Bioinformatics and mining the human genome
Stresa Italy, July 10, 2000 MDO2000 meeting 
David Nelson

Today, genome projects are the fashion.  The news is full of the race to finish the human 
genome.  Two weeks ago, on June 26, Francis Collins and Craig Venter stood with 
President Clinton in the White House as he heralded the completion of a rough draft of the 
human genome.  If one knows where to look, the progress can be monitored on genome 
meters like this one from the  Arabidopsis genome project, (92.9% 
complete on July 25) slide 1 NCBI has a similar meter for the  Human Genome Project 
 slide 2.  Here we see that on June 25, the human genome was about 21% finished 
and about 66% more was in draft form (meaning 99.9% accuracy or better).  With all this 
attention given to these genome projects, don’t be surprised if you get 
a notice in the mail for a new journal called Nature Genomics.

I don’t mean to downplay the importance of all this work.  It has been and continues to be 
an exciting time, one that will not be repeated.  Once the human and mouse genomes are 
done and the genes have been identified by comparison of the two genomes, much of the 
excitement will pass.  But for now, we are in the middle of it.  Bets are actually being 
taken on the number of human genes, and the number ranges from 34000 to 153000.  The human gene 
count is a surprisingly elusive and slippery number.  Here is a graph taken from the NHGRI showing  
sequencing progress. Slide 3  As you can see from this figure, most of the human genome sequence 
has been acquired in the past 15 months, since March 1999.  If you had looked for a human gene 
in Genbank before 1998 you probably would not have found it.  If you looked today the chances 
are about 90% that you would find it.  

How can it be that we have 87% of the human genome sequence, but we cannot agree 
on the number of genes within a factor of 4 to 5?  This paradox begins to reveal 
the difficulties of analyzing the DNA sequence.  For those of you that have 
looked at the human genome rough draft sequence, you have probably had some 
frustrations.  For those of you who have not, you may not know what is there.  
This morning I would like to talk about data mining in the human genome and what 
king of problems you may run into.  I will use examples from the cytochrome P450 
genes.  We are really faced with numerous problems when looking at the rough 
draft of the human genome.  First, most of the sequence is fragmentary.  The 66% 
that is in draft form is at four levels of completion.  Phase 0 sequence is the 
most preliminary.  Slide 4  A large BAC clone, is shotgun sequenced and all the 
unassembled reads from this sequence set are given one accession number and 
placed in Genbank in the HTGS (high throughput genomic) section.  The fragments 
are unordered and their correct orientation is unknown.  The clone size may be 
as large as 280000 bp and the number of reads in a single entry may be as high 
as 360.  The read lengths are about 600-750 bp. Because P450 genes are 
moderately long, about 10000 to 40000 bp, the short reads in phase 0 sequence 
cannot cover a whole gene. One read may only cover one exon or a part of one 
exon.  If there is only one P450 gene on a whole BAC clone it will be possible 
to assemble the gene based on comparison to other complete P450s and ESTs, but 
there are likely to be regions missing in the gaps between some of the reads.  
You also do not know if there is more than one P450 on a clone.  The pieces 
found may belong to two or more genes.  

The next level is phase 1. Slide 5  Here the reads have been assembled into 
contigs by searching for overlaps.  The number of contigs drops as the fragments 
are joined.  Phase 1 sequence is still composed of unordered pieces.  The 
accession number is given a version number when it is created.  As revisions are 
made and more fragments are joined the version number is increased.  I have seen 
version numbers reach 28, but they are usually in the 1-6 range.  It is still 
difficult and hazardous to assemble genes from phase 1 sequence. I’ll give 
you an example from chromosome 19 which has a CYP2 family gene cluster.  In late 
March 2000, this accession number was version 2 and it consisted of 16 unordered 
pieces.  Ten of them had P450 sequence on them, but there was evidence for five 
different subfamilies being present and probably 7 different genes.  At the time 
it was not possible to assemble all these genes with any confidence.  On May 4, 
the same accession number was at version 3.  This was a phase two sequence with 
only two fragments and they were ordered, so it was now possible to assemble the 
genes correctly assuming that none of them fell in the single gap or ran off one 
end or the other.  It is also interesting to note that the version 3 sequence 
was 137000 bp while the earlier version was 170000, so 33000 bp of sequence was 
discarded.  The P450 genes on this sequence look like this now. Slide 6.  Six 
genes are present, but the cluster is not complete.  In moving from version 2 to 
version 3, the 2A6 gene was deleted as well as the C-terminal exon of 2B7P1.  
The 2A6 gene has reappeared on AC025769.3, but this sequence is labeled as being 
from chromosome 5.  The other sequences seen in this slide are all present on 
this chromosome 5 sequence, so my guess is that there is a mistake in labeling.  
Chromosome 19 is probably correct based on other mapping data.  The genome 
sequence is thus in a state of flux.

