concept simply because I don't believe you could just add something like that to this game considering the foundation has already been clearly laid they've stated way too many times that this is not a multiplayer game to be seriously crying about such a thing right now.
if you want a multiplayer game go play a multiplayer game , stop asking of different concepts to be what you want when the developer has stated dozens of times otherwise.
It would seem that a lot of folks are dense. It was clearly stated what it was going to be. Wishful thinking can't change what it is.
What the hell are you idiots blabbering about. Multiplayer was never, EVER a component in this game. It has only ever been suggested that there is a slight possibility that you could run into another player but there was never anything suggesting that there was actual multiplayer gameplay. This is just another empty spin to try and make excuses to rip on a game because you illiterate morons couldn't comprehend the clear description of this game that has been there since day one. Everyone who paid attention to what this game was intended to be are loving it and everyone else expecting something different just continue to embarrass themselves.
The irony...
45 second mark the interviewer asks Sean: "Will you be able to play with your friends?"
Sean's answear: "Yeah"´
This either hints at multiplayer or split screen. There might be an update that adds multiplayer or split screen in the future but as of right now the people you're speaking of are FAR from being the "illiterate morons" you're portraying them to be.
'You could encounter other players, but the reality is the likelihood of that is timing' and yet you cling to a single word answer to a vague question that defies both common sense and every single description this game has ever had in which multiplayer never made sense. Look up the word irony before trying to insult me with it.
The double Irony: 2014:
"The only answer that I can think of for this is a really technical one. If we were to make a game where we synchronized every player, what they were doing with every other player, then that would be impossible and no one has ever done that."
"To be super clear — No Man's Sky is not a multiplayer game. Please don't go in looking for that experience," Murray wrote in the first of a series of tweets.
Vague question? What about the interviewer's question at the 40 second mark in the video I provided wasn't clear enough? What about Sean's answear to that question wasn't clear enough?
I don't need to look up on anything. You were the one standing on your imaginary pulpit, insulting other people thinking that you were oh so right and everyone else was oh so wrong but it turns out that you were the one that was oh so wrong, hence the óh so sweet irony.
BTW another video for you
@ thorstein
You mean the tweet that Sean posted in the 8th of THIS MONTH? A WHOLE day before this game was released after months of advertising the game as something it was not? The blame for expecting this game to have multiplayer is on the "illiterate morons" that bought the game 1 day after that tweet? I'm feeling the pain of the irony!!
The game does NOT have multiplayer which is something that Sean confirmed on the tweet that he posted 1 day before it released so I have no idea of what your 1st quote is for.
BTW I don't have a twitter account and the only reason why I know about that tweet and date it was posted was because I watched the video in this same exact comment 5 seconds before writting it :)
You've shown nothing but straw grasping nonsense. It's been very clear from day one that this game was single player exploration and multiplayer if any was completely circumstantial. Never once was it portrayed as even a viable option and all that gets thrown aside and you want to label the advertising as fraudulent. Once again I say, you have nothing and there is zero irony here.
Triple the irony. Now you are just lying. I proved to you that for 2 years he has said it isn't MP and you return with the same claim.
Why do you want the game to fail so badly? Why do you want a young vibrant studio to cease?
What is in it for you? Do you get off on stuff like that?
http://www.metacritic.com/g... I'll just leave that here.
So is it the shitty game? Or your taste in games? Or is everyone else just not understanding this "masterpiece"?
"You've shown nothing but straw grasping nonsense"
I showed Sean's words.
"multiplayer if any was completely circumstantial"
If any? So you went for no multiplayer at all to the possibility of having multiplayer? That is what I call progress :)
"Never once was it portrayed as even a viable option" Seriously? Even after those 2 videos? I can see that it's not worth continuing this discussion.
@ Thorstein
I'm lying? I thought that it was weird that you didn't provide links for the quotes you posted but I'm a curious guy and well, google is an amazing tool and it became apparent why you didn't provide BOTH links
"The only answer that I can think of for this is a really technical one. If we were to make a game where we synchronized every player, what they were doing with every other player, then that would be impossible and no one has ever done that. What we can do is, like many games that you have at the moment, where you are flying around with an open lobby. People are coming into that lobby and leaving it – like if you play Watch Dogs or something like that. Effectively, we have players joining your discrete space. We're not trying to make an MMO where you can play with literally 60,000 people on screen. We handle the case like where other people can fly past in your game or that you can bump into other players in the game."
