Computational protocol: Direct Mutagenesis of Thousands of Genomic TargetsUsing Microarray-Derived Oligonucleotides

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Protocol publication

[…] Samples for sequencing of the 12 individual strains were processed with Illumina TruSeq v2 sample prep kit and standard Illumina adapters. Samples for sequencing of the cell libraries were prepared with the NEBNext DNA Sample Prep Master Mix Set 1 kit for Illumina sequencing and manually ordered adaptors kindly provided by Luhan Yang (Harvard Medical School, George Church Lab).The libraries were sequenced in two separate lanes, whereas the 12 isolated strains were sequenced in one lane with multiplexing barcodes. The 12 prepared individual strains were sent to the Harvard Biopolymers facility (genome.med.harvard.edu) for Illumina sequencing and downloaded to the cbs.dtu.dk server and processed here. Sequences containing the T7 promoter sequence were extracted with the “grep” command. BLASTn was performed in CLC Bio main Workbench 6.0 with standard settings: “word size” 11, “match” 1, “mismatch” −3, “gap cost existence” 5, “gap cost extension” 2. Bowtie() was applied to perform the alignment of the reads to the reference genomes (see parameters in the script in ). Samtools() was applied to make a consensus reference genome and indexed BAM-file (for visual inspection of read alignment) from the bowtie output (see parameters in the script in ) […]

Pipeline specifications

Software tools BLASTN, Bowtie, SAMtools
Application WES analysis
Organisms Escherichia coli