Cooking Celebration Cake Princess | Games for Girls - Beautiful and Delicious Cake
-------------------------------------------
Handcuffed for handing out the Constitution - Duration: 1:23.WE JUST ANNOUNCED YESTERDAY,
AT KELLOGG COMMUNITY COLLEGE? STUDENTS WERE ARRESTED,
HANDCUFFED, HAULED OFF TO JAIL AND BOOKED FOR HANDING OUT
CONSTITUTIONS. >> AS WELL THEY SHOULD-NO, THAT'S WRONG, THAT'S
BAD, ISN'T IT? THAT'S- >> (LAUGHS) YEAH. >> FOR HANDING
OUT THE CONSTITUTION. 'CAUSE IT'S SUCH A HATEFUL DOCUMENT. >>
ABSOLUTELY. SO IT'S- >> THAT'S JUST STAG-OKAY, THAT KIND OF,
LIKE, MAKES YOU GO "BOING!" ANOTHER CASE WHERE IT WOULD BE,
LIKE, "YOU'VE GOTTA BE KIDDING," CAN YOU THINK OF ANOTHER? >> OH,
ABSOLUTELY. LIKE EXAMPLES TO WHERE YOU HAVE CHRISTIAN
BUSINESS OWNERS ARE BEING TOLD THEY HAVE TO PARTICIPATE IN
SAME-SEX WEDDINGS. >> RIGHT. >> AND IT'S, LIKE, THEY CANNOT, YOU
KNOW, REFUSE A CLIENT. IT'S NOT THAT THEY HAVE ANY ANIMUS
TOWARDS ANYONE IN THE HOMOSEXUAL LIFESTYLE, AND SOME OF THESE
INDIVIDUALS EVEN HAVE HOMOSEXUAL EMPLOYEES. THEY HAVE NO ANIMUS,
BUT THEY CAN'T PARTICIPATE IN THAT ACTIVITY BECAUSE THEY FEEL
IT'S A DENIAL OF THEIR FAITH. >> YEAH. BUT, SEE, THAT, THOUGH,
THAT LITTLE...RULE RIGHT THERE APPLIES ACROSS THE BOARD, WHICH
IS WHY THE CONGRESS PEOPLE WHO DIDN'T WANT TO ATTEND THE
INAUGURATION HAD TO GO. >> EXACTLY. (LAUGHS) >> YEAH.
ALRIGHT. KEEP UP THE GREAT WORK.
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Prime Minister's Questions: 1 February 2017 - Duration: 39:19. For more infomation >> Prime Minister's Questions: 1 February 2017 - Duration: 39:19.-------------------------------------------
Topeka votes to hire out of state firm to look for new city manager - Duration: 0:31.COUNCIL VOTED
TONIGHT
TO HIRE AN
OUT-OF-STATE FIRM
TO
SEARCH FOR ITS
NEXT
CITY MANAGER.
THE GOVERNING
BODY VOTED 7 TO 2
TO
NEGOTIATE A
CONTRACT
WITH EXECUTIVE
RECRUITING FIRM
"STRATEGIC
GOVERNING
RESOURCES" OUT
OF
TEXAS.
COUNCIL MEMBERS
"KAREN HILLER" AND
"ELAINE SCHWARTZ"
CAST
NO VOTES.
DURING
DISCUSSIONS..
HILLER
SAID SHE SHE
WANTED
THE CITY TO CHECK
REFERENCES
CHECKED
BEFORE MOVING
FORWARD.
SCHWARTZ SAYS
SHE
WANTS THE SEARCH
TO BE
CONDUCTED BY
SOMEONE
MORE FAMILIAR
WITH THE
NEEDS OF
TOPEKA.###
-------------------------------------------
HOT NEWS! 2017 Subaru BRZ Performance Package - Duration: 4:09.there's something to be said for using
the right tool for the job
it's not always possible but you know
that when you will have the vice grip
deep down inside it just feels wrong
their satisfaction joint even dignity
and using the proper instruments to
execute a task accurately on the first
tribe our attempt will be updated 2017
super brz on fuji speedway last year
were stymied by summer college
enrollment at the base of Mount Fuji
reducing visibility to just a couple of
car line but it's an extremely can't
circuit with a nearly mile-long front
straight so even on a clear day it's not
the ideal venue for the BRZ which is why
we accepted Subarus invitation to take
another go at it this time at Turkey to
aquatic and off-the-beaten-path track
near granada spain if you go trying to
pawn ella the performance packages the
highest spec available on the chassis to
date in the US and that includes toyota
and science presents the setup is
exclusive to the ER is a line and has no
toyota counterparts with the price of
1195 dollars on top and only available
lon the 20 8465 dollars limited trim
level with a manual it gets you a host
of upgrades to its unsprung component
all of which would cost several times
more is procured piecemeal in the
aftermarket sorry there are no power
upgrade save for the bump of five
horsepower and five pound feet of torque
on manual transmission 2017 modules the
most noticeable of the enhancements are
sharp gunmetal finish 17 by seven got
five inch wheel inspired by the same
Doris watanabe designed the door in
Japanese turning racers of the nineteen
seventies
the extra half inches with accommodate
larger grandma break four-piston
calipers upfront biting down on rotors
that have grown by 0.95 inches in
diameter and thickness to 12.8 by 1.18
and to pot calipers pinching 12 dot
forward by zero dot 79 inch rotors up
from 11.4 buying 0.71 how that these are
the same great dimensions that you'll
find on the car subaru still considers
the flag bearer of its enthusiasts
lineup the rally ready WRX STI
performance pack the are these are
suspended by fax EF tampers and curry
weight penalty of just 20 pounds over
the limited the series dot yellow seen
here takes all the goodies of the
performance package and adds exclusive
yellow paint this through is quick to
point out is entirely different from the
yellow that appeared on the 2015 lioness
RS release series 1.0 will concede that
it's less boy racer but only slightly
other visual cues include black versions
of the performance a callaway all black
side mirror housings and smokes imagine
-------------------------------------------
Things That Make Me Happy - Winter 2017 - Duration: 6:06.Lately, I've been feeling like I need to
add a little bit of positivity to my
life. It's easy to let your outlook get a
little bit darker whatever things aren't
really going your way so I thought that
I would bring back up this idea for a
series that I had a long time ago. We'll see if I
stick with it this time. Because I
think now more than ever, it's important
to focus on the things that make me
happy.
Hot cocoa!
I like tea. I've never really been a
coffee drinker but whenever I'm making a
choice regarding which hot drink i'm
going to have in the winter, it's hot
chocolate. My step-brother got me this
amazing hot chocolate that I can make
over a stove. It's these little blocks of spicy
chocolate that you melt in milk or in my
case, coconut milk and it is so good. So
especially when I'm having a bad day,
there are a lot of things that a cup of
hot cocoa will fix. [stressed out laugh]
Lately, I've been finding a lot of joy
in making art for people. I consider
myself an artist in a kind of broad
definition of the term. And I've enjoyed
making art but I've never really
considered myself to be much of a
traditional artist as far as drawing and
painting goes so I've been practicing a
lot lately and this past holiday season,
I made a bunch of custom portraits for
families that are important to me and
I'm actually really happy with the way
that they came out and more than
anything else, the reaction that I've
been getting to them has completely made
my stressful holiday season worth it.
There was something about watching them
look at this piece of paper that had a drawing
I had done of them. Before my dad even got
the bubble wrap off of his portrait, he
was crying. Giving gifts is always a
wonderful experience like that but I
feel like given how much time I put
into it and how personal it was, it
really just made me feel so good inside
to see how much they loved that so I'm
gonna try and do it more often if I have
the time because it really made me feel
good and I really needed that this
season.
Even though I'm all grown up, even
though I hate driving in it, I really love
big fluffy snowflakes. Just when there's
a blizzard and they're, the snowflakes are like
this big and they kind of float through
the air instead of falling down, they're
just so beautiful and I just want to take pictures of
them but the photos never quite capture
just how light and airy and fluffy and
wonderful they are and they make me very
happy.
I know it's a silly thing, but you have to
focus on the silly things sometimes.
Another thing that has made me very
happy
is being able to sleep through the night
for the first time since last summer.
When you have a baby, sleep just usually
doesn't happen and for a very very long
time
our son was not sleeping and lately we've been
working extra hard with him on
sleeping and he's done it a few times
this week
And it has been so nice! I've gotten to hang out at night
instead of going immediately to bed. I've had
the opportunity to wake up in the
morning and not feel like I've been hit
by a truck
it's been beautiful and wonderful. It's very
good. doesn't happen all the time but when it
does, I am very happy about it.
