Basic Bioinformatics

1
Basic Bioinformatics

• As it is applied to the Bacillus megaterium
  genome

2
What we are going to talk about

• Why we are doing all this DNA sequencing
• What genes look like and where they are found
• How we can compare sequences between different
  species
• How genes move between species

3
DNA Sequencing

• Bioinformatics is based on the fact that DNA
  sequencing is cheap, and becoming easier and
  cheaper very quickly.
• the Human Genome Project cost roughly 3 billion
  and took 12 years (1991-2003).
• Sequencing James Watsons genome in 2007 cost 2
  million and took 2 months
• Today, you could get your genome sequenced for
  about 100,000 and it would take a month.
• The Archon X prize you win 10 million if you
  can sequence 100 human genomes in 10 days, at a
  cost of 10,000 per genome.
• It is realistic to envision 100 per genome
  within 10 years everyones genome could be
  sequenced if they wanted or needed it.

4
Why its useful

• All of the information needed to build an
  organism is contained in its DNA. If we could
  understand it, we would know how life works.
• Preventing and curing diseases like cancer (which
  is caused by mutations in DNA) and inherited
  diseases.
• Curing infectious diseases (everything from AIDS
  and malaria to the common cold). If we
  understand how a microorganism works, we can
  figure out how to block it.
• Understanding genetic and evolutionary
  relationships between species
• Understanding genetic relationships between
  humans. Projects exist to understand human
  genetic diversity. Also, sequencing the
  Neanderthal genome.
• Ancient DNA currently it is thought that under
  ideal conditions (continuously kept frozen),
  there is a limit of about 1 million years for DNA
  survival. So, Jurassic Park will probably remain
  fiction.

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From DNA to Gene

• But extracting that information is difficult.
  How to convert a string of ACGTs into knowledge
  of how the organism works is hard.
• Most of the work is on the computer, with key
  confirming experiments done in the wet lab.
• The sequence below contains a gene critical for
  life the gene that initiates replication of the
  DNA. Can you spot it?
• We are now going to spend some time on what genes
  look like and how we can find them.

TTGGAAAACATTCATGATTTATGGGATAGAGCTTTAGATCAAATTGAAAA
AAAATTAAGCAAACCTAGTTTTGAAACCTG GCTCAAATCGACAAAAGCT
CATGCTTTACAAGGAGACACGCTCATTATTACTGCACCTAATGATTTTGC
ACGGGACTGGT TAGAATCTAGGTATTCTAATTTAATTGCTGAAACACTT
TATGATCTTACGGGGGAAGAGTTAGATGTAAAATTTATTATT CCTCCTA
ACCAGGCCGAGGAAGAATTCGATATTCAAACTCCTAAAAAGAAAGTCAAT
AAAGACGAAGGAGCAGAATTTCC TCAAAGCATGCTAAATTCGAAGTATA
CCTTTGATACATTTGTTATCGGATCTGGAAATCGGTTTGCGCATGCAGCT
TCTT TAGCAGTAGCAGAAGCGCCGGCTAAAGCGTATAATCCGCTTTTTA
TTTACGGGGGAGTAGGATTAGGCAAAACACACTTA ATGCACGCCATAGG
CCACTATGTGTTAGATCATAATCCTGCCGCGAAAGTCGTGTACTTATCAT
CTGAAAAATTCACAAA CGAGTTTATTAACTCTATTCGTGACAATAAAGC
AGTAGAATTCCGCAACAAATACCGTAATGTAGATGTTTTACTGATTG AT
GATATTCAATTCTTAGCAGGTAAAGAGCAGACACAAGAAGAATTTTTCCA
TACGTTTAATACGCTTCACGAAGAAAGC AAGCAGATTGTCATCTCAAGT
GATCGACCGCCGAAAGAAATTCCTACACTTGAAGATCGACTTCGCTCTCG
CTTTGAATG GGGCCTTATTACAGACATCACACCACCAGATTTGGAAACA
CGAATTGCTATTTTGCGTAAAAAAGCCAAAGCGGACGGCT TAGTTATTC
CAAATGAAGTTATGCTTTATATCGCCAATCAGATTGATTCAAATATTAGA
GAATTAGAAGGCGCACTTATT
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DNA

