Roche 454 Unit 3 Sequencing

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The Solid sequencing platform, produced by Technologies/Applied Biosystems (ABI), performs sequencing by ligation method. Similar like the Roche 454 library preparation, genomic double strand DNA were sheared into small pieces and ligated with two types of adatptors P1 and P2 on two ends. One end with P1 adaptor binds onto the surface of the magnetic bead and emulsion PCR takes place to amplify single nucleotide fragment. Then the oil was washed out and four fluorescent labeled di-bases probes were added into the beads mixture. By matching the 1st and 2nd position of the template by di-base probes, fluorescence was detected and the extra tail with fluorescent probe is cleaved out. After multiple cycles of ligation, detection and tail cleavages, the extended chain reached the end of the template. Then the whole extension chain is removed and a new starting primer switching down 1 nucleotide position binds onto the template for another cycle of reaction. Totally, five round of primer binding cycles are performed to complete the sequencing of each fragment. 3. Pitfalls and limitations of NGS Errors could be introduced in any step of the sequencing process, including library…show more content…
However, if reference genome is not available, denovo assembly will be performed to combine the overlapped reads to longer pieces, called contigs. Two classic algorithms used for denovo assembly are overlap graphs and de brujin graphs (26). Assembly errors could be reduced by adjusting the parameters of denovo assembly, such as K-mer (word size), bubble size, length fraction and similarity fractions when mapping reads back to contigs. Highly repetitive sequences are another challenge for denovo assembly as genomic assembler cannot differentiate two reads with similar repetitive pattern (26). N50, maximum length and average coverage are also essential factors to be evaluated for assembly

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