Phase 2 sequence  slide 7 has fewer contigs and they are ordered.  This 
is a significant improvement over the unordered pieces of phase 0 and phase 1 
sequence.  There are still gaps and a long human P450 gene could be missing some 
exons.  The longest human P450 gene I know about so far is CYP5A1.  This gene is 
on a completed genomic sequence [NT_001551] and it spans 197000 bp.  If this was 
in a phase 2 sequence it might be missing half the gene.  

After phase 2 the sequence reaches complete status.  There are no more gaps in 
complete sequences.  The last stage in processing the sequence is annotation, 
with identification of the coding regions, repeats, pseudogenes etc.  The 
annotation is always subject to change.  There are often differences of opinion 
about what constitutes a gene.  I have had discussions with genome sequencing 
experts that say they have found twice as many genes on the completed and 
annotated human chromosome 22 as the published article claims exist there.  In 
my own experience, I have found two P450 genes in a cluster joined together with 
the other parts of the two genes overlooked.  I have seen this in Drosophila, 
Arabidopsis and C. elegans, so it is a fairly common problem.  This underscores 
the need for expert human annotation in addition to computer generated 

So far, the problems I have mentioned with assembling P450 genes or any other 
genes have had to do with the incomplete nature of the sequence data.  This will 
vanish as the sequences are all moved out of phase 0, 1 and 2 to complete 
status.  The problems of gene identification and assembly do not end there.  
There is still the matter of fate in selection of the DNA to be sequenced.  On 
Chromosome 22, the first completed human chromosome, there is a small cluster of 
P450 genes.  These are the 2D6 gene and two pseudogenes near it.  This cluster 
has been well studied and documented.  However, the individual whose DNA was 
sequenced for the Human Genome Project carried an allele of CYP2D6 called 2D6*5 
where the whole 2D6 coding region was deleted and only parts of the pseudogenes 
remained.  So when I tried to find 2D6 on chromosome 22, I could not find it.  
This is the problem of polymorphisms.  I show a comment in this slide that 
emphasizes the problem.  Slide 8 There are numerous deletions.  This raises 
questions about P450s that might be real genes or pseudogenes.  On AC008537, 
there is a gene named CYP2G1P.  This gene is normal in every way except it is 
missing exons 4 and 5. Is this due to a polymorphic deletion in this individual 
or is CYP2G1P really a pseudogene.  We won’t know until the gene can be 
resequenced from other people.  The Celera genomic data is taken from 5 
individuals and sequencing on all five was completed June 23, so this question 
is probably answered in the Celera data set. 

Pseudogenes are also a difficult matter.  Currently there are 24 human P450 
pseudogenes.  These fragments of genes often flank real genes and one has to 
wonder if there might be alternative splicing in some cases.  Among human P450s, 
there seem to be a large number of 4F and 2C pseudogenes.  It is not clear why 
these parents should throw off so many more pseudogenes than other P450s, but 
they seem to be more abundant.  Pseudogenes may be a problem in humans in 
general.  Another gene that I work on is called the adenine nucleotide 
translocator or ANT1 and this gene is reported to have at least 9 pseudogenes on 
various chromosomes.  With the draft sequence quality at more than 1 error in 
10000bp, it is often difficult to tell a pseudogene from a sequence error.  It 
may take some time or some resequencing to clarify those P450 genes that have 
one or two in frame stop codons.  This is the case for the gene CYP2G2P.  
Otherwise it looks like a normal gene.  Is it real or not?

Another issue that comes up in such a large project is contamination.  There is 
one P450 sequence I found in human draft sequence that did not seem to be human.  
The best match to this human sequence is a mushroom P450 with 50% sequence 
identity.  Slide 9  I sent notice to Genbank that this was so and asked them to 
relay the message to the sequencers.  When I repeated the search for this 
sequence in June, I did not find it.  Calling up the accession number, I found 
that the version number had changed and the new sequence was shorter.  Slide 10 
Looking at the contigs in both the version 2 and version 3 sequence one can see 
that the first two small contigs have been removed.  The P450 sequence was on 
contig 2.  So the sequencers decided to drop those sequences in version 3.  I 
strongly suspect a fungal contamination of the library used in making this 
clone.  Unfortunately, this may affect other sequences in the library that are 
undetected so far.  This is especially hard if the sequence is chimeric, with 
human DNA on both ends, then it will look like a human sequence and be joined 
into larger contigs.  Errors like this will need to be checked by sequencing 
multiple individuals to eliminate contaminants.

Possibly the most difficult problem in assembling genes from the human genome is 
not due to sequence errors.  Even a perfect sequence would have this problem.  
It is the difficulty in assembling any gene from genomic sequence, finding the 
exons, especially the N-terminal exon.  One example of this is the new human 
P450 CYP3A43.  This gene is 38000 bp long and it is composed of 13 exons.  Slide 11  
The first intron is over 8000 bp and the first exon is only 24 amino acids 
long.  Seven exons are less than 40 amino acids long and one is only 18.  
Luckily the 3A subfamily is pretty well conserved and these short exon fragments 
can be detected by a systematic search.  This would not be the case if the 
percent identity between a new sequence and some other known sequence was low. 
 In that case one would need to depend on cDNA sequence to help locate the 
missing pieces, especially the N-terminal.  This is exactly the problem with the 
Dictyostelium discoideum P450 gene sequences.  Most of them seem to have a very 
short and poorly conserved N-terminal exon that just cannot be detected by 
examining the genomic DNA sequence.  