• DNA is just a long string of 4 letters
  (nucleotides, or bases) Adenine, Guanine,
  Cytosine, and Thymine.
• Which we will just refer to as A, C, G, and T
• and we are skipping lots of details
• Each DNA molecule has 2 strands, with the bases
  paired in the center
• A on one strand always pairs with T on the other
  strand
• G pairs with C.
• the strands run in opposite directions (like
  roads)
• Since the two DNA strands are complementary,
  there is no need to write down both strands

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Chromosomes and Genes

• each chromosome is a long piece of DNA
• B. megaterium genome is a circle (like most
  bacteria) of about 5 million bases.
• Human chromosomes are 100-200 million bases long.
  We have 46 chromosomes (2 sets of 23, one set
  from each parent).
• genes are just regions on that DNA. It is not
  obvious where genes are if you look at a DNA
  sequence.
• there is a lot of DNA that is not part of genes
  in humans only 2 at most of the DNA is part of
  any gene.
• Bacteria use more of their DNA 80 of the B. meg
  chromosome is genes.
• B. meg has about 1 gene per 1000 base pairs (bp)
  of DNA. About 5000 genes
• Humans have about 25,000 genes.
• We are far more complicated than bacteria
  regulation of the genes is very complicated in
  humans
• We use the same gene in different ways in
  different tissues

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Genes and Proteins

• Most genes code for proteins each gene contains
  the information necessary to make one protein.
• Proteins are the most important type of
  macromolecule.
• Structure collagen in skin, keratin in hair,
  crystallin in eye.
• Enzymes all metabolic transformations, building
  up, rearranging, and breaking down of organic
  compounds, are done by enzymes, which are
  proteins.
• Transport oxygen in the blood is carried by
  hemoglobin, everything that goes in or out of a
  cell (except water and a few gasses) is carried
  by proteins.
• Also nutrition (egg yolk), hormones, defense,
  movement

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The Genetic Code

• Proteins are long chains of amino acids.
• There are 20 different amino acids coded in DNA
• There are only 4 DNA bases, so you need 3 DNA
  bases to code for the 20 amino acids
• 4 x 4 x 4 64 possible 3 base combinations
  (codons)
• Each codon codes for one amino acid
• Most amino acids have more than one possible
  codon
• Genes start at a start codon and end at a stop
  codon.
• 3 codons are stop codons all genes end at a stop
  codon.
• Start codons are a bit trickier, since they are
  used in the middle of genes as well as at the
  beginning
• in eukaryotes, ATG is always the start codon,
  making Methionine (Met) the first amino acid in
  all proteins (but in many proteins it is
  immediately removed).
• In prokaryotes, ATG, GTG, or TTG can be used as a
  start codon. B. meg prefers ATG, but about 30
  of the genes start with GTG or TTG.

In bioinformatics, we generally ignore the fact
that RNA uses the base uracil (U) in place of T.
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Gene Expression

• How do you get a protein from a gene?
• A two-step process (called the Central Dogma of
  Molecular Biology).
• First, the gene has to be copied (transcribed)
  into an RNA form.
• The RNA copy (messenger RNA) is exactly like the
  gene itself, except RNA replaces T with U.
• Most gene regulation whether the gene is on or
  off happens here
• Second, the RNA is translated into protein by
  ribosomes, which are complex RNA/protein hybrid
  machines.
• With the help of transfer RNA molecules, which
  have one end that matches the 3 base codon and
  the other end that is attached to the proper
  amino acid.
• The ribosome starts at the start codon and moves
  down the messenger RNA, adding one amino acid at
  a time to the growing chain. When the ribosome
  reaches a stop codon, it falls off, releasing the
  new protein.

