Finding Protein and Nucleotide Similarities with FASTA

Abstract

The FASTA programs provide a comprehensive set of rapid similarity searching tools (fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local, and global similarity searches (ssearch36, ggsearch36), and for searching with short peptides and oligonucleotides (fasts36, fastm36). The FASTA programs use an empirical strategy for estimating statistical significance that accommodates a range of similarity scoring matrices and gap penalties, improving alignment boundary accuracy and search sensitivity. The FASTA programs can produce "BLAST-like" alignment and tabular output, for ease of integration into existing analysis pipelines, and can search small, representative databases, and then report results for a larger set of sequences, using links from the smaller dataset. The FASTA programs work with a wide variety of database formats, including mySQL and postgreSQL databases. The programs also provide a strategy for integrating domain and active site annotations into alignments and highlighting the mutational state of functionally critical residues. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons.

Keywords: E()-value; alignment annotation; expectation; homology; scoring matrices; similarity.

Figure 3.9.1. fasta36 search results

A simple fasta36 search using an E. histolytica putative phosphate transporter (UniProt C4M1E7_ENTHI) as a query sequence in a search of the UniProt reference human proteome. (A) Search summary and statistical output. The command line used to perform the search is shown, as well as the name and version of the program, the name and length of the query sequence as well as the name of the database searched. (B) The list of top scoring sequences, with their raw similarity scores (opt), the normalized bit score, and the expectation value. (C) The highest scoring alignment between C4M1E7_ENTHI and ABD1_HUMAN.

Figure 3.9.2. Alignment annotation

Annotations on the alignment of a putative E. histolytica protein ( C4M1E7_ENTHI) with human ABD1, a α/β-hydrolase protein family member (Figure 3.9.1C). The annotations on this alignment were produced by the scripts/ann_upfeat_pfam_www.pl script provided with the FASTA package distribution. (A) A text and graphical presentation of the annotation and domain data. UniProt annotated Variant: and functional ( Site: ) information is shown, as well as the conservation state, e.g. 166E=137E of the annotated site. Pfam domain boundaries Region: are also used to produce sub-alignment scores. The boundaries in the query ( 152-403) and subject ( 123-363) sequences are shown, as are the raw score, bit score, fraction identical, and Q-score (−10log(p)). (B) Compact alignment information, alignment encoding, and annotation information. The scores, and alignment start and end coordinates shown in Figure 3.9.1C and schematically in part (A) are reported here as tab-delimited fields. The single line beginning tr|C4M1E7|C4M1E7_ENTHI has been split into five lines to fit the page. The notations <cont> and parenthetical comments are not included in the single line output. Each of the fields in the -m 8CC output is separated by a <tab> character. The output fields match blast tabular output, with the addition of a CIGAR string and an annotation string. The annotation string includes all the annotation information shown in part (A).

Figure 3.9.3

Output from scripts/summ_domain_ident.pl.