Hello and welcome to today's NCBI minute on our updated ORFfinder, an open
reading frame prediction tool.
I'm Wayne Matten, with me today is Peter Cooper and you can see on this slide the
closed captioning information.
go ahead and put in your questions in the question pod as you think of them.
Peter may very well be able to answer those while I'm talking
but we'll put those in a document, with any that we get after I quit talking, into a
link "Materials" on the webinars and courses page. and i'll just go ahead and
show you that page.
so this is webinars and courses.
this is today's webinar but that will before very long move over to the
archive page, and there you see a link in our previous webinars to materials. so
those questions and answers will be there.
there are no slides from today. eventually there will be a video file in
materials and we'll also post a recording on our youtube channel.
I"ll probably get that up within the next several days.
if you have questions about ORF finder after we stopped the webinar feel free
to go ahead and email me at matten@ncbi.nlm.nih.gov or you can
send general
ncbi questions to that info email alias.
so this is intended to be a very short minute so let me go ahead and jump over
to the ORF finder page. you can find this in a web search if you search for ncbi
orffinder.
you may well come upon the old page but there's a link there to
this page. and the URL is simply the base
NCBI homepage URL, forward slash, orffinder,
all lowercase. so what's new in ORFfinder now? primarily it's the interface.
it looks more like many of our other resources, you'll see that particularly
on the results page, but we do now also have a standalone version. you can
download that binary
here at this linux x64 link. and the standalone version does not have the
limitation of processing 50 kb that the web version does. the other addition is
you can now send your translated orf directly to SmartBLAST, and we still
maintain the ability to send it to the full blastp service as well.
just a quick caveat before I demonstrate. ORFfinder is not intended to be, you know,
a gene prediction tool, particularly for eukaryotes. it works in many cases fine
as an ORFfinder in bacterial and archaeal genomes, viral genomes and also
for eukaryotic transcripts.
I'm not going to demonstrate the transcripts today but there's a link
here to demonstrate that for you if you want to look at that.
what I'm going to do today is enter an accession for an archaeal genomic
sequence.
there's that refseq accession [NZ_LT158599]. this sequence is actually close to three
million nucleotides long. i could put in some coordinates here and from and to
but ORFfinder will automatically stop the processing at about 50 kb so i'm not
going to bother with that. you have a few options here i'm going to go ahead and
select a slightly larger minimal orf length just to clean up my results a bit.
you may or may not want to do that depending on your goals and your
sequences.
i know i'm looking at an archaeal sequence so i'll go ahead and select
genetic code 11, and i'm going to choose to include alternative initiation codons.
you may in some cases want to use this "any sense codon"
option ,particularly if you have a feeling that your sequence may not
begin at the at the beginning of the open reading frame.
so if you choose any sense codon you'll be able to perhaps walk upstream to a
different initiation codon.
ok, I'm going to go ahead and check ignore nested ORFs, again primarily just
to clean up my output because this is a fairly long input sequence.
I'll click the submit button.
and this is running in real time, this is very fast. and given the conditions i had
found 115 ORFs, and it reminds me that it have calculated that on the first 50 kb.
I can also notice that by sliding over here in the viewer and see that the
processing stopped right about 50k.
ok so those of you familiar with our gene records and our genome browsers
will recognize this graphical viewer as our sequence viewer. and so it has the
same controls for zooming and a very similar list of tools, and i'll point out a
couple of those later that you might like.
and then below that graphical view is a table that's sorted initially by length
of the ORF.
so the longest ORFs on top, but these columns are all sortable. so for example
you might want to see the orfs in the order on the input sequence
so I'm going to go down here and select orf 46, it's the first longish one. and
when I select that it then gets highlighted up here with a marker in the
sequence viewer. now many of you are used to using a six-frame translation, and orf
finder allows you to do that here, you click this button to add the six frame translation
track because this is sequence viewer.
it adds that track and I'm zoomed out too far to see any detail here so let's go
ahead and zoom in on this first large ORF46.