Pokemon! Pokemon has been making me very
happy lately. Yes, I'm still playing
Pokemon Go. I don't get to go out for as
many walks because it's really cold
outside but I bought myself Pokemon Moon for
Christmas and I've been enjoying it
mostly because I actually have time to
play it once my baby goes to bed.
It's been really nice to just do
something for me that's not stressful, that
I've actually enjoyed since I was a
little kid and it's just
yeah! It makes me happy. That's been a thing
lately that has made my life just a little
bit better.
I definitely go through stages of my
life where I'm really obsessed with
Polaroid or Instax photos sometimes they just
come back into my life and I realize, man
they're cute! What is so cute about
these little instant photos with the little
white plastic frames?
I don't know! There's something about them. My
nephew brought an Instax camera out to
dinner with us and took a bunch of
pictures of everyone in the family and
gave them to us to keep and we have them up on
our fridge and I just, I love them! They're adorable.
The newly illustrated versions of the
Harry Potter books are beautiful.
We have the first two since that's all that's been
released so far and I have not read them
yet because I want to experience them for the
first time with my son when he's old
enough to understand what I'm saying
to him but I flipped through them and they
are spectacular. I just, we didn't have them
when I was younger obviously because
they're new and I am really excited to
get to enjoy this new version of the
stories I love so much with my own child.
I hope he likes them. I might be a little bit sad
if he hates Harry Potter.
another thing that makes me happy is
supporting handmade artists when I'm
financially able to. In addition to
being a handmade artist myself, I really
really love buying other people's handmade work.
It makes me super happy and knowing how
important it is for me to make a sale,
I really think it's important to support
the handmade artists that I love
Lately money's been a little hard to come
by but this past holiday season, whatever
anybody asked me what I wanted for
Christmas, I just basically sent them the
link to these artists that I love and some
people bought me some work that I've
been admiring for years and years and so
event though I didn't have the means to
support the artists myself, I was able to
direct somebody who did to check their
stuff out and now I have these wonderful
little handmade products and these
beautiful pieces of art and I get to
enjoy them and I love it! and to wrap up
this episode, the fact that I have a place
where I can share the things that I
create with people who will listen and
who will tell me nice things if they enjoyed
it is really wonderful and amazing!
I make these videos because I really
enjoy making these videos. I like having
something to look back on to document
different parts of my life but the fact
that all of you are here makes me
incredibly incredibly happy and I feel
really fortunate to have the opportunity
to show you the things that I'm making.
Thank you so much for being here and
supporting me. If any of these things
made you smile too, definitely like this
video and I'll see you soon!
-------------------------------------------
Games for Kids Talking Tom Gold Run Vs Despicable Me Vs Talking Hank Kids Video Android Youtube Kids - Duration: 9:04.Games for Kids Talking Tom Gold Run Vs Despicable Me Vs Talking Hank Kids Video Android Youtube Kids
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-------------------------------------------
When will the IPO floodgates open for tech's unicorns? - Duration: 1:06.The floodgates are probably open today.
Our team talks about a difference between a demand-side problem and a supply-side problem. Floodgates
suggest that the demand has not been there, I'd actually argue the demand
has certainly been there, but because of the preponderance of capital available in the
private markets for the last several years, companies have not needed to come public.
We've seen the private markets shift quite a bit from '15 to '16 and we've seen
activity pick up in terms of companies preparing to go public, so I would certainly anticipate
that '17 will be the year in which we address the supply side.
We've seen some really encouraging signs here in the latter part of '16 in terms
of investor interest in technology companies, in growth companies, that's very evident
to us today, it's really just a matter of companies coming to the fore and wanting to
be public companies.
-------------------------------------------
Wrangler Barricade Trailforce HD Front Bumper w/ LED Lights & Winch (2007-2017 JK) Review & Install - Duration: 8:26.I'm Ryan from extremeterrain.com, and this is my review and installation of the Barricade
Trailforce HD Front Bumper with LED Lights and Winch Combination, fitting all 2007 and
up JKs.
Today, we're gonna talk through the installation of this kit, which is going to be a fairly
simple one out of three wrenches.
Even though there are lights on this bumper and you have the winch, you're really only
making a couple of connections at your battery.
So again, one out of three wrenches for this install, but do give yourself around two hours
to get it done, maybe even a little bit more.
We'll talk more about the install in just a second.
We're also gonna talk through the construction and a few of the features of this kit.
This combination includes, of course, both your bumper and your winch.
You know that these two pieces are going to work together, you're gonna save a little
bit of money by buying them at the same time, and you're going to end up with a package
that is both very useful and will also drastically transform the look of the front of your Jeep.
Now, this kit is available with a couple of different bumpers, and this one, in particular,
has the LED lights built into it.
These lights are a little bit more of a marker light than they are an auxiliary light that's
actually gonna throw a lot of light in front of you on the road or on the trail.
But if you're looking for that, if you like that look of an integrated, built-in flush
mount light, and you want a winch that's gonna be very functional as well, all while saving
a little bit of money, buying them together in this combo pack is a really good option.
So obviously, there are two separate pieces here, and first we'll talk about the bumper.
This is the Barricade Trailforce HD Front Bumper with integrated LED lights.
This is going to be 5/32nds-inch steel covered in a textured black powder coat finish.
It does, of course, have the winch mount right up on top and your fair lead mount as well.
It has two D-ring mounts that are welded and bolt directly into your frame horns, giving
you a really solid connection for either a snatch strap recovery or using a snatch block
with your winch in order to double the pulling power of the winch.
And this does also come with the 3/4-inch D-rings as well, so it's not a piece that
you have to purchase separately, which is really nice, something you don't have to think
about.
This, of course, has a 2-inch hoop up on top of the bumper, which is going to offer a little
bit of protection to your winch from branches and brush.
And it also two light mount tabs up on the top, allowing you to mount any traditional
single post light.
Whether you're looking to mount some additional auxiliary lights or even a light bar up here,
you do have a couple of different options.
Now, this is a mid-width bumper from Barricade, so it is going to be slightly shorter than
that factory front bumper.
But even if you have a factory fender flare on your Jeep, I still think that this is going
to look pretty good.
Sometimes, when you go with a stubby bumper, you really want an aggressive fender flare
that jets in to give you a nice, clean look from the front.
With this, you can get away with running a bunch of different styles of fender flare
because it is a mid-width bumper, so you are going to get some protection and you're gonna
get those additional features that we just talked about.
Of course, this is the kit that comes with the winch as well, and this is Barricade's
9,500-Pound Winch with Synthetic Line.
Now, the general rule of thumb for the pulling capacity of a winch is that you want it to
be roughly double the loaded trail weight of your vehicle.
So if you have a really heavy, armored-up 4-door JK that you plan on burying up to the
axles in mud, maybe you wanna consider the 12,000-pound winch.
However, if you have a little bit of a lighter 4-door or certainly a light 2-door rig, 9,500
pounds is going to be more than enough for you.
Now, this is gonna be a 5.5 horsepower motor.
This does have a three-stage planetary gear set, so it is going to give you a good bit
of pulling power.
Now, in general, I really like a synthetic line.
It's going to be more expensive up front, you're going to have to replace it and maintain
it more often, and it is going to be more expensive every time you purchase a new one.
However, the benefit to a synthetic line is that it's lighter-weight, it's not going to
kink, it's easier to drag up those muddy hills, and it doesn't store energy like a steel cable
will.
This is going to stretch just like steel will, but when steel is under load and it breaks,
it snaps back.
It can hit somebody, causing an injury, or just some damage to your Jeep.
With the synthetic line, again, it doesn't store that energy, so if it were to break,
it falls directly to the ground fairly harmlessly.
You can mount your solenoid box right up on top here, or you could, of course, mount it
in back if you chose to.
Now, there are winches on the market that have some additional features, that if you're
gonna be doing some really long, hard pulls every single weekend, I might recommend spending
a little bit more on one of those other winches.
However, if you're going to be using this occasionally, this is going to be a really
nice winch that's gonna save you some money over those other options that are available.
This install is going to a one-out-of-three-wrench install that will probably take you right
around two hours, and the first step in getting this installed is going to be removing your
factory front bumper.
You'll do that by removing the splash shield on the bottom and the frame cover on the top,
removing the fog light sockets and the fog light wiring harness, and finally unbolting
that bumper and removing it from the Jeep.
Now, at this point, I would recommend installing the wiring harness for the lights on your
new bumper.
These are not going to plug directly into where your factory fog lights do, but this
does come with a really nice harness.
