the assembly of the human genome from whole - genome - shotgun sequence reads was once deemed impossible , and the probable outcome was dubbed the ' mad magazine version of the human genome ' .
this may have referred to mad 's fold - in back cover , where a drawing and the caption under it are folded twice to reveal a new picture and a new caption to go with it .
this fold - in back cover is rather like alternative splicing , or , more likely , the intent was to imply production of a crazy - quilt genome structure caused by the incorrect joining of the millions of repeat sequences in the human genome . in spite of these concerns ,
paired - end sequencing of three sizes of clones largely avoided the misas - sembly problem and resulted in a faithful rendition of the genome , now with only 498 contigs in build 34 version 2 .
the spectre of the mad magazine version of the genome is coming back in a new guise , however : the corrupt annotation of pseudogenes .
programs designed to scan genomic dna for genes are trained to look for gt and ag boundaries and some consensus regions around these boundaries .
when they encounter a pseudogene that is nearly intact , they try mightily to make it code for an intact protein .
the outcome is a frankenstein gene that never existed in reality , but was cobbled together from spare parts , beginning with the pseudogene carcass .
although it is thought that the number of pseudogenes in the human genome is 20,000 or more ,   these genome features are not annotated as completely as intact genes .
locus link has 21,382 protein coding genes , but only 2,592 pseudogenes are listed ( as of 13th february , 2004 ) .
complete annotation of the human genome must also include identification and naming of all the pseudogenes .
there are 57 intact and putatively functional huma cyp genes , as well as 58 pseudogenes .
these examples illustrate the trouble that the pseudogenes present for gene prediction algorithms . in the first example
, cyp2g2p encodes a nearly intact p450 ( figure 1a ) with two in - frame stop codons , one in exon 1 and one in exon 3 .
a frameshift or stop codon in exon 1 may trick the program into accepting the next downstream met as the starting met ; this happened in cyp2g2p , with a met immediately after the exon 1 stop codon being assigned as the start codon .
if the phase of the two intron - exon junctions bracketing the defective exon is the same , then the exon may just be skipped .
the result is a shortened protein ( figure 1b ) , missing part of exon 1 and all of exon 3 .
the ensembl prediction for this same protein is even shorter ; it is missing all of exons 1 , 3 , 8 and 9 .
figure 1c shows the alignment of the lower - case portions of the pseudogene with the functional mouse cyp2g1 gene , to show what was missed by the prediction algorithm .
the nine exons are shown , one per line , with the intron phase indicated in parenthese .
lower - case letters are the parts of the pseudogene missed in the genscan prediction shown in panel b. ( b ) genscan prediction of the cyp2g2p pseudogene taken from the university of california santa cruz genome browser .
the gene is shown in the same format as in panel a , with blank space for the missed regions .
( c ) alignments of the two regions left out of the genscan prediction against the mouse cyp2g1 functional gene .
a frameshift or stop codon in an internal exon may trigger several possible solutions to be created by the program .
figure 2 illustrates a second possibility not seen in figure 1 . in the cyp2ab1p pseudogene , a bad gt boundary at the end of exon 2 and a stop codon in exon 3
the result shortens exon 2 by 13 amino acids and replaces the first 25 amino acids of exon 3 with 17 amino acids derived from an alternative frame translation from the first part of exon 3 . later in the pseudogene
, a defective exon boundary between exons 6 and 7 and a frameshift in exon 7 are avoided by creation of a second cryptic exon .
this new exon replaces exon 6 and about half of exon 7 and bypasses both genetic defects .
this pseudopseudogene is now taking on a new artificial look , like a car that has been in a wreck and now has a different coloured door .
( a ) the human cyp2ab1p pseudogene , using the same conventions as in figure 1 .
