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    Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39

    Jelle Slager, Rieza Aprianto and Jan-Willem Veening

    A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Importantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1,015 transcriptional start sites and 748 termination sites. Using this new genomic map, we identified several new small RNAs (sRNAs), riboswitches (including twelve previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 92 new protein-encoding genes, 39 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 genetic repertoire to 2,151 unique elements. We report operon structures and observed that 9% of operons lack a 5’-UTR. The genome data is accessible in an online resource called PneumoBrowse ( providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies. 


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