D1212 Nucleic Acids Research, 2012, Vol. 40, Database issue reliable meta-information about the insertion alleles in the GABI-Kat collection. In detail, the recent improvements are (i) an update to the most recent Ath genome annota- tion dataset TAIRv10, (ii) an improved insertion site pre- diction and gene hit deﬁnition, (iii) enhanced information about conﬁrmation success and line availability. Together with the correction of problematic FST-to-line links using new experimental data, these enhancements result in an increased overall quality and reliability of the collection and its database. RESULTS AND DISCUSSION General database content The SimpleSearch database contains data about GABI-Kat insertion mutants, the derived FSTs and their mapping to the A. thaliana genome sequence. In addition and in contrast to other FST databases like SIGnAL (8) or FLAGdb++ (9), SimpleSearch focuses on metadata concerning GABI-Kat lines and insertion alleles. The database contains data about conﬁrmation attempts for predicted insertion alleles, genetic segregation data of the resistance phenotype provided by the T-DNA allowing an estimation of the number of insertion loci per line, and sequences of successfully conﬁrmed alleles from the off- spring generation that allow to determine the real site of insertion much more accurately than the crude FST se- quences. Moreover, the user is provided with information about line availability (alleles in dead lines are lost although the respective FST exists in databases), and about insertions that could not be conﬁrmed (failed inser- tions, which are predicted from FSTs but are not existing in following generations). The updated version of SimpleSearch offers signiﬁcantly improved data quality due to intensive and ongoing quality management and manual curation (see below). GABI-Kat FSTs are produced by an adaptor-ligation PCR method (4) (see also the Methods and FAQ pages on http://www.gabi- kat.de/) and are mapped to the A. thaliana genome using BLAST (10). Users can access the FST data, select alleles of interest and place insertion requests. Upon request the GABI-Kat lines are segregated on selective medium, genomic DNA is prepared and the predicted insertion sites are conﬁrmed by PCR and sequencing, using an in- sertion site speciﬁc primer and a T-DNA border primer. The obtained ‘conﬁrmation sequences’ are again mapped to the genome. If the insertion site deduced from conﬁrm- ation sequencing matches the position predicted from the respective FST, the insertion is regarded as conﬁrmed. Update of the database to TAIRv10 Until recently, the SimpleSearch database (7) was based upon the TIGR version 5 annotation dataset of the A. thaliana genome. The TIGRv5 annotation consisted of individual BAC sequences and contains an outdated set of gene annotation data (11). SimpleSearch has now been updated to the current genome annotation data, namely TAIR version 10, which is based on pseudochromosomes (ftp://ftp.arabidopsis.org/home/tair/home/tair/Sequences/ whole_chromosomes/). In this context all FSTs from the GABI-Kat collection were newly mapped to the genome sequence, and putative insertion positions were deduced. The increase in the number of successfully mapped FSTs results from using optimized mapping parameters, reduced gaps in the genome sequence and also from the generation of new FSTs for fractions of the GABI-Kat population with formerly low FST yield. About 20 000 new FSTs have been submitted to EMBL/ GenBank. These contribute to a total of now 130 000 FSTs at EMBL/GenBank, which are also accessible via SimpleSearch. The reliability of the insertion site predic- tion was improved by deducing the insertion position from the annealing site of the T-DNA border-speciﬁc primer and the nucleotide positions from the original trace-ﬁle of the FST (Figure 1) (12,13). Particularly in cases where no T-DNA sequence is detectable in the FST, the predic- tion of the insertion positions is more accurate now. This advantage is evident when compared to insertion positions deduced from the called and trimmed sequence only (8). However, deletions at the T-DNA border to genome junction still cause errors in the prediction, which can only be resolved by detailed analysis of the conﬁrmation sequences. Nevertheless, we now explicitly predict a deﬁned pseudochromosome position as insertion position, and not only a locus that is described by a gene code or BAC name. For a better assessment of the relevance of a predicted insertion allele, we used data from TAIRv10 to further qualify hits with respect to annotated genes. TAIRv10 contains information about the untranslated region of the mRNA (UTR) for the majority of the protein coding genes, as well as information about RNA-coding genes. Our former deﬁnition deﬁned a ‘gene hit’ as an insertion site prediction between 300-bp upstream of the ATG and 300-bp downstream of the stop codon of a gene, whereas a ‘CDSi hit’ had a predicted insertion between ATG and stop codon (CDSi for coding sequence plus Figure 1. Workﬂow of the improved insertion site prediction. The in- sertion position is determined using the best BLAST hit of the FST sequence vs. the A. thaliana genome sequence, and the location of the T-DNA within the FST sequence determined by pregap4. If no T-DNA is detected at the start of the FST sequence, the insertion site is located x bases upstream of the BLAST hit, where x is the number of bases before the start of the BLAST hit minus the distance of the T-DNA speciﬁc primer to the T-DNA border. Otherwise, the start of the BLAST hit is considered as insertion position.