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Reading Frames

• Here we get a bit subtle.
• Since codons consist of 3 bases, there are 3
  reading frames possible on an RNA (or DNA),
  depending on whether you start reading from the
  first base, the second base, or the third base.
• The different reading frames give entirely
  different proteins.
• Consider ATGCCATC, and refer to the genetic code.
  (X is junk)
• Reading frame 1 divides this into ATG-CCA-TC,
  which translates to Met-Pro-X
• Reading frame 2 divides this into A-TGC-CAT-C,
  which translates to X-Cys-His-X
• Reading frame 3 divides this into AT-GCC-ATC,
  which translates to X-Ala-Ile
• Each gene uses a single reading frame, so once
  the ribosome gets started, it just has to count
  off groups of 3 bases to produce the proper
  protein.

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Open Reading Frames

• Ribosomes are very obedient to stop codons when
  a stop codon is reached, the protein is finished.
  Thus, all genes end at the first stop codon in
  their reading frame.
• Since 3 out of the 64 codons are stop codons,
  random DNA has stop codons very frequently.
• However, genes do something necessary for
  survival, so natural selection keeps stop codons
  out of the middle of genes.
• That is, if a mutation arises that creates a stop
  codon in the middle of a gene, the organism dies
  and leaves no descendants.
• Open reading frames (ORFs) are regions with no
  stop codons. All genes reside in long open
  reading frames
• Note that stop codons in other reading frames
  have no effect on the gene.
• The start codon must occur upstream in the same
  reading frame as the stop codon. It is usually
  near the beginning of the ORF, but not
  necessarily the first possible start codon.
• Determining the exact start codon is not easy or
  obvious.
• But, the first stop codon in an open reading
  frame is always a reasonable guess

This is a map of the stop codons in all 3
reading frames in a stretch of DNA. The long ORF
in reading frame 1 is highlighted in black.
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Gene Placement

• Genes can occur on either DNA strand.
• If they are on the reverse strand, the DNA
  sequence needs to be reversed and complemented
• In bacteria, most of the DNA is part of a gene.
  Most long open reading frames (say 100 bp or
  longer) that dont overlap other long ORFs
  contain genes
• Most genes do not overlap each other.
• Sometimes there are very short overlaps (50 bp or
  less), especially if the two genes are
  functionally related.
• In bacteria, genes that affect the same
  biochemical pathway or function are sometimes
  adjacent to each other on the same DNA strand
  (not necessarily the same reading frame),
  allowing them to be co-regulated
• This group of genes is called an operon
• Operons only exist in bacteria they are not
  present in eukaryotes at all.

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Finding Genes

• First job is to find long ORFs, examining the
  longest ORFs first and putting together a set
  with minimal overlaps.
• It is also necessary to identify potential start
  codons, with the furthest upstream start codon as
  the easiest choice.
• Then, how do we know that the ORF contains a real
  gene? The most definitive way is to match it
  with a gene known from other species
• conservation of a sequence between species
  strongly suggests that the sequence has a
  function that is being conserved by natural
  selection
• We compare protein sequences, not DNA, because
  protein is more conserved in evolution than DNA
• The organisms survival depends on the protein
  being functional, which means having the proper
  amino acids sequence
• Since the genetic code is degenerate, many
  different DNA sequences will give identical
  proteins.
• The protein 3-dimensional structure is even more
  conserved, because it is more closely related to
  enzyme activity than the amino acid sequence is.
• However, we dont have good ways of determining
  3-D structure from a DNA sequence

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Sequence Comparison

• So, we compare our ORF sequence to a database of
  known protein sequences from many species.
• BLAST is the standard sequence alignment tool
  (BLAST Basic Local Alignment Search Tool)
• BLAST is based on the concept that if you compare
  the same (that is, homologous) protein from many
  different species, you can see that some amino
  acids readily substitute for each other and
  others almost never do.
• A substitution matrix, giving a score for each
  amino acid position in the proteins being
  compared.

