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Protocol publication

[…] Whole genome sequencing (WGS) was conducted among non-typable pneumococci isolated from mothers and infants in both study CSSs.Samples of genomic DNA from study pneumococcal isolates were sequenced using Illumina HiSeq, 100 bp paired end with 350 bp inserts. Kraken was used to differentiate between pneumococci and closely related species and to identify any potential contamination . MLST was derived from de novo Velvet assemblies . Detection of known serotype specific sequences in assemblies and mapping against known capsular loci was performed as previously described , .Sequence reads were mapped against the reference genome for S. pneumoniae ATCC 700669 using a pipeline developed in-house at The Sanger Institute that utilizes SMALT (http://www.sanger.ac.uk/resources/software/smalt/). In addition to the 34 non-typable genomes sequenced for this study a total of 64 isolates from independent pneumococcal sequencing projects were included in the analysis to give serological context to the phylogeny; six classically non-typable isolates , 52 Gambian isolates from a previous study and a further 6 Gambian isolates from the Global Pneumococcal Sequencing project (http://www.pneumogen.net/gps/). The resultant alignment was reduced to variable sites and phylogeny created using RaxML . Single nucleotide polymorphisms (SNPs) were reconstructed on the phylogeny using an in-house accelerated transformation parsimony method rooted on Streptococcus pseudopneumoniae. The phylogeny was visualized and annotated in ITOL . The data were deposited in the ENA with accession numbers provided in the supplementary data. […]

Pipeline specifications

Software tools Kraken, Velvet, SMALT, RAxML, iTOL
Applications Phylogenetics, WGS analysis
Organisms Homo sapiens