That includes the switch, the relay, the fuse and will plug directly into these lights,
so all you have to do is pick up power at the battery.
So you're gonna have to pull the switch wire through the firewall, get the switch mounted
inside the cab.
You're gonna run the two power wires, positive and negative, up to the battery where you'll
pick up power there.
And then you'll run the actual connection ends down to the front of the frame where
the bumper is going to be.
Once you get that into place, I would recommend bolting your winch up.
First, you're going to bolt the solenoid box on top of the winch if that's where you choose
to mount it, attach your wires from the box to the winch itself, so all you're going to
have are those two long leads that go to the battery, and finally bolt your winch onto
the bumper.
For that, you're gonna get your fair lead in place.
So once you have the winch bolted up to the bumper, you're gonna have a big, heavy package.
Why not install the bumper first and then the winch?
Well, it can be a little bit difficult to get to those winch mounting bolts if you do
it that way.
So again, I recommend the winch gets mounted to the bumper and the bumper and winch as
a package get mounted onto the Jeep.
Once you do have everything bolted up, you simply lift the bumper and winch onto the
frame rails and use the included hardware to get it bolted down tight.
Because you've already run all of the wiring for your lights, you simply plug those lights
in.
You'll want to stretch the two positive and negative wires from your winch through your
grille, up along the fender, and attach those to the battery as well as attaching the two
leads that you ran for your wiring harness to the battery as well.
And that's why I'm still giving this a one out of three wrenches.
Even though you do have the lights and the winch to wire up, it's really just four wires,
two that get attached to the positive terminal of your battery, two that get attached to
the negative terminal of your battery.
So even if you're not someone who's comfortable with automotive wiring, that's something that
really anybody can do.
As far as the tools necessary to get this installed, really just your traditional hand
tools.
No wiring tools or anything like that are necessary.
This is gonna be a pretty straightforward and a pretty easy install for anyone to do
in their driveway.
Overall, I think that this bumper and winch combination is fairly priced.
Of course, you're going to save some money over purchasing the winch and the bumper separately,
and if you're looking for the features that are offered in this kit, I do think this is
gonna be a really nice option for those of you who are going to be occasionally using
your winch.
This is gonna save you some money over those top-tier winches that are much more expensive
that has additional features, and of course, you're going to have the bumper for its features
and its protection.
Overall, these are gonna work really well together, they're gonna drastically change
the look of your Jeep, and I think that they're gonna be a pretty nice option for the price.
So if you're looking for a front bumper and winch combination for your Jeep to offer some
more function and drastically change the look of the front of your Jeep, I think this is
going to be a fairly-priced option.
So that's my review of the Barricade Trailforce HD Front Bumper with LED Lights and Winch
Combination, fitting all 2007 and up JKs, that you can find right here at extremeterrain.com.
-------------------------------------------
Hilarious Cruise Ship Pier Runners - ROASTED! - Duration: 4:37.
I love that he's like trying desperately to get this man up, but when he does, what is
his plan?
He's going...
I don't know...
Does he think he can disguise him?
I think it's "Weekend at Bernie'."
I think his plan is he's going to "Weekend at Bernie's" it, just kind of shove his arms
underneath and like "hey!"
Let me grab my phone... hold on, it's ringing... my phone's ringing.
Jimmy that's not a phone.
Mom!
Mom!
Jimmy, that's your shoe.
Mom!
Jimmy, that's your shoe.
Jimmy.
Jimmy.
Hang your shoe up.
Jimmy.
Now we've got some... they gave him a pillow to rest on, and just go to sleep.
If they could just let him sleep stand up...
Thank you, you're the best.
Your my buddy.
I love you Guinness shorts, hey Guiness shorts, you're my best friend.
Come on come on I gotta tell you something.
Can I tell you something? Hold on. Nah, forget it. Bye.
I feel like this is making the behavior acceptable.
Like hey, we're going to get you this tiny boat - we'll figure it out - we'll get you
this tiny oil rig... like, what is that?
If you had missed a boat, and that was the boat that came up to help you out, I'd be
like, no, I'm good.
No, this is the boat that Mark Wahlberg never came back on.
What was that movie?
The Perfect Storm?
Yes!
That is The Perfect Storm Boat!
Number 1, I was nuts enough to even get on that boat, then when they said to me what
we were about to do, like, not only are you going to get on this boat but hey, see that
guy that just opened this strange door on the cruise ship... this magical doorway appears...
we're going to figure out, we're going to throw you in that.
I'd be like, throw me in that?
And they're like, no, don't worry, we're going to give you this rickety strange ladder.
Oh gosh, she's flashing everyone. Wait, why would that be thing?
Also, we don't have... her husband must be like... uh, we don't have enough time to get
on this boat, we definitely don't have enough time for you to flash people.
She's probably one of those people that like she's really conservative when she's sober
but then you get a couple cocktails in Natalie and forget it, her bottoms come off.
That's just her instinct.
And her husband's so used to it, he's like pfff... that's my wife!
That's how she is! That's who I married!
They gave us extremely strict instructions and we said "I'm not listening! Phooey on you!"
They gave us instructions but they also let us get off on an island and 17 rum
cocktails.
Mama needs a minute.
I have a caftan, several caftans... that I need to purchase for discount prices at this
straw market.
Do you know how inexpensive?
First of all, my dollar goes twice as far, and they're knocking off, they're slashing
prices... left and right... in mid-conversation.
We're going to get to the edge of the dock and they're going to help from here.
I'm going to get to the edge of the dock and I'm going to let someone know who's standing
there "We're the group... they left... um, my dinner seating is at 5:30.
I don't wish to be late."
[WHISTLES & CAT CALLS FROM SHIP] "Here come the ***holes!"
"Stop!"
Stop!
Stop!
Stop!!
Stop!!!
Daniel, you're not going to yell curse words from our boat window, alright, Daniel?
This could go on the YouTubes and everyone's going to think suddenly that you're this guy?
-------------------------------------------
treatment for diabetes in urdu – hindi sugar ka ilaj - Duration: 3:37.Subscribe my channel for latest videos
-------------------------------------------
Kids First FY17 X01 Pre-Application Webinar - Duration: 47:33.hello everyone and welcome to the kids
first webinar for the x1 pre-application
discussion
my name is wretched boy and I'll be
doing introductory part of the webinar
um I'd like you to go over a few
instructions so that you understand how
we're going to operate the webinar on
the program is being hosted by the
Gabriella Miller kids first pediatric
research program and several of the
people involved in the program here at
NIH will be talking with you today right
now all the participants are music upon
entry for the webinar and we ask that
you please remain seated until the
question or discussion period at the end
of the presentation and that's how you
can unmute yourself by selecting start
six on your telephone or clicking on the
light symbol under the audio connection
on the webinar you can ask technical
questions at any time using the chat
service through the webinar to the host
if you are having technical difficulties
please feel free to use this means of
communication please save any questions
related to the program or funding
opportunity announcement for the
question period at the end and it's my
chance you have additional questions
after the webinar is over please feel
free to email them six his first Cody's
at iah not go and just a reminder we are
recording the webinar and the recording
will be posted to the kids first website
in case you want to review any aspect of
it or you have colleagues that were
unavailable this time to listen to live
so this is the pre-application webinar
for the discovery of the genetic basis
of childhood cancers and structural
birth defects the Gabriella Miller his
first TD after research programs
excellent one initiative and before we
get into the details of the funding
opportunity announcement but I want to
provide a little bit of background
information and introduce to you the
people that comprise the church
leadership group here at NIH we have a
number of programs involved isn't common
fund projects as you know it was
initiated in 2014 2015 with the overall
goal of establishing its first a
resource for the pediatric research
community we find we're very excited by
this opportunity both for Ali a chance
for the PDF research community programs
focusing on structural birth defects in
pediatric cancers and this is a comment
on program leadership responsibilities
are being shared across for NIH
Institute's Wallace college but the
people listed on this slide are the
folks that are involved in coordinating
the program as i mentioned i'm erection
boy i'm a program officer and I chg
Jonathan called mentions from NHLBI and
he and I are the points of contact for
special birthday sex questions of
themselves solve is an NHGRI and along
with James cool
nichd there the point of contact for
people in questions
James the project officer for the first
statement punters Nelson S&J goodra all
our program officers of the National
Cancer Institute and there are the point
of contact for ideas
PS answer questions
and Martin lurkey is the is involved
with our data resource project which
essentially funds this year will be the
process by which coordinating our
efforts from the common funds we have
very serious who is our program officer
and my instructor who is the operations
and budget person we also have a person
who handles policy planning evaluation
indication for us it was Josie honest we
recently lost them to another position
here at NIH more the process of getting
someone new and you all are probably
familiar with Valerie cotton who is our
administrative points1 coordinated
application and webinar today I just
want to take a moment to point out that
there's more than usual interest in this
program from both Congress and the other
50 communities partly because the way
the program change to be and today we
have stepped ishe of the Common Fund on
the line with us shoot the team leader
for the policy planning evaluation and
communication boxes and she's filling in
in the role of communications person
today during the webinar but you have
any questions on this aspect of the
program should be able to answer them
during our discussion period so the
people highlighted involved on this
slide myself and John Colin and Valerie
hot and will be the people talking with
