Making Sense of DNA and protein sequence
analysis tools (course #2)
http://www.ncbi.nlm.nih.gov/Class/minicourses/
Dave Baumler
Genome Center of Wisconsin, UW-Madison
dbaumler@wisc.edu
Todays session an overview
You have been given a 5 KB piece of DNA sequence
GeneScan: find any exons in the DNA sequence and generate a
predicted protein sequence
ScanProsite: scan the protein sequence for
domains/motifs/patterns found in the prosite database
BLASTP: run a BLASTP search against the Swissprot
database find some of the best matches (hits) and copy each
protein sequence into a word doc for the alignment
MultAlin: conduct protein sequence alignments from the
BLASTP search
In this session you will try out 4 different
tools, Lots of other tools exist
http://bioinformatics.ca/links_directory/
Where are the coding regions?
TCAGCGAAGATGAGATAGTTTTTAAAGGTGGGATTTCCCCACCTTTAAAAAGCGAGAAGTCCCGGTTTTAA
AGAGGAGTAAAATCCTCTTTTTCTAGCCCACTCAGGTGGTTTTTTTGGTTTTCGCTCCTTGCCGCATCTTC
TGTGCCTTTGATGGCGGCTGGTTGGGGTGAAAGGCTGCATATTCCAGAATTTCAGACAGTAGATTGTTTTT
GAAATCTTCCGTTTTATCGTTGACGAACTTAACCATCCTGTTGAAATCATCTTCCTTTGATACACCTTCAG
GAAATGCCTTAGGAACTGATGTTTGGCTATCCAAGGCATCTTGCAATATCTGCACGATCTCCGAATTCATT
GATCGCCCATTGGCCTTTGCTCTGGCGGCAACTGCGTCACGCATACCGTCAGGCATCCTAACTGTAAATCT
CTCAATGAAAGCTGGATCTTCTTTTTCAGTCATCATCTTAAACCATAAAAATTTATACAAAACACACTAGC
ATCATATTGACATTACCCACAATGACATCATAATGGTGTCAGGCATCAAAATGATGTCATCATGACAAGGG
GAAAGTAAATGCAAGATGTTCTCTATACAGGTCGTAAGAACGACAGCTTTCAGCTTCGTCTGCCTGAGCGA
ATGAAAGAAGAGATCCGTCGCATGGCAGAGATGGACGGCATTTCGATTAATTCTGCAATCGTGCAGCGCCT
TGCTAAAAGCTTGCGTGAGGAAAGAGTTAATGGGCAGTAAAAACAGCGAAGCCCGGAAGTGTGGGGACACT
AACCGGGCTTCTAATGTCAGTTACCTAGCGGGAAACCAACAATGACCAGTATAGCAATCTTTGAAGCAGTA
AACACTATCTCTCTTCCATTCCACGGACAGAAGATCATAACTGCGATGGTGGCGGGTGTGGCGTATGTGGC
AATGAAGCCCATCGTGGAAAACATCGGTTTAGACTGGAAGAGCCAGTATGCCAAGCTCGTTAGTCAGCGTG
AAAAGTTCGGGTGTGGTGATATCACCATACCTACCAAAGGTGGTGTTCAGCAGATGCTTTGCATCCCTTTG
AAGAAACTGAATGGATGGCTCTTCAGCATTAACCCAGCAAAAGTACGTGATGCAGTTCGTGAAGGTTTAAT
TCGCTATCAAGAAGAGTGTTTTACAGCTTTGCACGATTACTGGAGCAAAGGTGTTGCAACGAATCCCCGGA
CACCGAAGAAACAGGAAGACAAAAAGTCACGCTATCACGTTCGCGTTATTGTCTATGACAACCTGTTTGGT
GGATGCGTTGAATTTCAGGGGCGTGCGGATACGTTTCGGGGGATTGCATCGGGTGTAGCAACCGATATGGG
ATTTAAGCCAACAGGATTTATCGAGCAGCCTTACGCTGTTGAAAAAATGAGGAAGGTCTACTGATTGGCGT
ATTGGAAGGCGCAAAAAGAAAAGCCAGCAGATGGGCTGCTGGCATTCATTGGGTATATGAACTTTCGGAGA
ACATATGAAGTCAATTATCAAGCATTTTGAGTTTAAGTCAAGTGAAGGGCATGTAGTGAGCCTTGAGGCTG
CAAGCTTTAAAGGCAAGCCAGTTTTTTTAGCAATTGATTTGGCTAAGGCTCTCGGGTACTCAAATCCGTCA
Genemark.hmm a statistical model
Exon prediction in Eukaryotic DNA using
Genescan: Net result is a protein sequence
GeneScan looks for start and stop codons, promoters, splice
sites, polyA tails, provides statistics for coding potential
GeneScan results
GeneScan results
I have a protein sequence, now what?
