Similar protocols

Protocol publication

[…] GCCCAACGA-HEX (vector reads were not detected in the MDA product mixtures from cells). Amplified SCGs belonging to the OP9 lineage were further amplified by a second round of amplification, and library preparation and shotgun sequencing was carried out as previously described using custom bar-coded adaptor oligonucleotides to enable pooling of multiple libraries and the 454 FLX platform with Titanium chemistry (Roche, Branford, CT) or alternatively, using the Nextera 454 titanium kit (Epicentre, Madison, WI). In all cases, purified libraries were quantified by digital PCR and normalized prior to sequencing., All pyrosequence reads from SCG libraries were initially filtered for quality using mothur and reads corresponding to trace contamination with human DNA were identified by BLASTN against the human genome database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) and removed. Assembly of pyrosequence reads from individual SCGs was performed with the “Newbler” GS De Novo Assembler v2.6 (Roche), using default parameters with an expected coverage of 500 and the -urt option. FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to examine the %G+C distribution of reads, and the JCVI Multi-Dimensional Scatter Plot Viewer (http://gos.jcvi.org/openAccess/scatterPlotViewer.html) was used for TNF-PCA in assembled contigs. A subset of 10 SCGs with nearly identical %G+C read distributions and single, homogenous clustering of contigs by TNF-PCA, thus likely largely free of contamination, were selected. Contigs from these 10 SCGs were used to identify potential contaminants in four other SCGs using hierarchical average correlation clustering of TNF-PCA, with contigs cut into 2 kb fragments with 1 kb overlap. Reads mapping to contigs with one or more […]

Pipeline specifications

Software tools mothur, BLASTN, Newbler, FastQC