Wayne:High Throughput Sequencing Resources

High throughput (HT) platform and read types

• Illumina single-end vs. paired-end
• 454 Roche
• SOLiD
• MiSeq
• Ion Torrent

File formats and conversions

• bcl
• qseq
• fastq

Deplexing using barcoded sequence tags

• Editing (or hamming) distance

Quality control

• Fastx tools
• Using mapping as the quality control for reads

Trimming and clipping

• Trim based on low quality scored per nucleotide position within a read
• Clip sequence artefacts (e.g. adapters, primers)

RNA-seq analysis

• Quantifying and annotating aligned reads
• DESeq
• edgeR

A variety of additional R packages are available for normalizing RNA-Seq read count data and identifying differentially expressed genes (DEG):

• easyRNASeq (simplifies read counting per genome feature)
• DEXSeq (Inference of differential exon usage)
• DEGseq
• baySeq (also see: segmentSeq)
• Genominator (Bullard et al. 2010)

Basic server commands (for Sirius)

A suggested concentration for a general PCR cocktail:

Command	Usage
pwd	Print working directory (your current location
ls	List (all contents of current location)
ls options	-a (hidden files), -l (long/detailed list), -t (sorted by time modified instead of name)
cd	Change directories
mkdir	Make directories
mv	Move (akin to cut/paste), to remove the file in the current location; Usage: mv current/path/filename destination/path/filename
cp	Copy (also used to rename files if you keep them in their current path), keeps a copy in the current path; Usage: cp current/path/filename destination/path/filename
1.5-2μL	0.8μL
1μL	0.4μL
1μL	0.4μL
0.1μL	0.08μL
1μL	1μL

HT sequence analysis using R (and Bioconductor)

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