tissue con tains the highest percentage of mitochondrial genome derived transcripts, The estimate of the number of unique transcripts in the adult mouse heart derived from PD173955 the extrapolation of the results of SAGE experi ments exceeds 23,000, and a similar order of magni tude has been suggested for human heart, According to the UniGene, a gene oriented database of transcribed sequences, the number of transcribed genes in the mouse heart reaches 11,000, We detected expression of 80% these genes in the bank vole heart. Moreover, we found in our data sequences similar to over 15,000 other mouse UniGenes, with no heart confirmed expression. Thus, one the one hand, our gene discovery in the heart appears to be close to complete, but, on the other hand, public resources based mainly on Sanger EST sequencing may be very incomplete with respect to tissue specific expression.
Transcript completeness and evaluation of biases The length of nearly 1,000 transcripts was almost com pletely covered by our sequences, and when con sidering only coding regions, this number increased to over 2,000. However, genes with less than 20% of their transcribed length PD173955 covered constituted almost 34% of all transcripts detected Beta-Lapachone in EMTC, indicating that many tran scripts were only patchily reconstructed, a finding further confirmed by the fact that matches to more than 3,000 SwissProt proteins and over 4,000 ECMT were detected only Pyrimidine as singletons. Three factors apparently contributed to the variation in transcript Beta-Lapachone completeness of various genes.
First, transcript length was negatively PD173955 correlated with completeness, although this factor explained only a minor fraction of variation and many short transcripts were also very incomplete, Second, normalization was certainly not perfect, with variation still spanning orders of magni tude, Originally rare Beta-Lapachone transcripts would probably also remain rare after normalization, thus pro ducing a low number of reads and resulting in patchy coverage. Third, the sequence divergence from the mouse could have contributed to the less than complete recon struction of transcripts. We demonstrated this effect by comparing similarity of portions of contigs singletons matching UTRs and coding regions to mouse sequences. Higher sequence divergence in untranslated regions con tributed to the generally lower coverage of UTRs in com parison to the coding parts of transcripts.
The lowest coverage of 5UTRs may also reflect the bias against the 5 end of transcripts expected PD173955 if polyT primers are used for reverse transcription, although studies differ with regard to the extent of this bias, We expected a lower discovery rate for genes with long transcripts because our cDNA preparation method involved PCR with one primer anchored at the 3 end of transcripts. The reverse was true, with a higher propor tion of long transcripts detected than observed in the ECMT. However, many long transcripts were detected only as singletons, indicating that average coverage of long transcripts was poor. In a 454 study of the Arabidop sis transcriptome, Weber et al. obtained unbiased representations of short, medium and long transcripts.
In our data virtually no bias was observed for transcripts 1 2 kb long, The particu larly strong underrepresentation of transcripts Beta-Lapachone 200 bp was probably caused by the RNA extraction method, in which mainly fragments longer than 200 bp bind to a sil ica membrane. The two step approach for transcriptome characteriza tion requires that the expressed sequences be first char acterized using long read assembly. In our single Titanium run, we only achieved 45% average complete ness for CDS, which may not be enough for effective mapping of short reads to obtain information on expres sion level. The coverage was lower than expected for two reasons. First, the number of genes we targeted was larger than could have been expected on published information about the number of genes expressed in mouse heart. Second, despite normalization, there was
