lecture_notes:03-31-2010
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lecture_notes:03-31-2010 [2010/04/02 06:16] galt |
lecture_notes:03-31-2010 [2010/04/13 14:24] (current) learithe |
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| **Mate-Pairing** - The chances of damage to DNA increases with time. Hence, there is a high possibility of not getting the accurate sequence data from the other end of DNA. To avoid this, the Mate-Pairing process involves the determination of sequence of DNA from the end towards the beginning and then from the beginning towards the end.\\ | **Mate-Pairing** - The chances of damage to DNA increases with time. Hence, there is a high possibility of not getting the accurate sequence data from the other end of DNA. To avoid this, the Mate-Pairing process involves the determination of sequence of DNA from the end towards the beginning and then from the beginning towards the end.\\ | ||
| - | |||
| - | ==== Alternative Notes ==== | ||
| - | |||
| - | === High Level Overview === | ||
| - | |||
| - | The high level sequencing workflow for all next gen tools is as follows: | ||
| - | Fragment Sample -> Amplify Fragments -> Sequence Fragments | ||
| - | |||
| - | There are two main flavors of next generation sequencing technology: | ||
| - | * Sequencing by Synthesis (SBS) | ||
| - | * Hybridization | ||
| - | |||
| - | === Examination of Individual Technologies === | ||
| - | |||
| - | There are four technologies that were talked about today. | ||
| - | * Pyrosequencing (454) | ||
| - | * solid | ||
| - | * Illumina/solexa | ||
| - | * Ion Torrent | ||
| == Pyrosequencing (SBS) == | == Pyrosequencing (SBS) == | ||
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| After each nucleotide is added, you must wash away the nucleotides and start fresh before adding the next nucleotide and recording which positions on the plate light up in that case. | After each nucleotide is added, you must wash away the nucleotides and start fresh before adding the next nucleotide and recording which positions on the plate light up in that case. | ||
| - | There is a problem with multiple A's because of the modified nucleotides which are needed for this reaction. FIXME | + | There is a problem with stretches of identical nucleotides. Multiple identical nucleotides will be incorporated simultaneously, and the sequencer must use the degree of light intensity to estimate the number of nucleotides incorporated; this calculation is prone to error. This problem, referred to as the "homopolymeric read problem", is especially prounced for adenine (A). |
| You must get the amount of template on the beads just right. Must be all the same type. If too much template is present the read length is shortened (not sure why). If too few template is on the beads then the washing step will take away a significant amount of template and you won't have enough of a signal. | You must get the amount of template on the beads just right. Must be all the same type. If too much template is present the read length is shortened (not sure why). If too few template is on the beads then the washing step will take away a significant amount of template and you won't have enough of a signal. | ||
lecture_notes/03-31-2010.1270188968.txt.gz ยท Last modified: 2010/04/02 06:16 by galt
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