MCB 4320C Lecture 4: Lecture 4

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Long read sequencing becomes routine but with a low throughput. Fragment the dna - usually to a size of 20 kb. Hybridize a c2 sequencing primer to the adapter. Sequencing is done on a flow cell that contains many thousands of wells called zero-mode waveguides (zmws). Each base added is called a "phospholinked nucleotide" that contains a fluorescent dye. Dye is cleaved after detection of the added base. Very long read lengths can average 15,000 bases. Provides very high quality genomes when reads are assembled. Throughput recently increased to 1 million reads per run (sequel machine). Was just 65,000 reads on the rsii instrument. Pacbiosequencing another enormous advantage - epigenomics. Pacbio is the one of two ngs (post-sanger) sequencing platforms (the other is nanopore) that does not amplify the dna. Pacbio sequences raw, unamplified dna. unamplified dna still has methyl groups on some bases. Hence, along with the dna sequence, pacbio gives you the full.

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