Journal: Nucleic Acids Research

Article Title: Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39

doi: 10.1093/nar/gky725

Figure Lengend Snippet: Data analysis pipeline used for genome assembly and annotation. Left. DNA level: the genome sequence of D39V was determined by SMRT sequencing, supported by previously published Illumina data ( , ). Automated annotation by the RAST and PGAP annotation pipelines was followed by curation based on information from literature and a variety of databases and bioinformatic tools. Right. RNA level: Cappable-seq was utilized to identify transcription start sites. Simultaneously, putative transcript ends were identified by combining reverse reads from paired-end, stranded sequencing of the control sample (i.e. not 5′-enriched). Terminators were annotated when such putative transcript ends overlapped with stem loops predicted by TransTermHP . Finally, local fragment size enrichment in the paired-end sequencing data was used to identify putative small RNA features. α D39V derivative ( bgaA ::P ssbB -luc ; GEO accessions GSE54199 and GSE69729). β The first 1 kb of the genome file was duplicated at the end, to allow mapping over FASTA boundaries. γ Analysis was performed with only sequencing pairs that map uniquely to the genome.

Article Snippet: Analysis of SMRT sequencing data was performed with the SMRT tools analysis package (DNA Link, Inc., Seoul, Korea).

Techniques: Sequencing, Control

Journal: Nucleic Acids Research

Article Title: Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39

doi: 10.1093/nar/gky725

Figure Lengend Snippet: Multiple genome alignment. ( A ) Multiple genome sequence alignment of D39W, D39V, NCTC 7466, and clinical isolates SP49, SP61, and SP64 reveals multiple ter -symmetrical chromosomal inversions. Identical colors indicate similar sequences, while blocks shown below the main genome level and carrying a reverse arrow signify inverted sequences relative to the D39W assembly. The absence/presence of the pDP1 (or similar) plasmid is indicated with a cross/checkmark. Asterisks indicate the position of the hsdS locus. ( B ) Genomic layout of the hsdS region. As reported by Manso et al. , the region contains three sets of inverted repeats (IR1-3), that are used by CreX to reorganize the locus. Thereby, six different variants (A–F) of methyltransferase specificity subunit HsdS can be generated, each leading to a distinct methylation motif. SMRT sequencing of D39V revealed that the locus exists predominantly in the F-configuration, consisting of N-terminal variant 2 (i.e. 1.2) and C-terminal variant 3 (i.e. 2.3). ( C ) Motifs that were detected to be specifically modified in D39V SMRT data (see ). Manso et al. identified the same motifs and reported the responsible methyltransferases. α SPV_1259 (encoding the R-M system endonuclease) is a pseudogene, due to a nonsense mutation. β The observed C A C-N 7 -CTT motif perfectly matches the HsdS-F motif predicted by Manso et al.

Article Snippet: Analysis of SMRT sequencing data was performed with the SMRT tools analysis package (DNA Link, Inc., Seoul, Korea).

Techniques: Sequencing, Plasmid Preparation, Generated, Methylation, Variant Assay, Modification, Mutagenesis