( c ) alignments of the two regions left out of the genscan prediction against the mous cyp2ab1 putatively functional gene .
the cyp51p1 pseudogene has only 130 of 508 amino acids correctly predicted ( figure 3a , upper case ) .
curiously , the genscan prediction adds one bogus exon 72,022 base pairs ( bp ) upstream ; this distant exon has no significant blast hits other than itself in the nr section , of genbank , and no hits in the dbest section .
it is hard to imagine why this sequence is included as part of the cyp51p1 prediction .
( a ) the human cyp51p1 pseudogene . upper - case sequence ( 130/508 amino acids ) is correctly predicted by genscan .
( b ) the underlined sequence is a new first exon , plus an upstream extension from the start codon , to find a phase 2 junction .
most of the pseudogene is not predicted , although the pseudogene is 92 per cent identical to cyp51 over the entire length of the protein sequence .
the prediction for this pseudogene is 937 amino acids long , about twice the size of any p450 protein ( figure 4 ) .
investigation of the sequence shows that , after 211 p450 residues ( about 42 per cent ) , most of the transporter add1 gene ( major facilitator superfamily ) nm_181785 has been added .
the university of california santa cruz genome browser   shows another gene in this region with multiple mrnas , but it is on the opposite strand .
therefore , the genscan prediction for cyp51p2 fuses less than half of the true p450 pseudogene with another downstream gene and additional exon predictions that can not be real since they overlap a third gene on the opposite strand .
this problem of gene fusions is not rare . while annotating p450 genes in the drosophila pseudoobscura genome , a predicted triple - gene fusion was discovered which was 2,182 amino acids long ( contig3323_contig6140.179 ) .
this sequence sandwiched a p450 gene ( cyp9f2 orthologue , minus the last seven amino acids ) between a dna ligase iii homologue and an angiotensin - converting enzyme ( acer ) homologue .
erroneous gene fusions of multiple cyp genes in gene clusters have been observed in caenorhabditis elegans , arabidopsis thaliana and takifugu rubripes ( pufferfish ) .
( a ) the human cyp51p2 pseudogene . upper - case sequence ( 211/508 amino acids ) is correctly predicted .
this is a fusion of part of cyp51p2 with the add/ transporter gene and an additional sequence derived from the opposite strand of another gene .
cyp2u1 , a human p450 gene , has a verified gc boundary at the end of exon 3 ( figure 5a ) .
the genscan prediction misses the correct intron 1 gt boundary and jumps to the next one of the same phase .
genscan skips the gc boundary at exon 3 and extends exon 3 by 11 amino acids .
this gene is correctly annotated in genbank because the mrna sequence is known ; however , there are genes with no known mrna sequences that can be used to correct errors like this .
( a ) the human cyp2u1 gene known from full - length cdna has a gc intron boundary at the end of exon 3 .
the underlined sequence is not found in the true protein and the last two exons are missed after the gc boundary is read through .
a recent re - annotation effort on the drosophila melanogaster genome has predicted approximately 2,000 more genes than is the case for the current berkeley drosophila genome project ( bdgp ) annotations .
this result was obtained by relaxing the stringency of the fgenesh prediction algorithm ; 7,464 gene models were predicted above and beyond the bdgp annotations .
validation by spotting the gene model exon sequences in a microarray and probing for mrna sequences that bound to the array did give support for a large percentage of these predictions
. further analysis by reverse transcriptase polymerase chain reaction ( rt - pcr ) of a subset of the microarray - positive models confirmed that many of these genes are expressed and need to be added to the official gene count .
first , the number of weakly supported or unsupported gene models was large , but a significant fraction of these weak predictions were verified by microarray and rt - pcr experiments .
some of these genes had no blast hits in genbank and so represent new protein families ; this argues that the conservative estimates ( approximately 25,000 genes ) used by some human annotators   are missing many real genes .
there are an estimated 20,000 pseudogenes in the human genome and this number may be much higher .
the collection of these defective genes may be called the human pseudogenome . as discussed above
, what comes out of this set -- after genscan or ensembl gets through with it -- is pretty frightening .
