## Palindromes in Genomic sequences

### Due 28 Oct 2010 (beginning of class)

Note: this assignment was originally created by David Bernick, then edited by Kevin Karplus. Both are about equally responsible for the final shape of the assignment.

Feature prediction within genomic data is a powerful method for us to learn novel relationships armed with little more than a biological model and a way to express that model in terms of a score. You have seen already that when we can score natures choices for amino acid substitution, we can use that score as a means to align protein sequences. Similar models can be created to infer phylogeny, ancestral sequences or in searches for sequences that code for RNA structures (e.g., tRNA or snoRNA).

Proteins that bind specific regions of DNA, either as a regulation mechanism (transcription factors), or to recognize foreign DNA (phage DNA) need to make use of short sequences. These sequences need to be short in order to minimize the complexity of the binding protein, and they need to be specific in order to produce a specific regulatory effect. Consider, for example, what would happen if a regulatory protein that targets destruction of viral DNA happened upon that same sequence within the host genome. The benefits to the host of reserving these special words can now be seen. Since the machinery that makes use of these special words is also quite complex, we would tend to see them preserved over evolutionary time.

One class of special words appear to be DNA palindromes, which are reverse-complement palindromes (that is, the word is the same on both strands of the DNA, reversing the word and complementing it gives you back the word). For example,**TATA **is one of the more important palindromic sequences, used to signal start of transcription ( It can be written as: T A A' T', where A' means the reverse complement of A (T), and T' means the reverse complement of T (A). Palindromes are common recognition sites, because many of the DNA-binding proteins bind as dimers or with two copies of the same protein domain, with the two chains having the DNA binding sites facing each other and recognizing corresponding sections on the forward and reverse strands, as can be seen in this cartoon picture of the PDB file 1LMB:

Rocha et al.(http://genome.cshlp.org/content/11/6/946.full doi:10.1101/gr.153101), noticed that palindromes of size 4 and 6 seem to be under-represented in some prokaryotic genomes. They suggest that this may be due to particular gene-pairs (restriction modification systems) that exist in some prokaryotes and some phage (lambda for example). This may be a mechanism for establishing exclusive use or restricting use of these words within a genome.

So, armed with the possibility that there exist special words, and that these words are sometimes palindromes, can we now see if we can find them in our genome of choice?

For us to predict when a word is missing or under-represented, we need to have a model of the frequency with which we would expect a particular word, this will be our null model. We want to find the palindromes that occur significantly less frequently than would be expected.

There are many null models that are possible, and for this assignment we will make use of a Markov model that models extending the central palindrome in both directions. Note: nothing in this null model is restricted to palindromes we could use this model to look for any under-represented n-mer. The symmetry of extending in both directions from the central (n-2)-mer is particularly elegant for palindromes, but a very similar method can be made by using an order (n-2) Markov model as the null model. Somewhat surprisingly, the calculation for the expected count comes out the same in either model.

To compute the probability that an arbitrary n-mer is the word (W) that we are interested in, we will use 3 components: the probability that the core (n-2) bases match, and the probabilities of the first and last bases given that the core matches.

P(W) = P(W_{1} | W_{2}...W_{n-1}) * P(W_{n} | W_{2}...W_{n-1}) * P(W_{2}...W_{n-1}) .

If we check for the palindrome N times (roughly the length of the genome), we would expect to see N*P(W) occurrences of W. We can use counts and maximum-likelihood estimates of the probabilities in our null model to get an estimation formula:

E(C(W)) = C(W_{1}...W_{n-1}*) * C(*W_{2}...W_{n}) / C(*W_{2}...W_{n-1}*) ,

where E(C(W)) is the expected value for the count of the number of times W occurs in the genome, and C(W_{i}...W_{j}) is the actual count of the number of times the word W_{i}...W_{j} occurs. The '*' character on the subwords is intended to represent "any character". We are counting the number of words of length n that agree on the first n-1, the second n-1, or the middle n-2 positions. In practice, since our genome sequences are very long, we can ignore edge effects and just use

E(C(W)) = C(W_{1}...W_{n-1}) * C(W_{2}...W_{n}) / C(W_{2}...W_{n-1}) ,

If n=2, we are not counting empty strings on the bottom, but the number of 2-letter windows in our training data. (Actually, I counted the number of letters, which is slightly larger.)

Because our model for the count is the sum of N almost independent observations, each with probability P(W), it can be well modeled as a binomial distribution, with variance

Var(C(W)) = N* P(W) * (1-P(W))

= E(C(W)) * (1 - E(C(W))/N) ,

and the standard deviation is

sigma(W) = sqrt(E(C(W)) * (1 - E(C(W))/N)) . Note: because E(C(W)) is much smaller than N, the variance is approximately the same as the mean.

We can then compute a Z-score for each of our words:

Z(W) = (C(W) - E(C(W))) / sigma(W) . Using a Z-score is quite reasonable here, because we are expecting a distribution very close to a normal distribution.

