Use of Next-Generation Sequencing for the Identification of Y-Chromosome Derived Genomic Sequence Scaffolds from the Red Flour Beetle, Tribolium castaneum

We previously reported the use of microarray data (male-specific expression) and bioinformatic analyses (male-associated repeat content) to identify Y-chromosome sequence from the red flour beetle, Tribolium castaneum. In combination, these approaches allowed us to identified approximately 750 Kb of Y-chromosome sequence. Since the size of the T. castaneum Y chromosome is estimated to be approximately 5 Mb in length, we sought new methods for uncovering additional Y-chromosome sequence. Here we report the use of two next generation sequencing methods, Illumina and PacBio, for identification of Y-chromosome derived genomic sequence scaffolds. The first method, Illumina, provided millions of short sequence reads independently from T. castaneum males and females. The sex-specific sequence reads were independently aligned to the published (mixed sex) genome assembly and differences in relative abundance were determined. Candidate scaffolds were screened for Y specificity via sex-specific PCR. The second method, PacBio, provided thousands of long sequence reads from T. castaneum males. These long male-specific sequence reads are currently being used to extend Y-chromosome sequence scaffolds, as well as to generate superscaffolds. Of the ~1.5 Mb of Y chromosome sequence identified to date, we find that the sequence is enriched in highly repetitive sequence, including transposons and retrotransposons. In addition to repetitive sequences, some Y-specific scaffolds have also been shown to contain expressed, single-copy genes. However, thus far no gene known to be required for maleness has been found.