these are vivisected genes raised from the dead , as sure as shelley 's monster .
another chromosome 6 gene , tcba1 , contains an intron that is 479 kilobases long .
znf451 , a zinc finger protein , contains a single exon of 9,114 bp . these are all potential challenges for gene prediction programs .
unfortunately , prediction algorithms are not written to cope with pseudogenes , sequence errors or gc boundaries , giving rise to the results shown in figures 1 - 5 .
even the detailed manual annotation of chromosome 6 found only 633 pseudogenes , as compared with 1,557 genes .
other evidence suggests that the number of human pseudogenes should be closer to the number of intact genes .
perhaps the working definition for the chromosome 6 annotation did not include some small solo exons or detritus exons scattered near documented genes .
it is possible , however , that there might still be about 900 undocumented pseudogenes on chromosome 6 .
the genscan program is quite successful in predicting genes , especially the internal exons of real genes .
it has been optimised for different g + c compositions and different species , but not for pseudogenes . in order to be able to annotate all of the human genome and other genomes
, there is a need for a second program , which is optimised for detecting pseudogenes .
these pseudogenes need to be named and documented on the sequence records and genome browsers in order to avoid the aforementioned problem of creating frankenstein genes .
one program that may fulfil some of these criteria is gnomon , the national center for biotechnology information 's hidden markov model ab initio prediction program .
this program assumes that very close exon predictions ( less than 50 bp apart ) are separated by a frameshift , because very short introns are rare ( at least in vertebrates ) .
the hidden markov model used by gnomon allows non - consensus splice sites such as the gc boundary in cyp2u1 .
stop codons in the middle of an otherwise strong alignment are disregarded and the stops are included in the model , which is treated as a pseudogene rather than as a gene .
these features ought to allow the correct prediction of the pseudogenes in figures 1 - 4 . in practice
for example , all amino acids of cyp2g2p are correctly predicted , but 20 extra amino acids are added between exons 2 and 3 .
the cyp2ab1p sequence predicted by gnomon has 69 more correctly predicted amino acids than genscan , and includes two of the in - frame stops , but it misses exon 7 and adds the same cryptic exon as genscan did after exon 5 .
gnomon finds most of the c - terminal part of cyp51p1 , but misses the first two - thirds .
cyp51p2 coverage is improved , but both n- and c - terminal regions are missed , as well as two internal segments . in short , gnomon is better than genscan for these five genes but still misses the gold standard of hand curation .
finally , once pseudogenes are correctly assembled , nomenclature groups must devise a suitable nomenclature for them
this nomenclature could be applied to other gene families , or other proposals could be made , but this problem needs to be addressed . in the future , progress in genome annotation should make it increasingly hard to find the misleading and erroneous peptide predictions that currently abound , as these become replaced by accurate annotated and named pseudogene models .
 annotation of the human genome is inching forward . 
 seven human chromosomes have now been fully annotated , covering 17 per cent of the genome , and at least one chromosome has been re - annotated . 
 the enormity of the task forces a dependence on automated tools for detecting and assembling the genes , followed by hand curation to correct errors and polish the gene models . 
 the accuracy of gene prediction algorithms is very good for internal exons from intact genes , but these programs do peculiar and exasperating things to pseudogenes . 
 these programs can actually resurrect pseudogenes from the dead , making them into viable gene models for intact proteins , albeit science - fictional proteins . 
 this process is demonstrated for four human pseudogenes from the cytochrome p450 family and one putatively functional p450 gene , cyp2u1 , having a non - consensus intron boundary . 
 these examples are offered as a call - to - arms to improve pseudogene prediction as an art in itself , and not as a by - product of gene annotation . 
 failure to do so will flood the databases with thousands of false - positive predictions . 
 indeed , they are already there . 
Introduction
Pseudogenes live
Gene fusions
Stumbling on a GC boundary
Taking a lesson from the re-annotation of 
Conclusions