That brings up the flip side of the human genome project.  In addition to 
sequencing the genomic DNA, nearly two million EST sequences from human cDNA 
have been deposited in GenBank.  These can be very helpful in defining the 
intron exon boundaries of new genes.  More than 1.5 million of these sequences 
have been incorporated into UNIGENE.  Unigene scans the EST sequences and groups 
them in clusters that are from the same gene, or very similar genes.  The May 24 
version had 89632 clusters.  It is tempting to assume that this means there are 
about 90000 human genes, but there is not a one to one correspondence between 
UNIGENE clusters and genes.  Over 30000 of these clusters have only one EST 
sequence and it has been argued recently that many of these are probably 
accidental missprimings.  Serious attempts to estimate the number of human genes 
ignore these singleton ESTs.  That leaves almost 60000 clusters with two or more 
ESTs.  However, different clusters may be from different parts of the same gene.  

I have tried to catalog the UNIGENE entries for human, mouse and rat, though 
this is like shooting at a moving target. Slides 12, 13  see TABLE  The data 
changes so quickly that many of the UNIGENE numbers are retired due to splitting 
and mergers.  Of course pseudogenes that do not make mRNA will not have an entry 
in UNIGENE.  However, there are pseudogenes that do have ESTs such as the new 
CYP2T2P with 12 ESTs mostly from breast and placenta.  This makes one wonder if 
it is really a pseudogene.  A similar gene CYP2T1 has been found in the rat and 
it is not a pseudogene.  So perhaps humans have just recently lost a functional 
version of this gene.  Several other P450 pseudogenes have a UNIGENE entry.  
These include CYP2B7P1, 2D8P and 3A5P2.

There are also six human P450s that are not pseudogenes yet they have no ESTs 
associated with them.  These are CYP2A13, 2C19, 2F1, 4F22, 7A1 and 27C1.  
 Slide 14  This shows that the EST database does not have every gene represented 
even though there are 2 million sequences.  These 6 P450s represent 11% of the 
known human P450s, so gene coverage in the human EST database is about 89% based 
on P450 genes.  

Searching the human ESTs for a P450 will not always find a good hit, even when 
there are ESTs for that gene.  Some P450s have long 3 prime untranslated 
sequences and ESTs are often from this part of the gene. CYP26B1 has a 3000bp 
untranslated 3 prime sequence and no ESTs in the coding region.

I think I have given you a summary of the problems encountered in data mining 
the human genome sequence data.  I do not want to leave you with the impression 
that it is not worth the trouble.  On the contrary, in the past year, I have 
found several new P450 genes from the incomplete and sometimes fragmentary human 
genomic sequences.  slide 15  These include, CYP3A43, CYP2G1P, 2G2P, 2T2P, 2T3P, 
2U1, 4F22, 4V2 (related to a trout P450 fragment called 4V1), 26B1, and 27C1. 
This last gene is missing some sequence at the end and in the middle, but it 
may be an important new member of the mitochondrial P450s.  

The discovery of new human P450s is winding down.  With almost 90% of the genome 
in the databases, one might expect only a 10% increase in gene counts.  This is 
actually misleading, because there are some P450s that are known from cloning 
that have not been found yet in the genomic sequencing.  Slide 16  These make up 
a part of the missing 10%.  CYP2C9, 3A4, 11B1, 11B2 and 26A1 still need to be 
found in the genomic DNA.  (Note, on July 7 CYP3A4 was on AC069294.3.  This 
includes parts of 3A5, 3A7 and 3A43  see file 
)  We have 53 human P450s now.  If we subtract the five given above that are 
not yet found in the genomic DNA that gives 48 and a 10% increase would be 53 
again.  Based on these data, I don’t expect to find more than one or two 
more P450 genes in humans, and those are probably going to be in existing 
families and subfamilies.  There should not be any major surprises in store.  Of 
course, I like surprises.  Slide 17 

Note: 2C9 has been found on AL133513 joined to 2C19 to make a hybrid sequence.
The 2C cluster has four 2C genes close together.  I did a blast of the intron 
region at the joint of 2C9 and 2C19 and found that most of the intron region 
about 800bp of the 1160bp region was a line1 repeat element.  It had 91% 
identity to many other line1 repeats in the human genomic sequence.  If all four 
2C genes have this Line1 repeat in this intron, they could recombine to make six 
different chimeric P450s that would be functional genes.  The original 2C17X 
gene could have been caused by this phenomenon.  The person chosen for 
sequencing may have a deletion polymorphism of the 3 prime end of 2C9 and the 5 
prime end of 2C19.  This would be similar to the deletion of 2D6 on chromosome