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lecture_notes:03-30-2011 [2011/03/30 16:05]
eyliaw
lecture_notes:03-30-2011 [2011/04/01 12:20] (current)
svohr [Coverage] slight corrections
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 Inputs: ​ Sequencing data from various machines. ​ Some of the characteristics of these machines/​techniques:​ Inputs: ​ Sequencing data from various machines. ​ Some of the characteristics of these machines/​techniques:​
-===== Sanger capillary =====+==== Sanger capillary ==== 
 +  * ~800bp reads[(cite:​wikisanger>​http://​en.wikipedia.org/​wiki/​Microfluidic_Sanger_sequencing)]. 
 +  * Q (quality value) ~30 
 +  * ~$1/read, expensive because primers must be attached to each read. 
 +==== 454 ==== 
 +  * ~400bp reads[(cite:​wiki454>​http://​en.wikipedia.org/​wiki/​454_Life_Sciences)]. 
 +  * Pyrosequencing 
 +  * Q ~20 
 +  * $5000/​run/​1M reads, no downscaling (numbers approximate). 
 +==== SoLiD ==== 
 +  * 2x25bp or 1x50bp reads 
 +  * Paired end reads: ​ ligation with adapter, cleaves 25bp from adapter using restriction enzyme. 
 +  * Potential for double ligation: two unrelated sequences ligating. 
 +  * $2000/​run/​100M reads (numbers approximate). 
 +==== Illumina ==== 
 +  * 2x50, 2x100bps ? 
 +  * Paired end reads 
 +  * Potential errors: innies (ligated region not between sequenced regions) or chimeric (sequence passes ligated region) 
 +  * Cheaper than SoLiD, 10K Genomes project uses it. 
 +==== Ion Torrent ==== 
 +  * 2x100 base pairs 
 +  * ~50,000 to 5,000,000 reads depending on Sequencing Chip [(cite:​ionTorrent>​http://​www.iontorrent.com/​technology-how-does-it-perform/​)]. 
 +  * Ion semiconductor sequencing. No optics or modified bases are required. 
 +==== Pac Bio ==== 
 +  * Very long, single molecule reads (~10K) 
 +  * High error rates (~5%) 
 +  * Useful when mapping to a reference. 
 +===== Coverage ===== 
 +We briefly discussed how much sequence data would be required to assemble the genome. First, we considered the probability of seeing a particular base ''​i''​ in a single read ''​j''​. 
 +   
 +  P( seeing base i in read j ) L/G
  
-454 +where ''​L''​ is the read length and ''​G''​ is the total size of the genome. If we have ''​R''​ reads, then  
-SoLiD + 
-Illumina +  P( never seeing base i ) = (1 - L/G)^R 
-Ion Torrent + 
-Pac Bio+We can multiply ''​L/​G''​ by ''​R/​R''​ to get ''​((L*R) / G) / R''​ or ''​C / R''​ where ''​C''​ is our coverage of the genome. We take the limit of this as 
 +''​R''​ goes to infinity: 
 + 
 +  lim n->inf (1 - C/R)^R = e^-C 
 + 
 +Thus we can expect to miss ''​G*e^-C''​ bases. 
 + 
 +We cannot assemble an entire chromosome if we are missing bases. However, we can construct contiguous stretches of bases or //contigs// and later 
 +assemble them into //​scaffolds//​ using other information,​ such as long distance physical maps. 
 + 
 + 
 + 
 +===== References ===== 
 +<​refnotes>​notes-separator:​ none</​refnotes>​ 
 +~~REFNOTES cite~~
lecture_notes/03-30-2011.1301526357.txt.gz · Last modified: 2011/03/30 16:05 by eyliaw