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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 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. | + | 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 NanoDrop, as 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. | ||