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Practical BLAST

• BLAST itself is a bit of software that can be run
  on almost any computer, but the database needed
  for a good cross-species comparison is quite
  large
• the database is called nr for non-redundant,
  and it contains at least 20 Gb of sequence data
• We are going to use the BLAST service at UniProt,
  a European consortium that contains a
  comprehensive collection of protein sequences
• http//www.uniprot.org/
• Nearly all derived from DNA sequences direct
  sequencing of proteins is difficult
• Terminology your sequence, which you paste into
  the box on the web site, is the query sequence.
  Sequences in the database that match yours are
  called subject sequences.

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A Sequence to BLAST

• This is a more-or-less randomly chosen gene from
  B. meg.
• It is 174 amino acids long
• It is written in fasta format the first line
  starts with gt and is immediately followed by an
  identifier (ORF00135), and then some
  miscellaneous comments.
• After that the sequence is written without spaces
  or other marks.

• gtORF00135 chromosome 538197-538721 revcomp
  MKAKLIQYVYDAECRLFKSVNQHFDRKHLNRFLRLLTHAGGATFTIVIAC
  LLLFLYPSSVAYACAFSLAVSHIPVAIAKKLYPRKRPYIQLKHTKVLENP
  LKDHSFPSGHTTAIFSLVTPLMIVYPAFAAVLLPLAVMVGISRIYLGLHY
  PTDVMVGLILGIFSGAVALNIFLT

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Results
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BLAST Scores

• Results are arranged with the best ones on top
• The most important score is the Expect value, or
  E-value, which can be defined the number of hits
  any random sequence (with the same length as
  yours) would have in the database.
• E-values for good hits are usually written
  something like 3e-42, which is the same as 3 x
  10-42 , a very small number
• Bad hits are very common, and they have e-values
  in a more familiar form for example, 0.004 or
  1.2
• A really good e-values is less than 1e-180, which
  underflows the computers processing
  capabilities, so it written as 0.0
• E-values are affected by the length of the query
  sequence as well as the size of the database, so
  even perfect matches with short sequences give
  poor e-values
• In this case we see many hits with good e-values,
  and the top e-values all are quite similar.
• Before we can conclude that our protein is a
  homologue of the proteins BLAST matches it with,
  we would like them to have roughly the same
  length and have a high percentage of identical
  amino acids.
• the lengths of the query and subject sequences
  should be within 20 of each other
• There should be at least 30 identical amino
  acids
• In this case we can be quite sure we have a good
  match
• BLAST also returns a fourth value, the bit score,
  which we are going to ignore.

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Gene Names

• Mostly genes are named with the function of their
  protein.
• at some point, some related genes had their
  function determined through lab work by
  examining the effects of mutations in the gene,
  by isolating and studying the protein produced by
  the gene, etc.
• Enzymes (end in ase), transport across the cell
  membrane, genetic information processing
  (DNA-gtRNA-gtprotein), structural proteins,
  sporulation and germination, and more!
• Many genes (maybe 1/4 of them in a typical
  genome) have no known function, although they are
  found in several different species conserved
  hypothetical genes
• Every new genome has some genes that are unique
  no matching BLAST hits in the database.
• Are they real genes? Sometimes there is evidence
  in the form of messenger RNA, but usually we
  dont know
• call them hypothetical genes
• putative means that we think we know the genes
  function but we arent sure. Putative should be
  followed by the function name.

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More Gene Names

• One question of interest do the names of the top
  BLAST hits agree with each other? They should,
  but there are always annotation errors, and our
  knowledge of gene function increases over time.
• With some sloppiness due to different naming
  conventions practiced by different scientists
• Here we have a classic case of mis-naming. Why
  is the top hit ribosomal protein S2, with no
  other hit having this name?
• Ribosomal proteins are highly conserved in
  evolution
• Some checking on my part showed that no homology
  exists between this gene and the ribosomal
  protein S2 found in any other Bacillus species
• The other names are similar, although not
  identical.
• What is PAP2? A quick Google search shows that
  it stands for phosphatidic acid phosphatase,
  which fits the other names well.
• There is probably some uncertainty about its
  exact function, given the variety of names and
  the family protein designation in several of
  them.