you today during the course of the
webinar can XYZ I just want to go
through a bit of background as to how we
got where we are today so the program
was initiated and respond to the 2014
Gabriella Miller its first research act
which was signed into law and a third
2014 this law actually did three things
it ends attacks their contribution to
the presidential nominating conventions
and look at the small graphic at the
bottom of the slide visit the snap of
the other portion of your IRS 1040 and
the area highlighted in red was actually
a little checkbox that existed on
1040 back and your prior to 2014 and
2014 which allows taxpayers to
contribute tree dollars towards the
presidential nominating conventions fun
turned out a lot of money with cities
and the funds wasn't being best but the
times long past what it did was it
authorize the transfer of a hundred
twenty-six million dollars which were
sitting in the fun into a new pediatric
research initiative but it also
authorized congress to appropriate 12.6
million dollars a year for each of 10
years directly to the nih common fund
for a new pediatric research program and
the first appropriation was accomplished
in fiscal year 2015 think from our
perspective in bunny the program the
most important word on the slide is it
authorized the appropriation on an
annual basis so very important to
remember that this program there is a
mandate for 10 years of funding were
dependent upon congressional
appropriation annual basis to get the
funds for the program which artists back
to my earlier statement that there's a
lot of interest on the part of advocacy
group to see the captain's on an annual
basis
ok so if we look more closely at his
first working group as i mentioned as a
transit is effort supported by nih
common fund the Institute's that Sheriff
his first working group are the Child
Health Institute hard ones laws cancer
institute and the genome Institute it's
very important that you understand there
are a number of other is that you
working on this program and they're
listed here on the slide so just briefly
reiterated fidelis to choose the drug
Institute alcohol diabetes and kidney
Environmental Health Sciences the eye
and tissues are prized the
musculoskeletal diseases infectious
disease and the office research
instruction program
and we also have an ex-officio member
from the CDC's birth defects division
sitting on the working group so we've
got a lot of people involved in this
program and want you to be aware of this
because if you're interested in applying
to the exelon program it may be that you
your mission align more with one of
these other institutes and you should
feel free to contact program officers
you know what these Institute's that you
have questions about how relevant what
you want to do is to their mission for a
complete list of our working remembers
you can go to the kids first Common Fund
West fight and look at working group
members of the link there now I'm going
to turn the webinar over to John call
then who's going to review the program
goals
alright thank you so the overall goal of
his first program is to develop a data
resource which we are calling the
Gabriella Miller kids first p educated
resource where pediatric research
community can access well-curated
clinical genetic sequence data that will
allow them to identify genetic pathways
that underlies childhood cancer and
structural birth defects a greater
understanding of the genetic
underpinnings of these pediatric
condition is critical for developing and
improving preventive measures for
diagnostics is the character
intervention there are three main
edition is supported his first program
they include cohort identification DNA
sequencing the cancerous data resource
and data analyses
we'll talk about the first one goes late
today although i'll i'll briefly
mentioned what that the other two will
be doing swell cover identification the
DNA sequencing is accomplished during
the excellent program which will be the
topic of discussion to a rule is to
identify cohorts that children with
either childhood cancer or structural
birth defects more bone and their
families for whole-genome sequencing
concerns before the whole genome
sequence take of these cohorts to
elucidate the genetic contribution to
childhood cancers and a genetic etiology
of special birth defects the pictures
pediatric data resource will be awarded
an established later this year
the goal of this reasonable to develop a
web-based portal that will aid
researchers in identifying cohorts that
are available and have a sequence data
and that will also facilitate out cross
cohort analysis and it finally street
analyses conditions which is further
down the rows will be set up after the
resources of the naughty and will
further support analysis of kids
regenerated as well as knowledge it is
generated data to uncover new insight
into the biology of childhood cancer she
shows us now to talk about the extra
watches need a third cycle of our extra
1i it's currently called discovery of
the genetic basis to challenge cancers
and instructional 30 secs the first one
was released in 2015 and
jun 2015 x12 childhood cancer cohorts
five structural birth defects covert
sequence in 2016 three childhood
Cantrell chords and five structural
represent cool ones you want and then
the current exit one is par-70 dash 63
we hope you plan to continue sequencing
for several more years provided the
funds are appropriated as right
discussed and that cohorts are available
funding announcement has slightly
evolved over the three years of the
program in particular lesson emphasis
being placed on the minimum number of
samples also less than s is being placed
on trio design as long as there is a
valid analysis plan that accompanies the
overspeed proposed just a word about the
actual one back isn't the extra one is
not a typical award mechanism or typical
grant mechanism there is no award that's
given to the recipients and in fact
there is no notice of award recipients
are selected for the opportunity to have
their co-worker sequence again no funds
are provided more analysis but there is
an accompanied ro3 which is PR 16
dentistry 48 which originates from nichd
but has the number of partners that
supports analysis of data that is
generated by the excellent project also
these awards are not listed the is
reporter but abstracts from our prior
awardees are listed all the kids first
website the
web addresses on your street no
alternative about Valerie so i'm not
really tired and at laura is my a
program analyst 4-h program and a large
part of my job is to be able to speak
with the intent of fat as well as the
sln investigators will be selected
through PR 70 degrees i'm going to go
over some details that will hopefully
help you with your application to this
half fish and some of the information
about to cover is already addressed an
RFA Hughes if you are considering
applying for this at all i highly
encourage you to look at her if you
think you can find on our Facebook first
combines website at this link here we
can offer constantly updating faq will
update them after today of the questions
that we get the discussion that we have
and we see them on an ongoing basis as
we get everything for you see us
becoming centers that are providing the
UC printing services so that i described
in the so one away
they were selected for cooperative
agreements and we will be working with
such as for three years starting in
fiscal year 16 those cohorts have
already been selected and the samples
are being shipped and they're answering
the secret life around now I'm gonna be
working with them
physical you're 18 the first answer is
because been outside Institute provide
technology but I thought we'd be in
partnership with speak to children's
research hospital but by my exact the
other sensor is the grossest issue of
harvard and MIT like myspace Behavior
all photos of the sensors have the
capacity to sequence both cancer process
and birth defects projects
however our plan is to assign all kinds
of projects to the sequencing center
husband else's shoes to take advantage
of the analytical expertise for
identifying somatic variants that was
offered by the center and but depending
on the application as we can
exposes better proposed by with a
coworker left behind a process may not
work out exactly this way but this is
our general framework to see screen
centers are tests with 40 the sequencing
as well the variant calling I've already
ate things of DNA and RNA so that means
that will generate the variant calling
vials as well as bands and in some cases
that cute smile because cohorts will
receive holiness sequence paying an
average of thirty x coverage of cancer
called board will also receive 3x
coverage for whole-genome sequencing for
journalists sample but one change in
this my way that we are really not
introducing the submission of nucleic
acids from tumor samples I to go to the
husband elbows Thank You Spencer was it
was generate hold my own secret thing as
well as all that something RNA
sequencing because this is an
information thanks for their analytical
pipeline you may propose other methods
or technological approaches pretty one
thing in your applications for example
this past I stole someone's for proposed
higher coverage than 30s in the end
after working with us and our sequencing
centers they determined that 30s was the
most reasonable they would have free
sample size they wanted higher coverage
and they decided that the higher
simplifying the more valuable so on this
is an example of how you can propose
other things but the final product
design will be decided among discussions
with excellent investigators sequencing
center shaft and a nice program staff
another possibility that you might be
interested in proposing is a lynx long
new sequencing this is actually a
technological approach that was a pro
proposed by mostly sequencing centers
but since this is a new technology we've
actually asked them to work together to
run a small pilot study and this is so
that we can evaluate and better
understand the added value of utility of
this approach for these types of comfort
for birth defects and pediatric cancer
those they're doing this using 10x
platform so based on the results of a
pilot studies this is something that may
become more available for future
opportunities including this one so this
interests you at all
I encourage you to describe this in your
application and you invited application
for why you think it might be useful
your code for I just want to take a
moment and talk about where data lives
and how it's accessible so all this
hurts that you will have to be
registered in DB gap if you're not
familiar with unique a pin and I a
system for facilitating control access
of genetic data to other researchers so
you would be the time to research
research area generating the data but a
goal is to make this accessible to other
researchers as well and the end pts was
sort of a keeper out that process so I
projects well beyond the certain TV gap
sumx and i will also be submitted a gap
of sequence Dana will be submitted to
ncbi sequence read archive actually the
sequencing center will be responsible
for them but i just wanted to make sure
you guys need to wear daily living
intertitles of cancer process they also
have to be resurrecting PDF but all data