How big a palindrome can we use this method on? As the palindromes get bigger, the expected counts get smaller, and it is harder to tell whether the number is really significantly smaller than expected. Say we want to have 0.01 probability of a false positive. If we check 4^4 (256) palindromes (the number of palindromes of length exactly 8), we would need a p-value < 3.9E-05 to get an FPR < 0.01 (Bonferroni correction for multiple tests p/N (N = # tests). According to a normal probability table (http://www.math.unb.ca/~knight/utility/NormTble.htm), we would need a Z-score < -4 for this level of significance. For a zero count (the lowest we can measure) to be at least 4 standard deviations below the expected value, we would need the expected value to be at least 16. For a megabase genome, the expected number of counts for the 4^8 (64k) different 8-mers should be around 16, so 8-long palindromes are about as big as we can go. For longer palindromes, even a count of zero would not mean significant under-representation.

Note that if we wanted to check all 64k 8-mers for under-representation and not just the palindromes, we'd need a probability of 1.5E-07 of a false positive, or a Z-value around -5.1. This would require expected counts of at least 26 on a 1.7 megabase genome.

## Assignment

### Part 1:

Write a short piece of python code that generates all DNA palindromes for a given range of lengths. I found it useful to first write a (recursive) generator function yielding all k-mers over a given alphabet for a fixed value of k. I also found the following function useful:

# define reverse complement
complement_table = string.maketrans("ACGT", "TGCA")
def reverse_comp(dna):
return dna[::-1].translate(complement_table)

The definition of DNA palindrome above does not allow odd-length palindromes, since the middle letter is never the complement of itself. In practice there are often unimportant bases in the middle of a matching sequence, so let's add "odd palindromes" that have an extra letter in the middle: `dna+'A'+reverse_comp(dna)`

, `dna+'G'+reverse_comp(dna)`

, ...

### Part 2:

Write a program, named score_palindromes, that reports significantly under- or over-represented palindromes. The program should have at least the following options (**NOTE: **YOU MUST USE THE** ARGPARSE OR OPTPARSE **MODULE FOR THE FOLLOWING OPTIONS)**:**

- -e or --max_e=0.1 which specifies the maximum E-value to include in your report. The program should report both under- and over-represented palindromes with E-values < max_e.
- -k or --max_k=8 which specifies the maximum size palindrome to look for.
- -m or --min_k=2 which specifies the minimum size palindrome to look for.
- filename arguments any number of file names for FASTA files of genomes, any of which may be gzipped (indicated by the file name ending with ".gz" If none are provided, read (uncompressed) input from stdin.

Your program would then be executed as follows (for example):

search_nmer -e 0.01 --max_k=8 P.abyssi.fa.gz P.furiosus.fa.gz P.horikoshii.fa.ga

Note: you can open a gzipped file in python fairly easily using the `gzip`

module.

file = gzip.GzipFile(filename,'r') if filename.endswith(".gz")
else open(filename,'r')

Here are a few genomes for various prokaryotes as FASTA files (actually, as gzipped fasta files).

A.fulgidus.fa.gz

A.pernix.fa.gz

H.influenzae.fa.gz

H.pylori.fa.gz

M.jannaschii.fa.gz

M.thermoautotrophicus.fa.gz

N.meningitidus.fa.gz

P.abyssi.fa.gz

P.furiosus.fa.gz

P.horikoshii.fa.gz

S.acidocaldarius.fa.gz

S.solfataricus.fa.gz

S.tokadaii.fa.gz

V.cholerae.I.fa.gz

V.cholerae.II.fa.gz

V.fischeri.I.fa.gz

V.fischeri.II.fa.gz

V.parahaemolyticus.I.fa.gz

V.parahaemolyticus.II.fa.gz

V.vulnificus.I.fa.gz

V.vulnificus.II.fa.gz

These genomes are also available directly from /cse/classes/bme205/Fall05/ on the School of Engineering computers, and it is fairly simple to find other full prokaryotic genomes on the web. There will be one sequence per file, but your program should be able to handle multiple sequences in one file, so that it can be applied to multi-chromosome genomes or genomes that are not fully assembled. You could also apply the program to a set of genomes from the same genus, as the extra data would allow you to search for slightly longer palindromes. Note: the files take up about 45 Mbytes unzipped, so leave them in the compressed format. It is good practice to learn how to read gzipped files without having to uncompress them first, so that is a requirement for this assignment.

Hint: the program should use the k-mer counting module that you developed for the Markov chain assignment, modified to count all k-mers up to the maximum size specified. You can use a single dictionary to store all the different counts, since the k-mer keys are distinct. Note that your previous k-mer counting only counted one strand of the DNA. There is no need to run though the entire reverse-complement strand, as you can get the counts for the opposite strand fairly simply:

rev_counts = dict( (reverse_comp(dna),cnt) for dna,cnt in counts.items())
for dna, cnt in rev_counts.items():
counts[dna] += cnt

After you have counted all the k-mers, you can iterate over all the palindromes that you generated for part 1 and compare the observed counts with computed counts.

It is easy to compute the z-scores, since we have a model for both the mean and standard deviation that we expect. To turn the z-scores into p-values, we need the cumulative normal distribution function. This is now built into Python 2.7 as part of the math library, which includes the erf() and erfc() functions:

Use the math.erfc function to compute the desired p-value:

P(z > t) = erfc(t/sqrt(2))/2

P(z < t) = erfc(-t/sqrt(2))/2

and put them in another dictionary.

