LookSeq is (c) 2008 by Magnus Manske and released under the GPL. BASIC SETUP There are four locations on your system that are important for installing LookSeq: * a cgi-bin directory for the Perl scripts; needs to be accessible from the web, and allow Perl execution * a html directory for the HTML templates and the JavaScript; needs to be accessible from the web * a data directory for the reference sequence and the sqlite databases (unless you use a different database engine) * a directory for the LookSeq tools that can convert various formats into alignment databases CGI-BIN 1. Make a copy of all the files in the cgi-bin directory from SVN into your cgi-bin work directory. 2. Copy "settings.pm.sample" to "settings.pm" and edit according to your system setup. HTML 1. Make a copy of all the files in the html directory from SVN into your html work directory. 2. Adapt "lookseq.html" to your liking if you want; bear in mind that it might be accidentially overwritten with the next update of LookSeq 3. You can add custom localization strings for new languages or new interface items by creating e.g. "custom.en" (for English); keys in there take priority over "interface.en" keys 4. You can add new JavaScript functions by creating "custom.js". See "costum.js.sample" for an example. initialize_organism() will be called on script initialization (if the function exists) single_position_double_click(x,y) will be called when a single base (position) is double-clicked in maximum zoom view (if the function exists) TOOLS * "cigar2sqlite.pl" can convert a CIGAR alignment to a long-read alignment database. As it does not use the actual read sequence, it will not show SNPs, but only larger InDels (unless SNPs are marked as InDels) There seem to be two CIGAR variants (one with an additional first column); you might have to alter the script for your data accordingly. * "make_feature_list_from_embl.pl" can produce an annotation database from a modified EMBL file. Unlikely to be useful directly, but shows how such a database is to be constructed. * "make_snp_database.pl" can generate a sqlite database from a list of known SNPs. The input file has the format "chromosome-TAB-position-TAB-reference_base-TAB-alternate_base". * "mapview2sqlite.pl" can convert MAQ mapview files into sqlite databases. SAMTOOLS (BAM) * Use the C++-based renderer for increased speed (bam_c/render_image) * Insert header comment into your BAM file for highlighting expected fragment size : "@CO\tHIGHLIGHT 200-300" (for region 200-300) DATABASE The schema for read alignment storage consists of two tables for chromosome and meta information, and a set of four tables for each chromosome. The following schema works for sqlite, but should work identical or similar in other databases. If you decide to use a different DB engine, alter the scripts in cgi-bin accordingly. Chromosome and meta tables: CREATE TABLE chromosomes ( name VARCHAR[256] , size INTEGER ); CREATE TABLE meta ( key VARCHAR[64] , value VARCHAR[256] ); For each chromosome, there are four tables, each beginning with the chromosome name. The names must be identical to those in the chromosomes table. For a chromosome called "MAL10", the tables look like this: CREATE TABLE MAL10_perfect_match ( read_name VARCHAR[32], pos1 INTEGER, pos2 INTEGER ); CREATE TABLE MAL10_snp_match ( read_name VARCHAR[32], pos1 INTEGER, seq1 VARCHAR[64] , pos2 INTEGER , seq2 VARCHAR[64] ); CREATE TABLE MAL10_inversions ( read_name VARCHAR[32], pos1 INTEGER, seq1 VARCHAR[64] , pos2 INTEGER , seq2 VARCHAR[64] , dir VARCHAR[4] ); CREATE TABLE MAL10_single ( read_name VARCHAR[32], pos1 INTEGER, seq1 VARCHAR[64] ); The corresponding indices (technically not required, but they speed things up considerably): CREATE INDEX MAL10_perfect_index ON MAL10_perfect_match ( pos1 , pos2 ); CREATE INDEX MAL10_snp_index ON MAL10_snp_match ( pos1 , pos2 ); CREATE INDEX MAL10_inv_index ON MAL10_inversions ( pos1 , pos2 ); CREATE INDEX MAL10_sin_index ON MAL10_single ( pos1 ); The seq1 and seq2 fields should contain * a blank string if the respective read (part) perfectly matches the reference sequence * the read (part) sequence; perfect matches to the reference in lowercase, mismatches in uppercase Demo output: sqlite> select * from MAL10_single LIMIT 5; L16_917:1:123:273:487|28521|ggtGgtggtgaggaagataaagatgccaaaCatatgt L16_917:1:273:920:211|28521|ggtGgtggtgaggaagataaagatgccaaaCatatgt L16_917:1:61:34:512|28521|ggtGgtggtgaggaagataaagatgccaaaCatatgt L16_917:1:241:540:992|28522|gtGgtggtgaggaagataaagatgccaaaCatatgtt L16_917:1:93:28:252|28522|gtGgtggtgaggaagataaagatgccaaaCatatgtt sqlite> select * from MAL10_perfect_match LIMIT 5; L16_917:1:124:466:478|46|2865 L16_917:1:69:712:545|46|2862 L16_917:1:65:578:347|48|368 L16_917:1:241:948:401|77|165 L16_917:1:185:27:840|80|557 sqlite> select * from MAL10_snp_match LIMIT 5; L16_917:1:140:382:285|28195||28538|taaagatgccaaaCatatgtttgataggatagggCaa L16_917:1:224:586:508|28337||28532|ggaagataaagatgccaaaCatatgtttgataggata L16_917:1:304:692:165|29393|ccccacAtattttgactacgtgccgcagtatcttcgc|29564|tagagcacgaggtaagttgcgttatggtaatagatgT L16_917:1:192:361:596|29726||30054|ggcgagtcaggtactcctattAaaatccttaaaagtg L16_917:1:208:944:829|30342||30509|taataatgattgtggttgttttGaaaaatgggttAaA