my assessment make that and sequence
data will be submitted to NCIS genomic
data Commons this is a database that was
launched within the last year it's still
a little bit new show NCIS that working
with us and our investigators and see
sequencing centers to make sure that we
get the necessary information from each
project properly submitted so now i'm
going to address a few of our
expectations are requirements in terms
of what his first program goals are so
from the beginning we've been very
focused on whole genome sequencing to
discover Connecticut
is contributing to these pts condition
but we had the opportunity for full deck
so it's just transfer zone season of
humor as well based on the proposal and
the value of this argued by uzma thank
you
so given these are just because they
develop them be performed our efforts
expectation is that the DNA and in the
case of tumors are named as well
exceptions that you plan to submit are
sufficient quality and concentration of
these technologies will talk a little
bit later about how we ask you that
information is the application another
goal of the program is justin silicate
cross-court analyses base and these be
done based on common development one
very large task of our upcoming his
first date of resource will be to
harmonize phenotype across all of our
process so to this end we really need
some basic element but the better the
more details happy that you have the
better
um for birth defects projects we don't
have established set of standards for
phenotypes data but since the candidate
will be going into NCIC economic data
comments they have already clearly
established the elements that they
expect for my assessment and clinical
and you know Vegas so you can find this
swing here and also in our FAQ to look
at that you use data dictionary another
goal of ours is programmed out and so we
meet to run the diversity of both PSR
cancer and special birth defects data
says that will initially be part of our
kids first data resource this means that
if we get a lot of applications of me
hopes that we knew and we are faced with
the choice of cute very similar
applications but one of the proposals of
sequencing of the condition that we
haven't covered before we are more
likely to select that cord
this doesn't mean we can serve you broke
lying if you have a cohort of similar to
what we've got the board that we want to
be a parent
office and we in previous life if you
link to where you can find the list of
quickly statements before and we
encourage you to look at that huge goal
ours is data sharing so to make the
animated by our program as acceptable to
be this community as possible so in line
with NIH's no mysterious errand policy
cohort participants must have your
physics consent to allow the sharing of
individual level sequence and the
associated seen effective at through
high approved repository the classic
example EE gap another priority of ours
is to make sure this data to go to as
many as are constantly service possible
so we are most interested in doing that
is impressive to share as broadly as
possible though something that submitted
for general issues something that is
constructive for health medical or
biological research is preferred over
busy specific data use restrictions and
it's just because we want to promote
collaboration long investigators as much
as possible and then just because a
little bit more complicated with data
sets are restricted success researchers
again it doesn't mean we discourage you
from applying that you have is the
specific restrictions of but we do ask
that you previously that your
application and some of you like us is
recruiting you court or even be
confessing for every anyway as me and I
have guidelines two guys using who have
made with that allows for grass sharing
sample size and family structure as John
mentioned in previous years we really
focus on his trio base designs of what
we realized that there are other family
structures and designs out there that
can lead to discovery but this needs to
be very clearly communicated in your
application of obviously the larger the
sample size the better
um in some cases we have birth defects
of in Hawaii we talk about the birth
defects minimum 100 trios
but if you less than that I'm we would
like to see you so I clear scientific
justification for how you will be able
to identify the Americans if your sample
size not that large very strongly
encourage you to consider collaborating
with other investigators cool samples
together for larger sample size and of
course in the case that you don't have
trios use this week you on show us an
argument for how us that you have enough
program and toss that you will see Chris
enough effective and/or unaffected
family members to be in any discovery
and this type and very closely with
analysis Lance the goal asking for
analysis plans in these applications if
you have investigators seven days that
the proposed project has an adequate
research design for genetic discovery
and that usps one investigators are in
addition to responding analyses so one
of modification of this iteration of the
F away is that we do communicate that we
recognize the analytical power of each
dataset will increase once incorporated
with all the other datasets that are
part of his first no class it is
important that you demonstrate the data
will be usable on its own
if you feel that your team does not have
the analytical and little call our
statistical expertise to perform these
now is we highly encourage you to
partner with another group who may be
able to work with you on an analysis
plan so now i'm going to move on to
adjust a part of b funding announcement
called other attachments entire years
we've always discussed the institutional
education of indiscretion but from this
year we greatly expanded this part of
the application of to ask for sample
information description of the penis
penis
any description of the family structures
and even after this here that we want to
enable you to submit supplemental
additional material all the explaining
and response to make me a sport here
without really cutting into your limited
user strategy so we have also created a
optional downloadable form that is
available on earth a fuse and that is a
template where you can give us the
information that was asking or this is
awesome
you may choose to respond this
information that we need and in another
format whatever way is best for you by
the Board says as a place where both
applications and reviewers can look at
work with the uniform document
information can be organized and I'm
going in the next slide right over
easily use one by one so the first
attachment would be the official
certification or criminal certifications
or sample consent form institutional
certifications are documents that sure
is that these innovative new BFF pieces
and i use genomic data sharing policy of
many team cannot usually get a full
official official certification from
their ir e until after the board or the
state after you left yet so one program
so you need to remind the original
certification or the simple consent form
instead we use after the presentation
please use the current and I Swiss can
be found at this link that's where you
can find both institutional education
and the cell certain cases that's what
no matter what you submit it needs to be
clear need to cover all the sites that
contribute simple to your project nice
to specify whether being a competitive
presented the energy gap or other and i
use repository and organizations need to
be very clear bass playing
this snapshot below is a msg from the
institutional certification but you can
see how automatically you will have to
organize and label that spreads arms but
if you're not submitting a full official
certification with your application you
need to describe them somewhere else the
application and i just want to mention
that no matter what you submit with your
application if your cousin is selected
you will have to provide the school and
Cisco certification using this template
shortly after we notify you know your
project has been selected and this is
the first step of that he got
registrations I talked about we're not
going to get into the details here just
know that i SAT here to help you with
this but this would be the first step on
the other questions we asked for our
sample information so we really need to
understand the total number of samples
that you are proposing for sequencing
very clearly again you can modify this
table you can submit this the
information in the s4 whatever way works
for you but this is one way you can
organize these details though this table
of the rows are organized by sample type
or or DNA or RNA source this hot part of
this table would be for both birth
defects cohorts and cancer cohorts and
the bottom part is for tumor sample for
testing project we just need you to
describe the number of samples from heat
exhaustion message that was used the
concentration some never got quality and
tell us which metra could use a method
of quantification and then we need to
know how how many samples will be ready
to ship by August 2017 and if you slide
I'll talk more about the timeline of
this program but we anticipate that we
will be notifying crew being selected
under this so17 team iterations in the
life so we are hoping that we will be
getting bigger than our samples of
pipeline by august but we realized that
types there are some lingering
extractions pencil penis off and
sometimes there are some shipping little
script coordinate so we asked for how
many samples are ready now and have
examples already six months from now
just to get a sense of what is ready to
go again you can modify this slightly if
you know that you'll have everything
ready by November of 2017 that you can
change the January 218 number 170 and
tell us when all your will be submitted
by we also ask you to describe what you
no text information and demographic
information you collected from these
participants have minimum we expect a
good diagnosis maybe maybe you have some
other Asian formation sex race/ethnicity
and then just tell us what else you
collected describe you
primary uh clinical description and I
purchase a kind heart tumor spice up and
tell us what other phenotype information
whether it's clinical or otherwise that
you've collected over also interested in
knowing what family medical history you
have this is a very general table again
we just need to who you give us a sense
of what being a kata you have available
again for cancer code words this is a
little bit more clearly laid out of the
standards provided funds you see we
highly encourage you to look at those
and again for those cohorts that are not
feel designs we ask that you describe
what the family structure bar you might
have a program parents siblings of that
you might have multiple family might
have something with his family's we
don't ask me to submit enough
information to make it clear out any
samples for family you're planning on
sequence and whether they're a bit and
then he doesn't like to see simplest to
submit a pedigree information but again
tell us who you're proposing for
sequencing if you don't have a degree
information we ask that you find another
way
makes you really make it clear what
you're proposing is a handful of people
that makes us to work from with one