I'll mouse over that marker name and that gives me the option then to zoom to
sequence marker, so i'll click that and now I'm zoomed all the way into the
sequence. the shading here represents the beginning of orf 46 and you can see that
in fact it used the ctg as an alternate initiation codon to begin the ORF, and
down on the table you can see that this is in frame plus three, so let's take a
look at the six-frame translation,
+ 3, and we see here is an ATG and down just a little bit farther
another ATG. and so that would be a good use of blast perhaps to see if
we can get some information about what other related proteins actually start
with. and we'll do that in just a minute but first let's look at some of the
other options we have on this results page. so you notice a button here that's
grayed out at the moment that says Download Marked Set, so i could mark all
115 of these ORFs with this button, or i can pick and choose ORFs.
so how do you mark them? you have one selected here
you're looking at the translation here in the box on the left then you can mark
that one that you're viewing.
so that changes the color in the table and it actually puts a little gray box
around the ORF up in the viewer.
so let's say I want to select now orf 47.
I click on that in the table. now it's being displayed on the left,
the translation is, and then I can mark that one as well.
so now i have two marked ORFs up in the sequence viewer and so now my button to
download marked sets is active, and i can download then either the FASTA,
asn1, or feature table formats. you'll notice here
that's grayed out, you can not select it at the moment, is CDS FASTA.
that's because this is not implemented yet but hopefully will be before too
long, and that will allow you to download the nucleotide FASTA for that coding
sequence region.
ok and marking these also allowed us to use BLAST Marked Sets.
so these two buttons will send the translations off to the standard blastp
page. you can select a database here, but you get to the full page where you can
select other databases as well, and you also have all the algorithm parameters
you can play with.
I'm just going to demonstrate smart blast.
SmartBLAST does not have a SmartBLAST the marked sets button, because Smart blast
will take only the one query sequence at a time. so if you do want to analyze
multiple ORFs at once with blast, use the full blastp algorithm.
i'm going to go ahead and unmarked orf 47.
so I have this up here I can click unmark. now I have just one of these
marked but I want to blast 46 not 47,
so I click on that again in the table and that loads orf 46 up here.
so the question is which of these methiones might be the actual one that's
used as the initiation codon?
so note those up here and i'll go ahead and click on SmartBLAST . and again this
is also running in real time, and it's finished.
if you're not familiar with SmartBLAST we had a webinar on that back in
September
so you can check that out on our youtube channel. this did in fact search the NR
database but it uses a faster algorithm so that's why it comes back so
quickly.
it searches another database called landmark, with a much smaller set of
sequences, but that's detail we don't need to worry about today. and we got
some names here which means we probably got some pretty decent hits.
so I'm just going to mouse over that name and click on alignment in that pop
up menu. that takes me down the page to the alignment section.
so this in fact is a self, this is the genomic piece that I put in for this
particular strain. and we can see here, you can see this is actually that second
methionine, the first ATG methionine, and then if we scroll down a bit here's
another species that uses the same methionine, and scroll down to another
one
yet another species of the same genus, it's also using that one. so that's some
evidence anyway that it uses that first ATG as the start of that particular
coding region.
all right I'm going to go back to orf finder. i have just one more example here.
so i'll go back to the submitting page and here I'm going to input an accession
for a bacteriophage [af334111], and this is a much smaller sequence.
i'm going to just keep all the defaults and go ahead and click Submit. it found
21 ORFs
this time. and i just want to point out, if i had checked the box for
ignore nested orfs,
I would have seen this one, this one, this one, and this one. so I would have gotten
four orfs and it would have ignored all these smaller orfs that are completely
enclosed by a larger orf. and we're not going to do any analysis here
i'm at about over 10 minutes already, so i'm going to wrap this up pretty quickly.
i just wanted to point out since you're in the sequence viewer there's some nice
features here. for example you could download a pdf version of this graphics
view, you might want to get rid of the label first on this orf by removing that
marker. and if you wanted to send a link of this view to a colleague, you go over
here to the pull down menu next to the question mark and click on Link to View.
It tells you that this link will be active for one to two months, then you can send
your colleague that
tinyurl there. ok that's it for this quick demonstration. I remind you if you
have any questions you can write to me or send those into info.
do we have any questions online Peter? Not right right now.
alright i will close the webinar then. be sure to check out our courses and
webinars page and our YouTube channel to find out more about what we're doing
thanks, goodbye.