Prediction of Initiation Codons in cDNA Fragments
Using Statistical Information and Similarity withProtein Sequences
Tetsuo Nishikawa

Toshio Ota

Takao Isogai

nisikawa@hri.co.jp

ota@hri.co.jp

isogai@hri.co.jp

Helix Research Institute, 1532-3 Yana, Kisarazu-shi, Chiba 292, Japan

1 IntroductionMore than 100 millions of human cDNA fragment sequences have already been published by several
EST projects. These ESTs, however, are often incomplete in the 5'-region of full length cDNA sequences, whereas it is important to obtain the clones including the intact protein coding sequences(complete clones) if one is to analyze gene function. To obtain complete clones eciently, it is necessary to develop programs which select them eciently from given cDNA fragments as well as todevelop e ective methods for generating complete clones [1]. For this purpose, we have previously reported on a computer program, ATGpr [2], which estimates the reliability of the prediction that each
ATG is a true initiation codon in a given DNA sequence using statistical information. We evaluatedthe accuracy of the initiation codon prediction by ATGpr in complete cDNA sequences in that study.
In practice, however, it is also of importance to know whether a fragment sequence of cDNA, such asESTs, contains an initiation codon or not. Since ATGpr uses only statistical information derived fromthe cDNA sequence, the accuracy can be expected to improve when information on similarity withother proteins are used in the prediction. In this paper, we propose a new method which uses both
statistical and similarity information to obtain higher prediction accuracy for fragment sequences ofcDNA. We evaluate the accuracy of this method using UniGene data, a source of cDNA fragment
sequences.

2 Methods
2.1

Dealing with UniGene

The presence of initiation codons in 5'-EST sequences randomly sampled one from each human UniGene cluster (Build28, 6446 known gene clusters), representative 5'-ESTs, was judged by comparing
with the representative mRNA sequences of the clusters. Here, we de ne a sequence as \complete"
when it includes the initiation codon. The BLASTN program was used for this comparison.
2.2

Method for prediction of a fragment with the initiation codon1) The ATGpr prediction was performed for each representative 5'-EST. The sequence was judged
as complete when the maximum score in all ATG codons in the sequence was greater than ascore threshold.
2) The prediction using ATGpr combined with information on similarity with known protein sequences including those from other organisms was performed for each representative 5'-EST.
Each representative 5'-EST was compared with the OWL database (Ver.30.0) using the BLASTX
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program, and alignments with identities of 30-95% and with a consensus length longer than 100
bases were selected. We judged it as complete when the DNA sequence was longer than theprotein sequence at the 5'-end in at least one alignment and when the maximum ATGpr scorewas greater than a given threshold.
2.3

Evaluation Method

The speci city and the sensitivity of the two predictions described above were determined at scorethresholds varying from 0 to 1. The speci city and the sensitivity is de ned as Nhc/Nh and Nhc/Nc,respectively. Here, Nc is the total number of complete sequences, Nh is the number of the sequencespredicted as complete with the score greater than a given threshold, and Nhc is the number of correctlypredicted sequences in those sequences predicted as complete. The value where speci city equals
sensitivity as function of score threshold is de ned as Spn.

3 Results and Discussion
The Spn for fragment sequences when using only ATGpr was 35%. This is signi cantly lower thanthat for complete sequences in the previous report; this may be due to the fact that the portion ofthe \complete" ESTs in UniGene is very small (estimated to be 15%). The Spn when using ATGpr
combined with the similarity information was 55%, that is the Spn value increased by 20% by introducing the similarity information. When this method is applied to unknown ESTs the improvementin Spn will of course be less than 20%, because the number of proteins similar to unknown genes aremost often fewer than the case with known genes. However, with the rapid increase in the number ofprotein sequences, one can expect signi cant improvements in the Spn value.

References[1] Maruyama, K. and Sugano, S., Oligo-capping:a simple method to replace the cap structure ofeukaryotic mRNAs with oligoribonucleotides, Gene, 138:171{174, 1994.
[2] Salamov, A.A., Nishikawa, T., and Swindells, M.B., Assessing Protein Coding Region Integrity
in cDNA Sequencing Projects, Bioinformatics, 14:384{390 , 1998.

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