group they have a trios although they
also have situations where a program and
another person three family member was
infected with some defective family
members so because needed to be clear
how many of these family-type if you
have many different family types of and
how many samples from unaffected and
family number of your proposal when a
couple other things that didn't quite
fit into other slice
we just want to emphasize that another
change to lose this iteration of this
program is that we are cruising the
submission of application up guess who
has already sequence a case or programs
through other funding but have fun
sequencing play NASA's problem of
parents four parents and siblings for
tumor if you do this you ask that you
pretty quickly just ride the sequencing
technology that uses into the program
and I was but we are certainly open
because Google we also an SS opportunity
is open soon is real research program
I'm and that includes collaborators of
that is real research program is because
it doesn't come with formal fund is that
opportunity for pain so for the cycles
in 2017 cycle of you made for me how
about a lot of things that we prefer a
fact but really no matter what we heard
any investigators that have a co-worker
that meets the minimum requirements to
consider flying as you so we're working
with our current because others until
2018 on but we also have plans to
continue sequence being in later years
so we encourage you to start collecting
samples of your requirements now but
stay and just with us about this again
what will continue continue to
sequencing as long as funds are
appropriated and so forth are now also
be you
stay aware of what cohorts are out there
this is the timeline for this cycle it
is on an earlier time lines in our
previous two years but we wanted to make
sure but he was clear on this
this funny as that was published in
December today's category 5 30 this is
the weapon are the letters of intent is
to separate them as you know these are
not required but they do help us prepare
for their review of their helpful
mark 7 is the application deadline we
anticipate that some review will take
place sometime in April but we're not
going to be between unions alive we will
work with our working group to make a
selection there are no funds associated
with this program it doesn't go to an
institution helpful to work with our
working group and then we get a
recommendation approved by the Institute
directors at Coachella or a group to
make our dividend and then we plan to
know either as of yet sighs line and we
hope to get these comforts multiplying
by august so now I'm logins the floor up
for questions
I just want to reiterate that you can
continue to reach out to us this is the
analogous memberships for which we are
monitoring you also have the hot
information for program officer Barry
who are leaders of this program in the
study and ask myself my email addresses
on this life so we'll be publishing the
TV
PS of this side effects of all
publishing the webinar recording of the
webinar and just a reminder will take
the questions now we like you have some
vocally and so you'll have to unmute
yourself if you can call them through
telephone you can select start six on
your phone to unmute yourself and if you
call them through your computer you'll
have to go to the audio connection and
select the likes of all who won't need
yourself
sounds like something yummy to
themselves and her friends
so we're ready to receive your questions
hey thanks for the presentation my name
is Craig 1 i'm calling from the
university of new mexico i had a
question regarding existing coverage
have been already uploaded to DB gap but
with a net level genotyping data what a
doesn't make it simpler to have that
already done ahead of time if we want to
get a whole genome sequencing for this
data set go ahead
not sure I fully understand the
questions you don't get a full so I've
already done uh sniff genotyping and had
already uploaded cohort data to DB gap
however we would like to get whole exome
or whole genome sequencing for this
existing cohort on that and this is data
or this is DNA out of the DNA repository
so regarding the certificates and all
the paperwork and registering for the
you know DB gap and and and the kind of
administrative requests that were
presented earlier in the presentation at
zacks exact stuff gets grandfathered in
or do you have to go ahead and you know
we do a lot of the paperwork you
probably have to work with the folks who
helped you
registers as the original trial but
probably you can piggyback this data
onto that trial we've done that it
other other scenarios that are somewhat
similar where a cohort has already
undergone friend is X equal to and then
underway whole genome was a part of this
so so that data was sort of attached to
the original code word data and
obviously that will likely simplify the
registration process that you've already
done terminated and downloaded to take
it as well thank you very much
other questions
tight
Alex writing from Pittsburgh and the
question regarding the phenotypes does
it have to be one particular phenotype
or 10 and actually the collection
includes several phenotypes
right so yet the the short answer is yes
it can
are you talking about birth defects of
war camp every four right back on it it
certainly can be a part of the idea here
was that from a genetic standpoint you
take to find a fair bit of overlap
between genetics of different types of
birth defects obviously i think an
application would be stronger from
analysis standpoint if you could make an
argument about how these different
phenotypes might be analyzed together
but but certainly that possible
and what about prenatal sample and that
would be DNA obtained from what source
ubi it will be a million pieces and when
you have ultrasound data to support the
fact that the fetus has a deep at birth
because yeah the point is that it would
be the phenotyping would be down like
prenatal ultrasound that he would be a
very unique phenotyping data sets
it's not precluded from being such as we
haven't had anything I guess we had
really consider the possibility of
something prenatal oh yeah I they're
happy scientific justification for why
we would devote resources to a prenatal
cohort as opposed to pediatric cohort it
i can imagine reviewers might wondering
about the sort of validity / accuracy of
prenatal diagnosis of which obviously
could potentially be bolstered by
postnatal confirmation of the diagnosis
but you know I think you have to make a
strong argument for why be done this way
as opposed to our exit waiting
thank you
Daniella Silva Washington I was just
wondering you know you're really quiet
my I wonder if you're not coming here my
friend boner you find another way to
make your foot ladder her outright that
better
ok so i was i I was wondering if there's
an emo concentration for DNA the new
position yet you go through a huge we
have some information about what are you
can be under the best if you want more
details feel free to contact me Valerie
or distress that loss and we talked
about that
so there is some information on our
issues thank you and my other question
was about the results so when do you
expect we would get the results back so
and and are they going to be VCF files
or are you going to send us the damn
styles
yeah so like a few people generate BCS
and band and those will be somehow
transferred back to the investigator and
the investigator will have six months to
work with their own data before it gets
released to the public through DB gap so
yes we have ways to get the vaccine
investigators a timeline for data
generation
well depend on a number of factors i
have 15 days I mind but for example of
colors that are going into the pipeline
now are expected to finish the pipeline
between four and seven minutes four and
seven months
ok and so it is expected that we have
capacities to receive vampires we do I
just want to make sure if i have to
state that on the application that
emergency or not we weren't sure you can
handle a couple words in there so have
you spoken sure so we want to know if we
have to put in the application that we
have space to receive them files for
example here we weren't sure if we're
going to receive those fires are going
to access them promote yes you you will
need to talk about your capacity of your
institution to store those vampire
okay great thanks
yeah so that's a good point of one thing
as well as but once you are electric
service program and assigned to a
requesting better
there's a lot of room to collaborate
with the sequencing center on analyses
again your application you're going to
have to tell us what what your resources
you have but just know that I'm you know
this is a partnership and collaboration
and they're really great resources and
state-of-the-art sequencing centers that
were working with you know from
everything to analysis is mostly best
but so I'm you know we're really excited
about working with us and help that
makes you and and the road so you can
keep that in mind but don't forget to
have analysis plan
well it doesn't not like we have any
other questions
I'm so I think we're going to go ahead
and wrap this up but please continue to
ask questions of us three rules all of
our lives and resources and email
addresses again this webinar will be
posted on our website and as the slides
will be posted for the links and
information as well so i'm going to be
myself now and the housetop the webinar
thank you thank you
-------------------------------------------
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NCBI Minute: New Version of E-utilities Supports Accession.version Identifiers - Duration: 28:00.All right everyone.
Welcome to the webinar today.
We will be talking about a new functionality within the E-utilities API that supports accession.version
identifiers.
We have materials for this webinar, the PowerPoint and a few bells and whistles that will be
available for you at the FTP site and you can get to that through the coursesandwebinars
link on the screen here and there is a direct link also, the go.usa.gov URL.
With all of that said, I will start the webinar.
What we're talking about today is actually the fourth webinar in a series of webinars
about changes that have been going on with the way that NCBI deals with sequence identifiers.
Today we are happy to announce that the E-utilities, our main API for the Entrez system, is now
able to fully support accession.version identifiers, both as input and output.
What I want to do is give you a quick overview of how that works and point you to some examples.
As I said we have been doing webinars about this since the past summer starting back in
July.
there are three webinars we put out there about various topics relevant to the changes
in identifiers for sequence records here at NCBI.
Feel free to go back and take a look at those if you haven't had a chance to view them.
Very short and sweet, the change that is happening is that gradually NCBI is phasing out an identifier
called G.I. number.
These are integer identifiers that have been used for many years for sequences but for
a number of reasons we are phasing those out and transitioning to a situation where we
will be using accession.version identifiers as the primary identifier for sequence records.
