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Banana Slug Genomics Notes- Chris Eisenhart Kevin's notes on fixing the wiki Be more careful with meta-data, specifically the processing (FASTQC/preqc) results should be clearly linked to the data set they were made from. More discussion regarding results Make a page for each data set that was collected, linking relevant information The wiki needs more information analysis, less information dump. (Look at the old data for examples) Ed’s Lecture High-throughput sequencing techniques Get DNA (Complex DNA sample) -> Adapter Ligation -> PCR amplification & sequencing Often techniques differ in PCR amplification & sequencing Illumine Library Construction blunt end repair ligate adaptors fill in adaptors PCR amplification Sequencing attach primers to either side of the target segment Note that DNA can only be extended on the 3’ end (There is an -OH on the ribose at the 3’ end) PCR Biases Shorter strands are more amplified GC content Often Fastq sequences come based on the tile where they were sequenced 3 pg is roughly 3 gig abases, therefore 1pg = 1gb 2pg = 1 banana slug genome 1mg/2pg = 500,000 genomes there are 4 million 500 mers in a banana slug genome 2,000,000,000,000 total fragments for the MiSeq run Dilution parameter explanation Too dilute and not enough colonies form Too concentrated and colonies merge into each other, making sequencing impossible Sequencing by synthesis Add a new base (flourescent tagged) and watch for the light Limitations of Illumina Sequencing slow run time short reads Fluorophore overlap Out-of-phase accumulation
