Library: Folta Fragaria FA_SEa
The Fragaria EST data processed at CUGI utilized publicly available software
incorporated in a fully automated in-house developed script (ProcEST.pl). The
processing occurred in three stages:
Stage I: Trace File Processing
Sequence trace files were converted into fasta files and a quality score files using the
phred (Ewing et al, 1998) base-calling program. Vector and host contamination were
identified and masked using the sequence comparison program cross_match
(Gordon, et al, 1998). Vector trimming excised the longest non-masked sequence
and further trimming removed low quality bases (less than phred score 20) at both
ends of a read. Sequences were discarded if they had greater than 5% ambiguous
bases or less than 100 high quality bases (minimum phred score of 20). At this stage
of processing the script generated an overall summary report file, clone report
tables, a Genbank submission file and fasta formatted library files of the high quality
trimmed sequences and associated quality values. The fasta library was not filtered
to remove reads having significant similarity with the species specific mitochondial,
rRNA, tRNA or snoRNA sequences as no Fragaria RNA sequences are currently
available in GenBank.
Stage II: Assembly of High Quality Sequences
In stage II processing, the filtered library file was assembled using the contig
assembly program CAP3 (Huang and Madan, 1999). More stringent parameters (- p
90. -d 60) were used to prevent over assembly and help identify potential paralogs.
The assembly was refined where possible using homology to the swissprot database
to indicate contig accuracy. Homology was determined by running the contigs and
clones against Swiss Prot using the fastx3.4 algorithm (Pearson and Lipman, 1988)
with EXP < 1e -6. Contigs whose clones showed difference in homology were
deconstructed and contigs with the same homology to other contigs were joined
using default CAP3 parameters. The unigene data set was derived by joining the
contig and singleton data sets.
Stage III: Annotation
Annotation of the unigene data set consisted of pairwise comparison of both the
filtered library and the contig consensus library file against the Genbank nr protein
database using the fastx3.4 algorithm (Pearson and Lipman, 1988). The sequences
were also characterized by comparison with the Genbank Rosaceae EST dataset
(160,000 as of 072404) and the Genome Database for Rosaceae (GDR) mapped
peach ESTs using the BLAST software package (Altschul, et al, 1997). The most
significant matches (EXP < 1e -7) for each contig and individual clones in the library
were recorded. Simple Sequence Repeats (SSRs) were indentified in the unigene
data set using the CUGISSR.pl script and further filtered for optimal primer
development according to GC content. The sequence, assembly, homology and SSR
data will be stored in the GDR, facilitating efficient data querying and display. Users
can view contig assembly, clones and annotation, download the library and unigene
sequence libraries and search their sequences against the Fragaria EST database
using our BLAST/FASTA server facility.
References
Altschul, S.F., Madden, T.L, Schaffer, A.A., Zhang, J., Miller, W., and Lipman, D.J.
(1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res. 25(17)3389-402. Review.
Ewing, B., Hiller, L., Wendl, M. and Green, P. (1998). Basecalling of automated
sequencee traces using phred. I. Accuracy assessment. Genome Research 8, 175185.
Gordon, D. Abanjian, C., and Green, P. (1998). Consed: A graphical tool for
sequence finishing. Genome Research 8, 195-202.
Huan, X. and Madan, A. (1999). CAP3: A DNA sequence assembly program. Genome
Research, 9, 868-877.
Pearson, J.D. and Lipman, D.J. (1988). Improved tools for biological sequence
comparison. Proceedings of the National Academy of Science, USA 85,
Copyright © 2004 | Clemson University Genomics Institute. Last updated July 28, 2004.