-Amos Bairoch, (creater of SWISS-PROT),
created a collection of small well-conserved
segments (patterns) to classify and analyze
new proteins
-PROSITE is the name he gave to this pattern
database
-PROSITE also contains profiles which
describe every position of a protein family
-ScanProsite is a server that compares your
protein to the PROSITE database
-if your protein contains a PROSITE pattern,
it can give you a pretty clear indication of its
function
What does it look for on the
protein sequence?
-profiles of protein families
ScanProsite:
-conserved patterns in the
sequence ([RK]-x-[ST])
-cofactor binding motifs
-substrate binding motifs
Around the world there are ~8 other major collections of
domains, such as Interproscan, CD server, or Pfscan
ScanProsite results continued
Sequence Similarity Searches using BLAST
-If you have a region of sequenced DNA, and you want to know
what the protein encoded does
-If you can find similar sequences you can say, “if something is
true for that sequence, it is probably true for mine as well.”
-could take years in the lab, can take only seconds to search a
database for similarity
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between
sequences. The program compares nucleotide or protein sequences to sequence databases
and calculates the statistical significance of matches. BLAST can be used to infer
functional and evolutionary relationships between sequences as well as help identify
members of gene families.
This is an unknown gene sequence used in the next few slides
ATGGAACTGACTCCAAGAGAAAAAGACAAACTATTACTGTTTACCGCTGCACTGCTGGCAGAGCGTCGTCTGGCCCGCGGCCTGAAACTTAACTA
TCCCGAATCCGTGGCCCTGATTAGCGCTTTTATAATGGAGGGCGCTCGCGACGGCAAAAGCGTCGCTGCGCTGATGGAAGAAGGACGGCATGTC
CTGAGTCGCGAGCAGGTCATGGAAGGCATACCAGAAATGATCCCCGATATCCAGGTCGAAGCCACCTTTCCGGACGGCTCCAAGCTGGTTACCG
TCCATAATCCGATAATCTGA
The different types of BLAST
BLAST = Basic Local Alignment Search Tool
“The most popular data mining tool ever”
BLASTN
DNA sequence vs. DNA sequence database
BLASTP
protein sequence vs. protein sequence database
BLASTX
DNA sequence translated in 6 reading frames
vs. protein sequence database
tBLASTX
DNA sequence translated in 6 reading frames
vs. DNA sequence database translated in 6 frames
Steps to use Blast
#1) Paste
sequence
here
#2) Choose search
set
(Either nucleotide
collection or Protein Data
Bank)
#3) select program to use
#4 push
blast button
Blast output #1
The number of sequences in the database
The number of letters (base pairs) in the database
This is the length of your query (in
this case it was nucleotides)
Red, pink, and green
are good matches
How good is your BLAST hit?
The bit score: a measure of the statistical significance of the score (The
higher the score the better and matches <50 are unreliable)
E-value: it is the number of times that your database match may have
occurred by chance. The lower (closest to zero) the better, matches above
0.001 are close to the “twilight zone”
Click here next to get to this genbank entry
A GenBank file
Organism from which
the sequence was
characterized
List of annotated
features
Product
Structural
annotation
Function
Name of the
gene (ureC)
Once you find some protein sequences with BLAST,
copy and paste in word or a text editor
Note: each one will need a FASTA header with the
organism name following as the first line
MultAlin: conduct protein sequence alignments
from the BLASTP search
B Asx Aspartic acid or
Asparagine
Z Glx Glutamine or
Glutamic acid
Its your turn
http://www.ncbi.nlm.nih.gov/Class/minicourses/
Choose Course #2: Making sense of DNA and protein sequences
Questions to consider as they work through these exercises:
#1) What aspects of the tools/resources are confusing or
problematic? What questions do you think your students would
have?
#2) How can we design similar exercises for our classes that are
more compelling? How can we make the students more engaged,
invested and motivated to learn?
#3 Group compilation of additional resources/websites that might
be even better or more intuitive than the NCBI tools?