Since so many of our tools, including the E-utilities, have been dependent upon GI numbers
in the past, it was critical for us to be able to offer these APIs in a form that would
not be dependent upon GIs but would be dependent upon a different identifier set, accession.version.
The main thing that we introduced as a parameter called idtype that will control this.
It works a little bit differently in the various e-utilities.
I will go through esummary, efetch, esearch, elink . And then, the final prize package
at the end is how you get sequences that don't have GIs and what is that about and how you
can think about that problem going forward.
Let me review quickly again a little bit more detail about what's changing so everyone is
on the same page.
The GI numbers are being phased out.
What this means is they are not being deleted.
It's important for people to understand we are not deleting GI numbers but we are not
going to assign GI numbers to a growing number of newly deposited sequences.
If you have a sequence that has a G.I. number it will continue to have that G.I. number
and will always be retrievable by that number for the foreseeable future.
What's happening now is, as more and more new studies come in, those will not be assigned
GI numbers so if you stick with just G.I. space you won't have access to more and more
of the new data coming in.
A few things that have already been happening are that the GI numbers are no longer visible
on the flat file formats on the web or in FASTA definition lines, including in the BLAST
databases.
So you won't see the GI numbers in those presentations and again just to be clear those numbers are
still in the raw data files, for example, the ASN.1 or the XML.
They will not be shown in the more public web friendly presentations.
After March 15 of this year none of the releases of GenBank or RefSeq will contain GI numbers
in their presentations in the flat file or in the FASTA that you get off the ftp site.
That's another thing to be aware of.
That's what's happening.
Let's talk about E-utilities and what effect it has on them.
For those of you not as familiar I wanted to go over what the utilities are and what
they do and their function and how they depend upon the unique identifier.
The E-utilities work in all of the Entrez databases, from PubMed to the sequence databases,
medical databases, whatever, the e-utilities function across a wide variety of identifier
spaces.
What they do is a few functions.
ESearch can take a text query and return back to a set of these unique identifiers, that
correspond to the result set.
Those unique identifiers, uids, can be passed to additional E-utilities to retrieve those
records.
For example, I can pass a UID to ESummary to get the record summary, or docsum, which
is a short abbreviated metadata about a record that you commonly see as search results on
the web.
Alternatively I can take a UID and pass it to EFetch which would download the full data
record in a variety of formats.
PubMed you can get abstract, citation, Medline, etc., ASN.1, XML.
For sequences you can get FASTA, flat file, XML, ASN.1, etc.
Efetch is a great utility for getting the data down once you have the identifiers in
hand.
ELink is another powerful utility that lets you take identifiers from a given database
and find linked records in another database.
For example I want to find the PubMed literature associated with a sequence.
I can link from the sequence records to PubMed.
I can link from nucleotide to protein, etc., etc.
Elink also talks in this UID space.
One set of UID's in and another out.
Finally, epost is essentially an upload utility that takes UIDs and puts them onto a temporary
storage facility that we call the Entrez history.
And this is a server that stores temporarily your search results.
Then I can take the results from that history and pass those to fetch or to summary or to
link for further processing.
I think it's pretty clear as you look at the diagram that all of these utilities either
output UIDs or accept UIDs as inputs.
It's critical to the function of this API that the UID space be well determined.
For sequence records, talking about nucleotide, protein, EST and GSS databases, and Popset,
the UID has always been the G.I. number, the integer identifier.
Not anymore.
You can continue doing that but now all of these utilities will do the same thing for
accession.version.
It's a very powerful upgrade in the functionality of these APIs, it extends their scope significantly
and we'll talk more about that towards the end.
The set of UIDs is now a set of accession.version identifiers.
You have the option, it's important to understand that you can continue to use GI numbers but
you have the option to use accession.version.
I listed there the primary databases that this affects.
Let's step through them and talk about what would happen.
Luckily ESummary and EFetch get an upgrade that is silent.
You use the ID parameter as input for the UID's.
So in the first URL showing there, ESummary, and the database is nuccore, there are two
UIDs there.
That's a traditional ESummary call.
For EFetch it would be the same kind of call, same UIDs, but I might put rettype equals
fasta to specify I want the FASTA format.
What you can do now is instead of using those integer identifiers, the gi's, you can put
accession.veresion.
If you put in accession without a version you get the most recent version of that accession.
That just works.
You don't have to do anything else to your call.
You don't have to add an ID type parameter or anything else.
It just works.
This is great and you can just start using this
For those of you who've been using these APIs, you may realize that EFetch has been able
to do this for many years.
It has been able to accept accession.version for several years along with GIs, it continues
to do that.
In this regard EFetch has not changed.
ESummary has changed.
It has never been able to accept accessions as input but it can now.
That's what's happening with ESummary and EFetch.
Pretty simple.
You don't have to do anything, you can just use accessions now.
ESearch is more interesting because what ESearch can do now is output data as accession.version.
What ESearch does is takes a text query, like foxp2[title] AND human[organism].
I want the human sequences for foxp2.
Before, I would assemble this call.
What that would give me is a list of GI numbers back.
What it does now is you have the option, if you add the ID type parameter, and give it
the value acc, so &idtype=acc is appended to that same search call, you will get something
different.
This is what you get different.
The default without using ID type is a list of the top 20 GI numbers.
Unique identifiers that match your query.
Those should be nucleotide GIs.
If you use ID type equals acc, you get back the accession.versions.
This is cool.
You get the accession.version as if the GIs don't exist.
You get them back directly and can use them for further processing.
Elink is going to behave in much the same way.
It takes sets of identifiers as input and gives you sets of identifiers as output, in
the same or a different database.
A very simple example is here's a nucleotide G.I. number and I want to find the protein
that it encodes so I say dbfrom=nuccore, db=proetin, and there is my ID.
I would expect to get back the protein G.I. that is associated with this.
If I use ID type equals acc, append that to the URL, now it allows me to do two things.
I can have an accession number as input.
So I don't have to use a G.I. number and I will get back accessions in the output.
Here's a comparison of the XML that I would get back from those two calls.
If the ID type is not set, the default is essentially GI numbers.
Here's my nuccore dbfrom, here is the id in nuccore, the link is to protein, here is the
ID number for protein, 34577063.
That's the G.I. number of the protein linked to the incoming nuccore record.
But if I use ID type equals acc.
What you see on the right side, is that the NM_ record, 001126.3, is now linked to NP_001117.2.
so I get the accessions back immediately.
ID type equals acc is required for ELink and ESearch if you want to get the accessions
in the output.
Before we do a little bit of playing with this I want to talk about some expanded scope
that the e-utilities now have access to because of this change.
It's important to understand this so you don't run afoul of the way the data are restructured
now and be aware that these changes will continue happen as things progress.
we have sequences that don't have GIs.
That's an issue for a number of reasons because a sequence without a G.I. is not part of the
Entrez system.
It's not part of Entrez Nucleotide, nuccore, or Entrez Protein or any other Entrez database.
That has several consequences.
ESearch cannot find them.
They are not indexed in Entrez, so esearch has no access to them.
They do not have document summaries, they are not in Entrez so ESummary cannot retrieve
them.
They have no Entrez links so ELink cannot perform links between gi-less objects.
Also they cannot be placed on the Entrez history.
For example, EPost cannot handle or process them.
For those of you who are e-utility aficionados, if you use ESearch with usehistory=y, it won't
work for a gi-less sequence because they can't be put on the history, they are not part of
Entrez.
But EFetch can now download them.
EFetch has become an odd man out in that it now has a special function, that it has access
to the whole world of GI-less sequences through their accession.version.
All NCBI sequences, whether they have a gi number or not, have a accession.version identifier
and EFetch now can access all of them.
This is brand-new functionality that we have never had before, that API access to all of
these GI-less sequences that still have a accession.version.
So what are we talking about?
A lot of WGS and TSA.
So whole genome shotgun datasets and transcriptome shotgun assembly datasets.
Many of these data sets do not have GIs for their contigs.
They may have a G.I. number for their master record but they will not in many cases have
GI numbers for all the congtigs.
Ultimately most new sequences, eventually almost all, will be like this.
They will not have GI numbers but they will have accession.version.
It's important to understand that this is the future and where things are moving so
that accession.version becomes the main identifier that you can depend on.
A cardinal example currently is the Norway Spruce project, CBVK.
This is a very large project with over 1 million contigs.
Because of the size it was not assigned GI numbers.
It has one G.I. number that represents the master record.
So CBVK and all those zeroes, that single representative record, which has no data in
it except metadata, that one record is indexed in Entrez.
