Good morning! I'm the genomics outreach coordinator for NCBI. And I'd like to introduce you to
the NCBI Assembly database. We'll have a closed captioned recording of this video on our YouTube
channel. You can see the URL below. And please subscribe to our YouTube channel.
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If your question does not get answered, please either e-mail webinars@ncbi.nlm.nih.gov.
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Great. All right, so today I'm going to talk about obtaining genome sequences and annotations with NCBI
Assembly. In my personal view, right now, Assembly is the fastest, easiest way to get
genome sequences and annotations from NCBI. And I'll talk about why that is over the next
few slides.
First I want to talk about the new human genome assembly, GRCh38. It fixes almost 1,000 loci
from GRCh37, and there are about 178 alternate loci. These are particularly important for
immunologically relevant regions of the human genome. For example, if you're talking about some sort
of immunoglobulin locus, it does not make sense to have a human reference immunoglobulin
locus, and that's why we have alternate loci.
So please consider using GRCh38 for your analyses in this realm. More details on the differences
between 37 and 38, as well as mouse 37 versus 38, can be found at the Genome Reference Consortium
website shown here. Just Google genome reference consortium.
If I go to assembly and I [sic]Google (search) homo sapiens, I will see the latest patch for
GRCh38, that's obviously p3. I can click on that, and then go into the FTP site. I'll
show you how to bridge those two things in just a couple minutes. And what I see here
is I can grab nucleotide FASTA, the gff3 annotations. By the way, we're going to be released gtf
annotations probably in the next 6-9 months. I can also see protein sequences as well as
RNA sequences and their annotation.
Many people also want to get sequences from other organisms, particularly bacteria. So
we have about 20,000 bacterial genomes that are available for download in RefSeq, and
about 30,000 available for download in GenBank. For those of who you are new to this, the
big difference between GenBank and RefSeq, GenBank are sequences and annotations that
have been submitted by the user community. We do not adulterate them in any way. RefSeq,
we don't change much for genomes, except to put in mitochondrial sequences. That's for
the pure genomes; for proteins things can change a little bit. But sometimes we do put
in new annotations based on our annotation pipelines, which I will show you in a little bit.
So one might think you would want to go to Genomes at NCBI to download genome sequences.
But what the genome database does right now is it shows genomes in their genera context
with respect to each other. So what I have here is -- actually more likely species context
with respect to each other. Here I have the several thousand Mycobacterium tuberculosis
species for which we have sequences.
And you can see a tree here with obviously quite a few of the nodes collapsed substantially.
Our intention over the next nine months to a year is to make this tree totally interactive,
going down to the individual strain level from which you will be able to link to assembly.
But we have not enabled that functionality yet. Please be on the lookout for a webinar
where we will actually solicit user input on how that resource should be laid out.
Right now, like I said, I think the best place to get sequences for individual strains is from
the Assembly database.
So if I search Assembly for Mycobacterium tuberculosis, I get the very well studied,
for example, H37Rv strain up at the top. If I want to look at all of the H37Rv strains
we possess, I can certainly do that in the Assembly database. We can see where they are submitted
from and when they were submitted. If I go to an individual H37Rv record, the really
nice thing is these two links to the right, the GenBank and RefSeq FTP sites. I apologize
that this slide got a bit distorted. And then I can go and I can -- if I click on that link,
particularly for RefSeq, I can get the FASTA, I can get the annotations, the protein FASTA
and annotations.
The thing we've done with the FASTA, and I’m proud to say we’ve done this, is we have
changed our genomic FASTA files such that they will work with popular RNAseq analysis
software, such as bowtie, hi set and star. So, if you index these genomes for bowtie,
hi set or star, and then you use cufflinks, cuffdiff, or DEseq, you will get actual transcript
expression values or gene expression values, depending on what you're trying to do.
So that's a fairly useful thing to know right now.
One thing to remind you guys who are doing that kind of work, remember the accession
in the FASTA has to match exactly with the accession in the gff3, or gtf file. So right
now I would suggest not mixing annotations from UCSC or Ensembl with genomes from NCBI.
Although we do intend to make a converter in the near future.
Also if things have not been submitted as full genomes, if they have been submitted
just as contigs, you can get those out of the Nucleotide database. The easiest way to
dump those sequences is to go to the "Send to" button. This is an often underestimated
button in the NCBI pantheon. You go to that button, you can send them to a FASTA file,
as shown here.
I wanted to talk a little bit about our genome annotation pipelines. These are user questions
I get quite a bit. If you have other questions about our annotation pipeline, please e-mail
us at webinars@ncbi.nlm.nih.gov.
This is our prokaryotic annotation pipeline. If you submit bacterial genomes as contigs,
scaffolds, or preferably as whole chromosomes, we will annotate your bacteria for you.
Also we have a eukaryotic annotation pipeline. That's something that is obviously quite a bit more complex, and you can read
very extensive documentation about that at this URL shown here. Also this is a basic
diagram, showing that we essentially throw the kitchen sink at these annotations. These
are annotations based on perceived need from the community — the rankings are based on
perceived need from the community, so if you believe that there is a genome that really
needs to be annotated by NCBI, please e-mail genomes@ncbi.nlm.nih.gov and we will consider
your request.
If you have any other questions about eukaryotic genome annotation, please e-mail genomes@ncbi.nlm.nih.gov.
I wanted to talk just a little bit about de novo RNAseq assembly. We do house quite a
bit of data in that realm, although not typically in mouse or human. But in non-model organisms
we house millions of records for de novo RNAseq assembly and those can be found in the [sic]Transcription
Shotgun [sic]Archive. (Transcriptome Shotgun Assembly) which is part of the Nucleotide
database. I can talk about that more extensively if anybody has questions.
Finally, I would like to thank a large amount of people for their contributions to these
databases, as well as their help putting together this presentation. And for the next 5-10 minutes
or so, I'll be available to take any questions.
Thank you very much. I hope all of you have a fantastic day, and for those of you on the
East Coast, welcome to Spring. Thank you.
All right. This is a pretty good question. As a rank beginner to assembly, where would
you suggest I begin? What I would do is go to the Assembly database and dive in head-first.
I would go to the Assembly database, and I would look for your genome of interest, and
I would try to download the FASTA file and do whatever it is you want to do.
One thing -- I teach a lot of bioinformatics courses — and one thing I often tell my
students is, it is best to start with a biological question when you're doing computational things.
Because you are most likely a biologist.
Do we have any assemblers on NCBI? We do not have assemblers on NCBI . It’s a good question.
There are a couple of websites that outline assemblers. I cannot endorse anybody's product,
so I cannot speak to the pros and cons of different assemblers. But I can say that omictools.com
is a pretty good place to look for just a listing of tools for this type of thing, including
assemblers. Some popular ones are velvet, SOAPdenovo, ABySS; there's a bunch of them.
Also Broad has an assembler comparer called MetAMOS, so you might want to check that out.
Can one download plasmid genomes? Yes you can. We don't have a ton of them. And often
they will come with whatever bacterial species they were discovered with. So they will be
listed separately in the FTP site. Some of my colleagues may wish to weigh in on that;
that's something we've been talking about a lot. So if they have more, we'll put them
in the FTP site linked from the video. That's a great question.
If there are no other questions... Oh, by the way, please subscribe to our YouTube channel.
And, by the way, the NCBI minute is going to change from 9:45. We're going to move it
to noon to make things a bit easier for our friends on the West coast. So the next NCBI
Minute will be May 6th. We'll actually talk about PubMed Commons, it will be fantastic,
and it will be at noon Eastern time. Thank you very much, everybody! And have a great day.