Extracting System of Accurate ORFs
Tetsuo Nishikawa, Katsuhiko Murakami, Koji Hayashi, Hiroyuki Sato, Tetsuji Otsuki,
Naoko Kasahara, Tomohiro Yasuda, Kouichi Kimura, Keiichi Nagai, Ryotaro Irie,
Tomoyasu Sugiyama, Takao Isogai.
An extracting system of accurate ORFs from cDNA sequences.
Genome Informatics, 13: 545-547 (2002).

1. Introduction
The development of the Human Genome Project has brought about a rapid
expansion of the range of databases concerning gene sequences. EST sequences
registered in high volume [1] are sequences that are collected with an emphasis on
throughput, and therefore the precision of these sequences is not very high (It isreported that approximately 3% of EST sequences is error [2]). The frame-shift errors
make it difficult to extract reliable amino acid sequences from DNA sequences.
Furthermore, reverse transcription is known to be an error prone process [3]. It istherefore required that amino acid sequence with high precision is extracted fromthese sequences by identifying these artifacts.
Conventionally, an amino acid frame display has generally been used for theextraction of amino acid sequence from a cDNA sequence. In the frame each positionof initiation and termination codon is displayed and a segment that starts at aninitiation codon and terminates at a termination codon is identified; the obtained
segments are identified as possible open reading frames (ORF), and among them, thelongest ORF is identified as an amino acid sequence extracted from the cDNA. In thecase where a frame shift error exists on a cDNA sequence, an ORF is split anddisplayed over 2 frames. Further, since the border of the split ORF is not clear, anamino acid sequence is, in general, identified with an error of tens of bases. It has
been reported that statistical information included in a DNA sequence, such as coding
potential, can be used to identify cloning errors including frame shifts [3]. Dr. Hirosawa
showed that the application of a modified GeneMark program for detection of artifacts
in cDNA clones. This program serves to provide a warning when any spurious split ofprotein-coding regions is detected. Though this method is effective for detecting thesplit of protein-coding regions, it is difficult to detect the strict location of theframe-shift, because of the limitation of the statistical analysis. The most reliable
method to identify the frame-shift errors in a DNA sequence is to use similarity
information to known amino acid sequences. Methods of comparing a cDNA sequencewith amino acid sequences in consideration of the occurrence of frame-shift errors inthe DNA sequence have been developed including FASTY [4] and TRANS series
developed by our laboratory [5]. Using these methods, even where a frame shift error
exists, a single alignment can be obtained and it is possible to identify the frame shift
site.
Thus, to extract an amino acid sequence from a DNA sequence, there are threemethods, a method of using amino acid frames, a method of using statisticalinformation, and a method of using similarity information to known amino acidsequences. However, in order to extract a highly reliable amino acid sequence even
where a frame shift error exists on a cDNA sequence, the application of either one ofthese methods is not sufficient. Here, we propose a new method, mRNA check systemthat integrates these three types of information, amino acid frames, statisticalinformation, and similarity information with known amino acid sequences, in order toobtain a highly reliable amino acid sequence from a DNA sequence.

2. Methods and Results

ATGpr Score

Amino acid sequence determined by similarity
the longest ORF in the framesa) Amino acid frame display
Frame shiftb) Coding potential displayc) Graphic display ofamino acid alignments withframe-shift informationd) Window of DNA-amino acid alignment
840 :
850 :
860 :
870 :
880 :
890
Query: 830 ctggtacggattgaagttgtggacatcaatgacaacg-tcccagagttgcaaagctgctctat
LeuValArgIleGluValValAspIleAsnAspAsn ProArgValAlaLysLeuLeuTyr| . | | | | | | | | |:. :| |Target: 223 ThrValHisIleGluValValAspIleAsnAspAsnSerProGlnPheValGlnSerLeuTyr

Frame shift
Figure 1 Window of analysis results and window of DNA-amino acid alignment.
Window of analysis results consists of a) Amino acid frame display, b) Coding
potential display, c) Graphic display of amino acid alignments, and d) Window ofDNA-amino acid alignment.

The mRNA check system performs the following analyses, ORFs extraction, similarity
analysis to amino acid sequences by using BLASTX and TRANSQ [5], and statisticalanalyses, ATGpr and coding potential analysis, for a given cDNA sequence. The
analyzed results are displayed on the window shown in Figure 1. This window consistsof the following four sub-windows, a) amino acid frame sub-window, b) coding potential
sub-window, c) sub-window for graphic display of amino acid alignments, and d)window of DNA-amino acid alignment. In the amino acid frame sub-window, initiation
codons, termination codons, and the longest ORF in each frame are displayed on threeframes. On the initiation codon mark, ATGpr score that is a measure for likelihood ofinitiation codon is displayed. In the coding potential sub-window, coding potentials of
three frames are plotted along the given DNA sequence. In the sub-window for graphic
display of amino acid alignments, the multiple alignments results with amino acidsequences are displayed graphically. The location of the frame-shifts detected by
TRANSQ are indicated in the graphical alignment lines and local identity in thealignment discriminated by colors provides the reliability of the detected frame-shifts.
For the selected amino acid sequence, alignment between the given cDNA sequence
and amino acid sequence is shown in the sub-window of DNA-amino acid alignment.
The amino acid sequence determined by this alignment is displayed in the frames inamino acid frame sub-window. Users can inspect the frame-shifts by observing ORFs,
amino acid sequences obtained by similarity, coding potential, and alignment withmultiple amino acid sequences, graphically at the same time. This observation makesusers judge the reliability of the frame-shifts effectively. The mRNA check systemenables the effective detection of incomplete DNA sequences such as truncatedcDNAs, frame-shifts in database amino acid sequences and chimera.
The next target is to develop automatic detection method for frame-shifts with higher
reliability. This work was supported by a Grant from NEDO Project of Ministry ofEconomy Trade and Industry of Japan.
[1] Boguski, M.S., Lowe, T.M., and Tolstoshev, C.M., dbEST--database for "expressedsequence tags", Nat Genet, 4, 332-323, 1993.
[2] Nishikawa T., Nagai, K. EST Error Analysis in a Large-scale GenBank Search ofESTs using Rapid-idenyity-searching Progrm, Genome Sequencing and Mapping,
1996.
[3] Hirosawa, M., et al., Detection of Spurious Interruptions of Protein-Coding Regionsin Cloned cDNA Sequences by GeneMark Analysis, Genome Res., 10, 1333-1341,
2000[4] Pearson W.R., Wood T., Zhang Z., Miller W., Comparison of DNA sequences withprotein sequences, Genomics, 46, 24-36, 1997.
[5] Kasahara, N., et al., Highly Sensitive Homology Search Methods on Parallel
Computer, GIW ’97, 294-295, 1997.