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part of cDNA and introduced additional mutations disrupting further polyA T, such that in double stranded cDNA only runs of no more than four identical nucleotides were present. Double stranded cDNA was normalized using the TRIMMER Lomeguatrib kit, equal amounts of normalized cDNA from four selected lines were combined into one pool, and normalized cDNA from four control lines formed the other pool. 5 ug of each pool were sequenced in the separate half of a single 454 Titanium run in the Functional Genomics Center, Uni ETH Zurich. Sequence analysis and assembly All bioinformatic procedures used publicly available soft ware. Custom Python and Perl scripts were used in sequence analysis pipelines.
After adapter trimming, we used SeqClean for identifying and removing low com plexity regions, overly short reads, remains of polyA tails, and reads with high similarity to mammalian repetitive sequences in RepBase ver. 14. 09. The Lomeguatrib cleaned sequencing reads produced in this study have been deposited in NCBIs SRA database Trimmed reads were searched for microsatellite repeats. Dinucleotide repeats of at least 10 units and tri and tetranucleotide repeats of at least 8 units long were identified using Msatcommander, Cleaned and trimmed reads were assembled de novo with the CAP3 Sequence Assembly Program, After preliminary tests, 25 bp overlap and 90% identity were chosen as assembly parameters. All other options of CAP3 were set to default values. Functional annotation of the transcriptome To annotate the transcriptome, we performed similarity searches against both protein and transcriptome genome databases.
A AZD2858 well annotated general protein database, UniProtKB Swiss Prot, was searched with BlastX at an E value threshold of 10 5. The best hit for each contig singleton was based on the lowest E value and highest bitscore. If multiple genes produced identical bit scores with a given contig single ton, ties were broken as follows. i if exactly one of tied genes gave unambiguous best hit with some other con tig or singleton, this gene was selected, ii in the remaining cases ties were broken randomly. The ENSEMBL collection of mouse transcripts was searched with BlastN using an E value threshold of 10 5. If more than one tran script was available for the best hit gene, we conserva Page 12 of 14 tively used the longest transcript Messenger RNA for downstream analyses.
For each result, we assigned AZD2858 Gene Symbol and CDS coordinates, using available ENSEMBL Lomeguatrib API and custom Perl scripts. Mouse UniGene was also BlastN searched with an E value threshold of 10 5. For sequences that did not produce hits in ECMT, we performed a BlastN search against the mouse genome, rat AZD2858 genome, and AceView non redundant mouse transcript base, with the same threshold value as above. All results were stored in a MySQL data base for further data mining. Using the CORUM Ruepp and BioCyc data bases we estimated Lomeguatrib the completeness of gene discovery for selected macromolecular complexes and basic meta bolic pathways which are expected to be present in all nucleated cells Completeness of transcripts To evaluate the completeness of transcripts of genes detected through ECMT similarity searches we used Spi dey an mRNA to genomic local alignment program.
As a refer ence, we conservatively took the longest transcript for each identified mouse gene and aligned all bank vole sequences with significant hits to that gene. In the Spidey analysis, we set inter species alignment flag to allow for sequence divergence, as the reference was mouse tran scripts. AZD2858 Parsing Spidey result files and incorporating information about transcript length and coding sequence CDS location, we computed the fraction of the mouse transcript length covered by the bank vole sequences, both overall and separately for untranslated regions and CDS. Identification of SNPs Single Nucleotide Polymorphisms were identified in GigaBayes on the basis of CAP3 generated ace files utilizing raw reads and associated quality values. We used the minimum total