Zhang et al. BMC Genomics 2011, 12:63 http://www.biomedcentral.com/1471-2164/12/63 Table 1 Summary statistics of MPSS signatures in the precocious trifoliate orange (MT) and its wild-type (WT) MT library WT library Total reads 16,564,500 16,235,952 Total nucleotides (nt) 1,242,337,500 1,217,696,400 High-quality reads 16,067,565 15,701,789 High-quality reads (%) 97.00 96.71 Low quality reads 496,935 534,163 Low quality reads (%) 3 3.29 GC percentage of high-quality reads (%) 44.64 45.06 Reliable significant reads 10,576,876 10,527,303 Number of contigs 83,412 81,148 Number of singletons 46,046 43,119 Number of unigenes 31,468 29,864 not 1) reliable, i.e., observed in only one sequencing run, and 2) not significant, i.e., never observed at or above 3 transcripts per million (TPM) in either library. Of the 16,067,565 signatures for MT, 34.2% (5,490,689) did not meet the reliability criterion (Table 1). Meanwhile, of the 15,701,789 signatures of the WT library, 33.0% (5,175,486) did not meet the reliability criterion. After filtering, 10,576,876 reliable signatures were observed in the MT library, with 10,527,303 reli- able signatures in the WT library. The final set of reli- able and significant signatures comprised 31,468 unigenes for the MT library and 29,864 unigenes for the WT library. When the data from the two libraries were combined, a total of 36,523 nonredundant uni- genes were observed. Of these, 6,859 were not observed in the WT library and 5,055 were not pre- sent in the MT library (Table 1). We found that MPSS was able to detect many tran- scripts expressed at low levels. Figure 2 shows the distri- bution of MPSS signatures at different abundance levels in TPM. The distribution of signature abundances across both libraries was generally quite similar. Three signatures in MT and four signatures in WT were expressed at a high abundance (more than 0.1%, or > 1000 TPM). While the number of signatures increased dramatically with a decrease in abundance, 74.88% of the total signatures in MT and 73.56% of the total sig- natures in WT had an abundance of less than 0.0001% (Figure 2). Moreover, of the signatures present in a given library, the vast majority, 93% of the WT library and 98% of the MT library, were below 0.001% abun- dance (< 100 TPM) (Figure 2). This illustrates the sensi- tivity of next generation sequencing technology in identifying transcripts with low expression. Of the total signatures, 1.6% of them were not found in the MT library and 1.7% of them were not found in the WT library (Figure 2). Page 4 of 20 Differential expression of MPSS signatures between MT and WT plants The frequency of signatures was regarded as the relative expression level of each transcript in MT and WT libraries. Comparative analyses of the signature frequen- cies between MT and WT revealed that the expression ratio (MT/WT) varied greatly from 0.009 to 318. Only 3.6% signatures were species specific, in that they were found only in one library and were absent from the other (expression ratio = 0, Figure 3). Of the common signatures in both libraries, only 26.5% of all signatures (9670) showed a twofold or greater (ratio > 2 or < 0.5) expression difference between MT and WT, and were regarded as differentially expressed transcripts according to the criteria defined by Meyers et al. [33]. Of these, 19.9% had expression ratios between 2 and 5, and only a small percentage of signatures (3.6%) showed more than a fivefold difference in expression level between the two libraries (Figure 3). Signature frequency was also com- pared statistically between the two libraries using the Z- score method according to Kal et al. [34], which uses the p-value and a statistical significance value. The expression of 2,735 signatures was significantly different at p < 0.005, at the same time their expression ratios were greater than 2 or less than 0.5 (Figure 4). Among these, 1000 genes were down-regulated in the MT com- pared to the WT and 1735 genes were up-regulated. Of these 2735 signatures, 1855 (23%) were significant at p < 0.001. Length distributions and functional annotation of unigenes The average length of unigenes was 674.41 and 665.35bp for the WT and MT library, respectively, which is shorter than those of Arabidopsis (1445bp) and soybean (1539bp) [35,36]. The median value for the length of the unigenes was 1020 and 1002bp for the WT and MT library, respectively, which is shorter than Arabidopsis (1459bp) and rice (1548bp) [37], but longer than poplar (990bp) [38]. The average length of the open reading frame was 718 and 711 bp, corresponding to an average polypeptide length of 239 and 237aa, respectively, which was also shorter than those of Arabi- dopsis, rice, and soybean, and longer than poplar and maize [39,40]. Additional File 1 provides the distribu- tions of cDNA length and the CDS length from 31,468 and 29,864 nonredundant unigene sequences, respectively. Our annotation method was based on sequence homology searches and the annotations that accompa- nied them. Its aim was to capture the most informative and complete annotation possible. Table 2 shows the hit numbers and percentages relative to those of the major public databases. These annotation statistics show all