Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 million per sample. Approximately ~65% of the total reads uniquely mapped to the Btau 4.0 reference gen- ome http://www.ncbi.nlm.nih.gov/genome/guide/cow/ index.html. There were ~10% non-specifically mapped reads and 25% unmapped reads. Only the uniquely mapped reads were considered in the analysis. As described by in Bentley et al. [16] and Ramskold et al. [17] a threshold RPKM (reads per kilo base per million mapped reads) value was established to balance the number of false positives and false negatives. Detailed analysis of unique gene reads (number of sequence reads that match uniquely to the genes) and unique exon reads (number of sequence reads that match uniquely to the exons, including exon-exon junctions), revealed a threshold value of 0.2 RPKM for detectable gene expression in MSC [18]. For the genes with < 0.2 RPKM, a detailed analysis was conducted to identify the number of unique reads aligning outside the exonic regions due to annotation errors and the exclusion of new exons in the Btau 4.0 assembly. Using this strategy genes with RPKM values lower than 0.2 with more than 3 unique gene reads were also considered as genes expressed in MSC (Table 1). All the published gene expression studies conducted on mammary gland up to this point have used microar- rays, and this is the first publication of RNA-Seq analy- sis of tissues related with lactation. Compared to microarray which is limited only to the probes on the array, RNA-Seq analysis considers all the genes expressed in a given tissue. A recent study conducted by Maningat et al. [19] on gene expression profiling of human milk fat globule using the human Ref-8 Illumina Bead-Chips with ~22,000 gene sequences, showed expression of 14,070 of these genes in human milk fat globule. This represents 63% of genes in the bead-chip. Considering that 14,070 genes were detected expressed in milk fat globule using 22,000 genes as reference, it was not unexpected that a higher number of expressed genes were detected by RNA-Seq in MSC. In our analysis, 12,083 genes (44%) in transition (day 15) milk samples had RPKM values ≥ 0.2 and 4,809 genes (17%) had RPKM values ≤ 0.2 with more than 3 Page 3 of 14 unique gene reads, therefore a total of 16,892 genes (61%) were considered as expressed in transition lacta- tion MSC. In peak lactation (day 90), 13,350 genes (49%) had mean RPKM values ≥ 0.2 and 5,744 genes (20%) had RPKM values ≤ 0.2, therefore a total of 19,094 genes (69%) were considered as expressed in peak lactation. In late lactation (day 250) 12,610 genes (46%) had mean RPKM values ≥ 0.2 and 5,460 genes (20%) had RPKM values ≤ 0.2, therefore a total of 18,070 genes (66%) were considered as expressed in late lactation. Peak lactation MSC had the highest number of expressed genes and transition lactation MSC had the lowest number of expressed genes. The 10 most highly expressed genes in transition lactation had very high RPKM values (3,039-265,630 RPKM), and they contribu- ted to ~61% of the total gene reads. In peak lactation, the 10 most highly expressed genes (4,007-21,636 RPKM) contributed to ~11% of the total gene reads, and in late lactation, the 10 most highly expressed genes (3,011-31,398 RPKM) contributed to 19% of the total gene reads. Therefore, transition lactation milk showed the lowest complexity in the transcriptome with a smal- ler number of genes contributing to a larger fraction of the total mRNA while peak lactation milk showed the highest complexity. Analysis of genes with high expression in each stage of lactation In order to categorize the genes with different level of expression, a multiphasic graph was obtained by plotting the log2 transformed RPKM values versus the expressed genes. According to the phases in the graph, gene expression RPKM values were categorized into three groups: high (≥ 500 RPKM), medium (≥ 10 to 500 RPKM) and low (< 10 RPKM) expressed genes. There were 86 (0.5%) highly expressed genes, 2,850 (17%) medium expressed genes and 13,956 genes (82.5%) with low expression in transition lactation MSC (Table 1). Genes with the highest RPKM values in transition lacta- tion were LGB (b-lactoglobulin), CSN2 (b-casein), CSN1S1 (a-S1-casein), LALBA (a-lactalbumin), CSN3 Table 1 RNA-Seq gene expression results for three stages of lactation Category1 Day 15 Highly expressed genes (≥ 500 RPKM) Medium expressed genes (≥ 10 RPKM to 500 RPKM) Lowly expressed genes (< 10 RPKM) RPKM values ≥ 0.2 RPKM values ≤ 0.2 with more than 3 unique gene reads Total expressed genes Non expressed genes 1Genes were annotated using Btau 4.0 with total number of 27,368 genes 86 2850 13956 12083 4809 16892 10476 Day 90 140 5379 13575 13350 5744 19094 8274 Day 250 150 5038 12882 12610 5460 18070 9298