CLC Sequence Viewer 6.5.2 was exerted to align sequences and the alignments for the representative specimens were represented in Fig. 3.
We aligned these to the TAIR9 genome and filtered the alignments for quality and for evidence of splicing, yielding nearly 500,000 ESTs that showed evidence of having been spliced (Table 1).
In order to estimate the magnitude of this redundancy, we aligned the Onthophagus non-redundant sequences to Drosophila proteins, filtered the alignments for highly similar matches (BLASTx, E-value < 10-60), and then manually examined the alignments for separate Onthophagus sequences that align to distinct regions of the same Drosophila protein.
The alignments for each identified extended gene family were created using the ClustalW [47] program.
First, we discard all alignments or single genome regions that were used to compute the alignments for the current guide tree node.
The chicken was not represented in about 33% of the alignments for both maxspe and maxgen and was therefore excluded from all analysis based on single genes.
However, more detailed examination of those alignments and trees disclosed problems in the alignments for all of the candidate novel sequences.
In the second stage, FastBLAST quickly finds the top homologs for a given gene by inspecting the alignments for the families that the query belongs to (both known families and ad hoc families).
The alignments for the 70 sequences in the related data set are extracted from the STAP database, and the query sequence is aligned to them using the CLUSTALW profile alignment algorithm [40] as described above for domain assignment.
Since the alignments for STAP analysis typically contained 70 sequences, 2000 random columns were generated for an alignment of 70 sequences of that average nucleotide composition and each column was assigned a quality score.
To test if the noise from the fragmented metagenomic samples overwhelms the phylogenetic signal of the reference set sequence from UniRef, a reference tree based on the alignments for the HMMs was built for each domain.
