Table 4. Sequence retrieval and processing for SILVA 91 SSU Parc LSU Parc Candidates <300 Bases >2% Ambiguities >2% Homopolymers >5% Vector contamination Insuﬃcient relatives <300 Gene bases <30 Alignment quality or base pair score Total sequences in Parcs 900 573 320 327 8018 19 240 14 973 49 063 25 961 6583 461 823 417 217 297 218 2193 4772 2573 13 081 7510 3390 85 689 Operating systems and programming languages The SILVA core system was written in C++ and runs on an Ubuntu GNU/Linux 6.06 LTS based 64bit Dual Dual- Core Opteron cluster with at least 16 GB of main memory on each node. The database server runs MySQL 5.0 and features 32 GB of main memory. The Sun-grid engine (N1GE 6.0) is used to distribute jobs, such as importing, quality check, and aligning on the cluster. The web server is a LAMP system running Ubuntu GNU/Linux 6.06 LTS, Apache 2, MySQL 5.0, and PHP 5. It is connected to the internet via a synchronous 34 Mb connection. The website was written in PHP and Ajax and it is managed using the typo3 content management system in version 4.1. Due to the complexity of the system and the high hardware requirements the system is currently not intended for local installation. RESULTS AND DISCUSSION Data retrieval and processing The total numbers of retrieved sequences and the number of and reasons for rejected sequences are listed in Table 4. Cross checks with RDP II and greengenes indicated a sensitivity of our search procedure of >99%. For ambiguities, homopolymers and vector contamination the numbers are non-additive, since some of the sequences may be aﬀected by two or three parameters. Cut-oﬀ values have been determined based on a statistical evaluation with relaxed parameters (data not shown), and are intended to balance the quality of the databases with any loss of information. Manual inspection of the sequences rejected by the aligner showed that most of these sequences were not ribosomal RNA sequences. A comparison of the length distribution immediately after importing the SSU sequences with the length distribution of aligned sequences conﬁrmed that no unexpected loss of sequences in certain length classes occurred (Figure 1). Partial sequences between 300 and 800 bases were more frequently rejected than longer ones. A closer comparison of sequence quality versus sequence length conﬁrmed that sequences below 700 bases tend to be of low quality. These ‘problematic’ sequences may be generated in diversity studies based on single strand sequencing. The high number of rejected sequences with less than 300 bases is evidence for the increase in short length tag sequencing using e.g. pyrosequencing machines. Nucleic Acids Research, 2007, Vol. 35, No. 21 7193 Figure 1. Sequence length distribution of rRNA genes in the SILVA 91 SSU database. The dotted line represents the sequence distribution directly after importing, the solid line after quality checks and alignment. The huge amount of sequences around 100 bases reﬂect the ﬁrst impact of tag sequencing approaches. Figure 2. Sequence length distribution in the SILVA 91 LSU database. The dotted line represents the sequence distribution directly after importing, the solid line after quality checks and alignment. The huge amount of sequences around 100 bases reﬂect the ﬁrst impact of tag sequencing approaches. The LSU database shows a similar distribution for rejected sequences as the SSU database (Figure 2). As expected, the SSU sequence length distribution follows the prominent primer sets used for sequencing speciﬁc conserved regions on the 16S/18S rRNA gene (24). A distinct peak exists around 500 bases, a small one at 900 bases, and a peak between 1300 and 1500 bases. The large number of sequences with 300 and 600 bases is typical for diversity studies that use single reads or ﬁngerprint techniques like DGGE (25). A text search for ‘DGGE’ across all ﬁelds of the SSU Parc database using ARB showed that 8241 (93%) out of 8889 ‘DGGE’ sequences found belong to the 300–600 nucleotide length class. A taxonomic breakdown for the 300 to 600, 600 to 1000, and 1300 to 1600 bases length classes revealed that 80 to 90% of all sequences per class were of bacterial origin. Nevertheless, from the shortest to the longest length class, the relative numbers for Eukarya decreases, whereas Archaea and Bacteria peaked in the 600–1000 and 1300– 1600 length classes, respectively. This again reﬂects the application of the typical universal primers for Bacteria (24) and Archaea (26). A comparison of the number of sequences hosted by the SILVA, greengenes, and RDP II projects revealed that the