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

Songbirdshave anunusual genomic elementwhich is only foundin theirgermline cells, knownas the germline-restrictedchromosome (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 canbeused to confidently assigncontigs totheGRC. Usingthis comparative coverage analysis, we successfully identify 733 contigs as high confidence GRC sequences (720 newly identified in this study) and 51 contigs whichwere validated using quantitative polymerase chain reaction. We also identified two newGRC genes, one hypothetical protein and one gene encoding an RNaseH-like domain, and placed 16 previously identified but unplaced genes onto their host contigs. With the current focus on sequencing GRCs fromdifferent songbirds, ourwork 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).

Automated summary

The study introduces a new transcriptomic method for identifying GRC sequences in songbirds, successfully identifying 733 contigs as high confidence GRC sequences. By using comparative coverage analysis, two new GRC genes and 16 previously unplaced genes were identified.