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Horizontal and Vertical Gene Transfer

• We are accustomed to thinking of genes being
  passed from parent to offspring, always staying
  within the species, with very occasional
  splitting of one species into two.
• This is called vertical gene transfer.
• But, we know that some genes are transferred
  across species lines, not by the standard genetic
  mechanisms.
• This is called horizontal gene transfer
• It is rare in humans and other higher organisms
• In bacteria 10 or more of genes have been
  transferred in horizontally.
• B meg genes that come from vertical descent have
  other Bacillus species (or another closely
  related species) as the closest BLAST hit
• Horizontally transferred genes can come from
  almost anywhere other bacteria, Archaea,
  eukaryotes plants, animals, fungi
• The general mechanisms are well known, including
  conjugation (direct transfer of DNA between two
  bacteria), transduction (transfer of DNA using a
  virus as a carrier), and transformation (the
  bacteria pick up DNA molecules from their
  environment.

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Bacillus Phylogeny

• Kings Play Chess On Fine Ground Sand
• Bacteria is the domain
• Firmicutes is the phylum
• Bacilli is the class
• Bacillales is the order
• Bacillaceae is the family
• Bacillus is the genus.

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Our Example

• Most of the top hits are from various Bacillus
  species there is little doubt that this gene is
  the results of normal, vertical gene flow.
• What about Anoxybacillus flavithermus?
• Click on the accession number to get more
  information, including its phylogeny.
• Taxonomic lineage Bacteria gt Firmicutes gt
  Bacillales gt Bacillaceae gt Anoxybacillus.
• Same family as B meg.

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Aligned Sequences

• You can see the aligned sequences by clicking on
  the Local alignment diagrams
• Query sequence on top, subject below
• Identical amino acids are in the middle of the
  alignment, and similar ones have a sign.
• Gaps regions where one sequence has amino acids
  not found in the other sequence, are indicated
  with ---.
• This protein is very typical in that the best
  matches are in the middle of the protein, with
  fewer identical amino acids near the ends.
• Also, the match doesnt quite make it to the very
  beginning of the proteins, although they are
  almost identical in length.
• The active site of most enzymes is in the middle
• The ends of proteins are often not well conserved

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Local Alignment Result
27
Graphical Overview

• Click on Graphical Overview (just under the BLAST
  box on the left) to get an overview of all the
  aligned sequences
• The extent of the matching region is shown with
  the colored boxes, with non-matching regions
  drawn as a line.
• Color indicates percent of identical amino acids
• You can see that mostly our query and the various
  subjects (matches) line up along almost all of
  their lengths.
• This is a good way to check whether our start
  site is reasonable.
• A few odd ones lower down.
• Genes, and pieces of genes, can move to new
  locations in the genome, fuse with other genes,
  break apart, etc. Always subject to natural
  selection if the altered gene doesnt work, the
  organism will die and we wont see it.
• And of course, sequencing and annotation errors
  occur.

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The Basic Points

• DNA can be read in 3 different reading frames, a
  consequence of the genetic code (3 bases 1
  amino acid)
• Genes are found in long open reading frames,
  areas where there are no stop codons.
• BLAST is the tool we use to compare sequences
  between species
• BLAST scores (e-values) describe the probability
  of finding a random sequence in the database
• Gene sequences are conserved between species by
  natural selection
• DNA sequences outside of genes are much less
  conserved
• Most genes are transferred vertically, from
  parent to offspring, but a significant number are
  transferred horizontally, from unrelated species).

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End
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Other Stuff

• Within-species BLAST--are there duplicate genes?
  Do their names match? What is most closely
  related species? Present in both strains?
• Are nearby genes related by subsystem?