When we run the command

score_palindromes --max_e=0.01 --min_k=6 --max_k=6 ~/.html/bme205/f05/H.influenzae.fa.gz

We get

# Reading from /cse/faculty/karplus/.html/bme205/f05/H.influenzae.fa.gz
# Reading from H.influenzae.fa.gz
# There are 64 palindromes being considered,
# and about 3660050 positions where palindromes could be.
# Reporting palindromes from length 6 to 6 ,
# which are under- or over-represented with E_value < 0.01
#kmer observed expected Z_score E_value
GTTAAC 38 673.62 -24.49 5.716e-131
GAATTC 604 1337.26 -20.06 5.812e-88
TAATTA 1832 2820.60 -18.62 6.870e-76
GATATC 352 897.61 -18.21 1.295e-72
GTGCAC 282 760.93 -17.36 4.972e-66
AAGCTT 718 1302.25 -16.19 1.804e-57
ATGCAT 510 955.62 -14.42 1.292e-45
GGCGCC 54 279.78 -13.50 5.084e-40
GTCGAC 8 186.88 -13.09 1.274e-37
CATATG 348 674.85 -12.58 8.387e-35
CTTAAG 244 517.74 -12.03 7.780e-32
TGCGCA 700 1092.76 -11.88 4.640e-31
AAATTT 5808 6760.86 -11.60 1.330e-29
CAATTG 1348 1840.40 -11.48 5.269e-29
AATATT 4070 4819.54 -10.80 1.056e-25
TTCGAA 352 604.80 -10.28 2.770e-23
GGTACC 144 323.15 -9.97 6.845e-22
CGATCG 284 505.93 -9.87 1.844e-21
TTTAAA 4568 5171.36 -8.40 1.476e-15
ATATAT 850 1131.41 -8.37 1.883e-15
AGCGCT 94 211.23 -8.07 2.322e-14
GCTAGC 122 241.76 -7.70 4.274e-13
GTATAC 114 229.59 -7.63 7.583e-13
GAGCTC 128 243.79 -7.42 3.860e-12
GGATCC 192 323.87 -7.33 7.482e-12
GGGCCC 50 133.22 -7.21 1.791e-11
CAGCTG 620 822.28 -7.05 5.527e-11
CACGTG 472 636.72 -6.53 2.126e-09
CGTACG 234 357.26 -6.52 2.226e-09
GACGTC 70 146.63 -6.33 7.933e-09
ATCGAT 774 961.08 -6.04 5.071e-08
TTATAA 1776 2029.53 -5.63 5.793e-07
CTATAG 204 285.75 -4.84 4.238e-05
TGATCA 978 1138.01 -4.74 6.710e-05
TTGCAA 2518 2740.44 -4.25 6.817e-04
TGTACA 554 651.77 -3.83 4.099e-03
AACGTT 1694 1849.17 -3.61 9.825e-03

If you sort your output by length of word and alphabetically within that, you may see helpful patterns, such as palindromic sequences that have a don't-care region in the center. (My report did not do that sorting, but sorted by E-value.

### Part 3:

Run your program as in the example above (*H. influenzae *for palindromes of length exactly 6. Also run your program for the three pyrococcus genomes taken together, for length 4 to 8 palindromes:

score_palindromes --min_k=4 --max_k=8 --max_e=0.01 P.abyssi.fa.gz P.furiosus.fa.gz P.horikoshii.fa.gz

Write up a short discussion that includes your data, along with an interpretation of their meaning. Is there anything special about these sequences? Try Google, or the following links: http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cross_index_recognition_sequences.asp and http://rebase.neb.com/rebase/rebadvsearch.html The first page has table, the second has a search site for finding known restriction enzymes by organism or sequence (among other methods). (Use 'N' for wildcard, not 'X' on this search page.) Do your finds match up with known restriction enzymes?

### Extra credit:

- It might be useful to have an option (
`--all`

) for looking at all n-mers, and not just palindromic ones. Make sure you change the number of hypotheses when computing the E-values!
- Several of the odd-length palindromes have a don't-care base in the center, but our current algorithm only checks for the exactly matching base. You could merge the counts for abcdxd'c'b'a' for all values of x to do the same sort of analysis for palindromes with a don't-care base in the center. Because you are merging counts, you could go up to one longer palindromes this way. Make sure that you generate the appropriate counts for the needed subwords also. It may be easiest to do this
*after *generating the table of k-mer counts.
- You could go even further, looking for palindromes with 2, 3, or more don't-care bases in the center. Since the DNA-binding mechanism does not require that the two protein domains bind adjacent to each other, it is quite common to have a pair of sites that are separated by a few bases.
- If you are feeling really ambitious, try coming up with a different way to estimate the probability of a given palindrome. Does it produce better results? If so, you may have found something worth publishing. It seems fairly clear that the method in this assignment is not completely right, because almost all 3-mers or 4-mers are significantly under- or over-represented.

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