Illumina sequencing pipeline 1 sample preparanon 15

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Unformatted text preview: cing (2001) •  •  •  •  •  300 ABI DNA sequencing plaXorms 50 producNon staff 20,000 square feet of wet lab space 1 million dollars / year for electrical service 10 million dollars in reagents Current cost of human genome: < 10,000 $ Second/Next GeneraNon Sequencing •  Second GeneraNon sequencing techniques overcome the restricNons by finding ways to sequence the DNA without having to move it around. •  You sNck the bit of DNA you want to sequence in a liple dot, called a cluster, and you do the sequencing there; as a result, you can pack many millions of clusters into one machine. Sequencing a strand of DNA while keeping it held in place is tricky, and requires a lot of cleverness. Illumina Sequencing Pipeline 1. Sample preparaNon (1 ­5 days) 2. Cluster generaNon on flow cell (1.5 days) ligate adapters 4. Analysis (days, months, years…) 3. Sequencing and Imaging (1 week) Illumina Sequencing: Video We mulNply up the template stand, i.e. the bit of DNA that we are sequencing, and sNck on a few bases of ‘adaptor sequence’; this sequence sNcks on to complementary bits of DNA stuck to a surface, which holds the DNA in place while we sequence it: We then flood the DNA with RT ­bases. We also add a polymerase enzyme, which incorporates the RT ­base into the new strand that is complementary to the template strand: We then wash away all the RT ­bases, leaving just those that were incorporated into the new strand; we can read off what base this is by looking at the color of the dye: •  Finally, we send in the cleavage enzyme, which cuts off the terminator region and the dye, leaving a normal base pair. We can then start again to sequence the next base pair. •  In a single Illumina machine we have hundreds of millions of these clusters; cameras look at all of these dots and record how they change color over Nme, allowing you to determine the sequence of bases of millions of bits of DNA at once. •  Sequencing method is actually prepy inefficient, however, the machine is capable of sequencing millions of fragments of DNA at once. Inside the Illumina Machine 51 Flow Cell Imaging A flow cell contains 8 lanes Each lane contains three columns of Nles Each column contains 100 Nles 20K to...
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