If you search for Norway Spruce in Nucleotide you would find that record but all of the
million contigs are not available to you from entrée and have not been able to be available
to from the API e-utilities.
They are now.
One question that folks might have is how do I check if a sequence has a G.I. number
or not?
The easy way to do this programmatically is to use ESummary because it will immediately
return to you in error tag telling you that this accession does not have an identifier,
a gi identifier.
And you can do that with a mixed bag of input and esummary will very politely tell you which
ones work and which ones don't.
At this point that's our recommendation as to the best way to check.
Esummary is fast and it can process thousands of records in a given URL, so it's a good
way to parse through a big list if you want to find out what has a G.I. number or not.
There are other approaches if you have massive projects and you are concerned about using
ESummary, write to us, talk to us, we can give you some additional help.
What I'm going to do now before I go to this slide is do some demo.
We will go through a few calls so you can see how they work.
Also this little Perl script, an incredibly simple little script, I've put on the ftp
site in case any of you want to play with it.
I'm just going to explore the calls a little bit.
I'm going to actually show you what the Perl script looks like.
That's it.
All it does is post a URL and I have an array here of eight sample URLs.
You can explore this and edit as you want and play with this functionality.
The first URL is an ESummary and that will get back two sequence records specified by
an accession.
This is something we haven't been able to do in the past, so to run this little script,
I just say perl geturl.pl with index one.
I review that and I get some data.
What you see here are two docsum records.
The first one here.
And the nice thing is it works.
In the past this would have given an error.
I get my accession back and get my record back.
For those of you that are parsing docsums you might want to be aware we have a new tag
within the docsums called Accession.version.
That thing is brand new and it has the accession.version in it.
That is something you can parse out directly and that's where the identifier is now.
Of course the GI numbers going to be there also.
And again, if it doesn't have a G.I. number, ESummary can't find it.
That's how esummary's going to work.
Now esearch.
Two ESearch calls, one will look for foxp2[title] and the other one has human[organism] without
$idtype=acc.
The other one as $idtype=acc appended.
So let's do the first one.
As I was showing in the slides, this is what I get back; I get 48 things back.
Here is the top 20; I get gi numbers.
All I have to do is add that ID type equals acc in that URL and i get back the accessions.
It is quick and simple.
All right, going on, ELink.
We can do elink just as we did esearch, linking from a nuccore record to a protein record.
Here is the id.
It's also NM_001126, so we will do it both ways.
Just know you have to include idtype=acc at the end, if I'm going to do the accession.
So my first elink call, again, has the G.I. number as input and gives me the gi number
for the output for the protein.
If I do idtype=acc, here is the NM_ as the input, the NP_ is the output.
You can do this if you are linking across from non-sequence to sequence databases.
So if I'm going from pubmed to nuccore or pubmed into protein, you will use idtype=acc.
And, of course, PubMed will not have an accesion number.
That's fine.
You'll use pmid's as your input to ELink but the output from ELink will be accession numbers
in protein space.
That will all work just fine.
All right.
Let's take a look at the last ones here.
Here is EFetch.
Here is some of that Norway Spruce data.
This is WGS data that we've not been able to give you before with EFetch.
That URL, here is the ID, that long accession, the accession for the contig.
And I'm going to get FASTA back.
So I do that.
Boom, there is the FASTA for that wgs contig and if I scroll up to the top, there it is,
Picea abies.
There is the data, it has a contig name.
All of this little FASTA definition line is something we are providing.
It doesn't exist in Entrez, but we are providing one to you.
And so here it is, you can get those data.
It's very likely if you want the entire project, ftp might be a better method.
But, this is a way you can get contig by contig if you want to get a set of contigs down from
one of these sets.
Last but not least, ESummary as a check.
How do I check to see whether my sequence has a G.I. number?
I'm going to try ESummary with the CBVK.
The same accession of just retrieved with efetch.
Let me see what happens.
But I'm going to try it in a mixed list of other things; an NM_ record and a GenBank
record.
How would it look if I had a mixed list and a bomb in their that doesn't have a G.I. number,
what is esummary going to do?
ESummary in the case where I just give it that Norway Spruce wgs contig, it very politely
tells me, ERROR Invalid uid at this position.
I know that is the contig that had no UID.
If I give it the three identifiers, two of which have GI numbers and one doesn't.
This is the data I get back.
At the top you see, ERROR Invalid uid at this position.
And then I get the two valid docsums back as expected.
So this should be a fairly rapid way to quickly check a list of data to say what has gi's
and what doesn't.
This little Perl script, this almost brainlessly simple Perl script.
It is on the FTP site and you can play with it if you want.
If you have questions about any of that, we will be taking them in a couple moments or
if you have questions later, write to us.
That will be my last statement before I be quiet and we start discussing.
Continue to follow our social media posts, blog, news, Facebook and Twitter accounts
for more updates and announcements about things that will be happening with these sequence
resources, the changes with sequence identifier space going forward.
The NCBI Learn page is a great place to come for events like this if you want to have more
awareness of the webinars and other courses that we are offering.
If you want to find out about the e-utility updates, it's a great idea if you are using
them, please subscribe to utilities-announce@ncbi.
That's where we send out all of the announcements about these upcoming changes, how you can
prepare for them.
Where we announce in advance that changes changes are happening.
So it's a great place to look for those kinds of things and keep yourself aware and not
be surprised.
Finally, info@ncbi is our general helpdesk.
Please don't hesitate to write to us if you have questions or concerns.
All right I'm going to be quiet and open the floor to questions.
This is Peter Cooper.
We do have a couple questions.
Eric.
Several of them I answered and you sort of answered as you went through, so I won't go
over those again.
Those are the ones I answered in the questions pod.
Interestingly people asked those questions then immediately you said the answers to them.
Here's a few others.
The first one is about downloading WGS sequences from the FTP site.
Given a top-level WGS project ID, so that's essentially that prefix, what's the best way
to programmatically get the ftp location for the entire assembly?
One way you can do that is to use the BioProject database.
That has a good place to search for these data sets.
That has metadata in it that will allow you to find the project that is the source of
the WGS data.
You can link from BioProject into nuccore and that will give you the master record,
if it's not in the BioProject data itself.
That's one avenue.
You then would be able to construct an FTP URL down to the FTP site to get that data
from Genbank.
Another way you could approach that task of getting the appropriate accession is through
nuccore itself.
At this point my understanding is that all of our WGS projects -- or at least the vast
majority -- will be assigned a G.I. number for their master record.
And that master record would be indexed with some metadata.
So if you're trying to search organism or a title or something like that, you may be
able to find it that way.
Then you can construct an FTP URL into Genbank.
I hope that helps.
If you want more detail on that, let us know and we can provide you with some additional
suggestions.
I think the structure of the FTP site is a formal way of doing that based on those accessions.
What I will do is put that -- I'll have to look at it so I can describe it properly -- I
will put that in the Q&A document so everybody can take a look at that.
The other question that I see here is can esummary maintain the order of the input UID
even if there is a GI-less accession.version in there?
That's a good question and one that we're thinking about ourselves.
At the moment the order should be maintained for those records that do have GIs.
It appears what's happening is we are writing the errors at the top of the output screen.
So there's a disruption in the current order of the input.
That's a good suggestion.
We will take that up with the development staff and see if we can do something to address
that.
At the moment you're going to be stuck with finding the errors at the top and the rest
should be in the order in which you submitted them.
All right.
Thank you all very much for your time and for attending.
Good luck out there and let us know if you run into problems or have concerns or suggestions
about these new functions.
Thank you very much and have a great day.
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PHRASAL VERB SIGN - Duration: 6:09. For more infomation >> PHRASAL VERB SIGN - Duration: 6:09.-------------------------------------------
Margaret's Weather Picture for February 1, 2017 - Duration: 0:30.ARE CAUSING MAJOR
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-------------------------------------------
Legacy Challenge Kickoff – A campaign for UF College of Medicine scholarships - Duration: 2:21.DUKE: Hello!
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ROSENBERG: Awesome
(Cheering and Band Playing Music)
-------------------------------------------
Daily Tarot Reading for 2 February 2017 - Duration: 16:50.subtitles
-------------------------------------------
Team Bonding is Key for Slovenia's Women's Team | Patagonia Dreaming - Duration: 8:22.I really like the place here.
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(PATAGONIA DREAMING)
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with the Skidoos and walking,
and some lunch in the nature.
-------------------------------------------
BofAML provides a forum for tech leaders to discuss the latest trends - Duration: 1:05.This is the eighth time we've run this summit, and in the seven prior years what we've
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