As you can see, he didn't say that multiplayer was impossible so who is the liar now? Add this to Sean saying point blank that it would have multiplayer AFTER that interview and I'm definetely not an "illiterate moron"
"Why do you want the game to fail so badly? Why do you want a young vibrant studio to cease? "
I said that? No, I just replied to someone that for some reason felt the need to insult other people but if you ask me I have no sympathy whatsoever for devs that lie in order to sell their games.
Wow. You are persistent. And meticulous. I admire that. Perhaps a tad too meticulous? Funny how you left out the part after " bump into other players in the game" Where he said "But that's okay for us because it will NEVER happen." (emphasis added)
That video where he said yes to being able to play with friends (I'm genuinely curious why he said yes) would definitely make someone think it has multiplayer, as long as that's their only source of info on the game. Otherwise I find it hard to fathom why anyone would have expected multiplayer-unless maybe they hadn't bothered to get any info on the game at all.
Yeah, one called me illiterate moron and the other called me a liar.
"Never happen"
It was so impossible that 1 or 2 days after the game released, 2 gamers tried to meet on the same exact planet.
No because the game doesn't have online. They were on the same exact planet but obviously they couldn't see each other.
So, I guess he was telling the truth when he said it'd "never happen"
What is the point of multiplayer in this game? Its a game about exploring the limits of your own boredom. There's nothing to do cooperatively but except trade resources.
Everytime Sean was asked about multiplayer, he said it was extreme unlikely you'd run across another player.
So how do people get "You can explore the galaxy with your friends" from "extremely unlikely to run across another player"? It's wishful thinking. In the best case scenario where you run into someone else once in a blue moon would hardly make this what most people called a multiplayer online game


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LOS ANGELES (AP) — Naked Donald Trump could be yours. Celebrity auction house Julien's Auctions The statue is expected to fetch at least $10,000 at the Oct. 22 sale. A portion of the auction proceeds will benefit the National Immigration Forum, an immigration policy organization. The statue originally appeared on Hollywood Boulevard in Los Angeles. Similar Trump statues popped up in San Francisco, Seattle, Cleveland and New York. The naked Trump will be on view at Julien's Auctions beginning Oct. 17. Other lots available at its "Street Art and Contemporary Art" sale include Shepard Fairey's "Hope" portrait of President Barack Obama and a Banksy print. … [Read more...] about Naked Donald Trump could be yours! Statue up for auction
Donald trump 007
Kim Jong-un might lose 'special relationship': Donald Trump warns after North Korea conducted 'important' test
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GOP Is 'Running for Cover' Over Donald Trump's 'Deeply Disturbing' Alleged Attempt to Force Ukraine to Investigate Biden, John Kerry Says
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Wicklow County in Ireland has just experienced a massive fire, leaving countless people at risk of losing their homes. However, and probably rarely can say this: this wildfire brings an interesting fact. People discovered a message hidden between the trees, only visible when burned. The message has existed since World War II.
The fire at Bray Head revealed the letters EIRE made of stone, placed along the coast during World War II to signal the bomber that they were flying over neutral territory. In Irish, Éire thought Irish .
The fire at Bray Head revealed the letters EIRE made of stone.
The Irish Air Force Police Unit discovered a message that could be seen from above when flying across Wicklow territory, to investigate the situation after the fire took place.
And this is not the only "sign" of the EIRE in Ireland. Along the coast, there are many similar areas.
"Signs posted by the Coast Guard Service in the summer of 1944, to warn war planes are not active in a neutral country , " Michael Kennedy from Ireland's Neutral Defense Organization told. Dublin Live. "Up to 150 tons of stone were used to make a total of 83 signs along the Irish coast".
This "signage" helps American bombers cross the Atlantic Ocean.
Kennedy also added: "The US Air Force has demanded to make these signs, making them a means of positioning for pilots. They have helped American bombers cross the Atlantic."
Although the number of "signposts" is very much, but the Irish Air Defense Police unit calls this an unusual discovery, because there are very few such places "surviving" through World War II. .


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