A Transcriptomic Pipeline Adapted for Genomic Sequence Discovery of Germline-Restricted Sequence in Zebra Finch, Taeniopygia guttata

Songbirds have an unusual genomic element which is only found in their germline cells, known as the germline-restricted chromosome (GRC). Because germ cells contain both GRC and non-GRC (or A-chromosome) sequences, confidently identifying the GRC-derived elements from genome assemblies has proven difficult. Here, we introduce a new application of a transcriptomic method for GRC sequence identification. By adapting the Stringtie/Ballgown pipeline to use somatic and germline DNA reads, we find that the ratio of fragments per kilobase per million mapped reads can be used to confidently assign contigs to the GRC. Using this comparative coverage analysis, we successfully identify 733 contigs as high confidence GRC sequences (720 newly identified in this study) and 51 contigs which were validated using quantitative polymerase chain reaction. We also identified two new GRC genes, one hypothetical protein and one gene encoding an RNase H-like domain, and placed 16 previously identified but unplaced genes onto their host contigs. With the current focus on sequencing GRCs from different songbirds, our work adds to the genomic toolkit to identify GRC elements, and we provide a detailed protocol and GitHub repository at https://github.com/brachtlab/Comparative_Coverage_Analysis (last accessed May 12, 2021).