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guide_to_amplicon_sequencing_experiment [2013/04/11 11:17]
anniearchambault
guide_to_amplicon_sequencing_experiment [2013/04/30 13:16] (current)
anniearchambault
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 ==== From Soil to Sequence Submission - A guide to amplicon sequencing experiments ====  ==== From Soil to Sequence Submission - A guide to amplicon sequencing experiments ==== 
  
-A guide prepare by Terrence Bell, postdoctoral fellow at [[http://​www.irbv.umontreal.ca/​recherche/​initiatives-majeures/​genorem|projet GenoRem]], [[http://​www.irbv.umontreal.ca/​etudiants|Institut de recherche en biologie végétale ]]+A guide prepare by Terrence Bell, postdoctoral fellow at [[http://​www.irbv.umontreal.ca/​recherche/​initiatives-majeures/​genorem|projet GenoRem]], [[http://​www.irbv.umontreal.ca/​etudiants|Institut de recherche en biologie végétale]]
  
 Terminology note: the sequence data output by massively parallel sequencing instruments (Roche 454, Illumina HiSeq, IonTorrent…) are commonly termed “reads”. In this guide, “sequences” and “reads” are synonymous.  ​ Terminology note: the sequence data output by massively parallel sequencing instruments (Roche 454, Illumina HiSeq, IonTorrent…) are commonly termed “reads”. In this guide, “sequences” and “reads” are synonymous.  ​
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 == DNA/RNA extraction== == DNA/RNA extraction==
  
-To make things easy, use MoBiodistributed by [[https://​ca.vwr.com/​|VWR in Canada]]. ​ It’s well-tested,​ and results compare easily to most of the literature. ​ You can even do dual extractions of DNA/RNA if you need both.+To make things easy, use MoBio distributed by [[https://​ca.vwr.com/​|VWR in Canada]]. ​ It’s well-tested,​ and results compare easily to most of the literature. ​ You can even do dual extractions of DNA/RNA if you need both.
  
 If you have a ton of samples and money is a concern, I have also used the following protocol successfully:​ If you have a ton of samples and money is a concern, I have also used the following protocol successfully:​
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 Roche 454 Roche 454
  
-  * At [[http://​gqinnovationcenter.com/​services/​sequencing/​technoMPSRocheGSFLX.aspx?​l=e|Genome Quebec]] ​standard ​use for pooled amplicon experiments+  * At [[http://​gqinnovationcenter.com/​services/​sequencing/​technoMPSRocheGSFLX.aspx?​l=e|Genome Quebec]] ​ 
 +  * Standard ​use for pooled amplicon experiments
   * Usually 1.5 million or more reads per run   * Usually 1.5 million or more reads per run
   * Plate can be split into 8 regions   * Plate can be split into 8 regions
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   - PCR amplify your samples   - PCR amplify your samples
-  - Purify the amplified sample by extracting only the band of interest from a gel, and processing with a gel purification kit. +  - Purify the amplified sample by extracting only the fragments of the expected size. This can be done by extracting ​band of interest from a gel, and processing with a gel purification kit, but other methods for size fragmentation exist
-  - Quantify your samples using Picogreen or Qubit (not NanoDrop as it is not precise).+  - Quantify your samples using Picogreen or Qubit (not NanoDropas it is not precise).
   - Pool your samples in an equimolar ratio. ​ Currently Genome Quebec requests a bare minimum of 450 ng in 30 ul for an MID library. Be sure to check with them for changes in their protocol.   - Pool your samples in an equimolar ratio. ​ Currently Genome Quebec requests a bare minimum of 450 ng in 30 ul for an MID library. Be sure to check with them for changes in their protocol.