Sacks:RAD-seq - Revision history

Lindsay V. Clark: /* References and additional reading */ basics sites

Lindsay V. Clark — Mon, 21 Jan 2013 20:13:58 GMT

References and additional reading: basics sites

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	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]		* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]
	* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]		* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]
		+
		+	===The basics===
		+	* An overview of restriction digestion and ligation: [http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/index.html]
		+	* [[DNA ligation]]
		+	* [[Restriction digest]]

	==Contact==		==Contact==

Lindsay V. Clark: /* References and additional reading */ hohenlohe

Lindsay V. Clark — Thu, 29 Nov 2012 14:46:18 GMT

References and additional reading: hohenlohe

←Older revision		Revision as of 14:46, 29 November 2012
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	* Davey, J. W., Cezard, T., Fuentes-Utrilla, P., Eland, C., Gharbi, K. and Blaxter, M. L. (2012), Special features of RAD Sequencing data: implications for genotyping. Molecular Ecology. [[doi: 10.1111/mec.12084]]		* Davey, J. W., Cezard, T., Fuentes-Utrilla, P., Eland, C., Gharbi, K. and Blaxter, M. L. (2012), Special features of RAD Sequencing data: implications for genotyping. Molecular Ecology. [[doi: 10.1111/mec.12084]]
	* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]		* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]
		+	* Hohenlohe PA, Catchen J, Cresko WA (2012) Population Genomic Analysis of Model and Nonmodel Organisms Using Sequenced RAD Tags. In: Data Production and Analysis in Population Genomics, Pompanon F and Bonin A, eds. 235-260. [[doi:10.1007/978-1-61779-870-2_14]]
	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]		* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]
	* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]		* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]

Lindsay V. Clark: /* References and additional reading */ MEC

Lindsay V. Clark — Fri, 16 Nov 2012 18:01:59 GMT

References and additional reading: MEC

←Older revision		Revision as of 18:01, 16 November 2012
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	* Catchen JM, Amores A, Hohenlohe P, Cresko W, and Postlethwait JH (2011) Stacks: building and genotyping loci de novo from short-read sequences. G3: Genes, Genomes, Genetics 1:171-182. [[doi: 10.1534/g3.111.000240]]		* Catchen JM, Amores A, Hohenlohe P, Cresko W, and Postlethwait JH (2011) Stacks: building and genotyping loci de novo from short-read sequences. G3: Genes, Genomes, Genetics 1:171-182. [[doi: 10.1534/g3.111.000240]]
	* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]		* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]
		+	* Davey, J. W., Cezard, T., Fuentes-Utrilla, P., Eland, C., Gharbi, K. and Blaxter, M. L. (2012), Special features of RAD Sequencing data: implications for genotyping. Molecular Ecology. [[doi: 10.1111/mec.12084]]
	* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]		* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]
	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]		* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]

Lindsay V. Clark: /* References and additional reading */

Lindsay V. Clark — Fri, 16 Nov 2012 16:49:49 GMT

References and additional reading

←Older revision		Revision as of 16:49, 16 November 2012
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	* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]		* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]
	* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]		* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]
-	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and	+	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]
-	Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]	+
	* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]		* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]

Lindsay V. Clark: /* References and additional reading */ peterson

Lindsay V. Clark — Fri, 16 Nov 2012 16:49:31 GMT

References and additional reading: peterson

←Older revision		Revision as of 16:49, 16 November 2012
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	* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]		* Davey JL and Blaxter MW (2010) RADSeq: next-generation population genetics. Briefings in Functional Genomics 9(5):416-423. [[doi:10.1093/bfgp/elq031]]
	* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]		* Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, and Mitchell SE (2011) A robust, simple Genotyping-by-Sequencing (GBS) approach for high diversity species. PLoS One 6(5): e19379. [[doi:10.1371/journal.pone.0019379]]
		+	* Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and
		+	Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. [[doi:10.1371/journal.pone.0037135]]
	* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]		* Serang O, Mollinari M, Garcia AAF (2012) Efficient Exact Maximum a Posteriori Computation for Bayesian SNP Genotyping in Polyploids. PLoS ONE 7(2): e30906. [[doi:10.1371/journal.pone.0030906]]

Lindsay V. Clark: /* Quality control */

Lindsay V. Clark — Tue, 13 Nov 2012 19:29:43 GMT

Quality control

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	[[Image:LVCbioanalyzerExample.jpg \| Expected bioanalyzer results on RADseq libraries using this protocol]]		[[Image:LVCbioanalyzerExample.jpg \| Expected bioanalyzer results on RADseq libraries using this protocol]]
	* Calculate the concentration of the PCR product in nM. Keck supplies a worksheet for this calculation. If <math>x</math> is the concentration in ng/μL, <math>y</math> is the average size in base pairs, and <math>z</math> is the concentration in nM, then <math>z = \frac{10^6*x}{649y}</math>.		* Calculate the concentration of the PCR product in nM. Keck supplies a worksheet for this calculation. If <math>x</math> is the concentration in ng/μL, <math>y</math> is the average size in base pairs, and <math>z</math> is the concentration in nM, then <math>z = \frac{10^6*x}{649y}</math>.
-	* Dilute the purified PCR product to 10 nM.	+	* Dilute the purified PCR product to 10 nM in EB (10 mM Tris).
	* Give 20 μL of 10 nM library to the core facility (Keck). They will use real-time PCR to confirm a concentration of 10 nM. Using Illumina Hi-Seq, do one lane of 100 bp single-end reads.		* Give 20 μL of 10 nM library to the core facility (Keck). They will use real-time PCR to confirm a concentration of 10 nM. Using Illumina Hi-Seq, do one lane of 100 bp single-end reads.

Lindsay V. Clark: /* Bioinformatics */ initial content

Lindsay V. Clark — Tue, 13 Nov 2012 19:20:49 GMT

Bioinformatics: initial content

←Older revision		Revision as of 19:20, 13 November 2012
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	==Bioinformatics==		==Bioinformatics==
		+	We are aligning ''Miscanthus'' sequences to the ''Sorghum'' genome, which can be downloaded at [http://www.phytozome.net/sorghum phytosome.net].
		+
		+	We are primarily using the software package [http://creskolab.uoregon.edu/stacks/ Stacks] for processing the data.
		+
		+	Computation is done on [http://help.igb.uiuc.edu/Biocluster Biocluster], a Unix cluster at the University of Illinois.
		+
		+	Example script for initial processing of the data: [[Media:LVC121113preprocess.txt]]

	==Notes==		==Notes==

Lindsay V. Clark: /* DNA quantification and dilution */

Lindsay V. Clark — Mon, 12 Nov 2012 16:46:04 GMT

DNA quantification and dilution

←Older revision		Revision as of 16:46, 12 November 2012
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	** Allow to dry on the lab bench.		** Allow to dry on the lab bench.
	** Resuspend the DNA in 20 μL TE.		** Resuspend the DNA in 20 μL TE.
		+	** Requantify with Picogreen, then dilute to 50 ng/μL.

	===Restriction digestion and ligation===		===Restriction digestion and ligation===

Lindsay V. Clark: /* DNA quantification and dilution */ precipitation instructions

Lindsay V. Clark — Mon, 12 Nov 2012 16:44:36 GMT

DNA quantification and dilution: precipitation instructions

←Older revision		Revision as of 16:44, 12 November 2012
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	'''Based on the Picogreen concentration estimates, dilute the DNA to 50 ng/μL in 10 mM Tris (and 0.1 mM EDTA, optional).'''		'''Based on the Picogreen concentration estimates, dilute the DNA to 50 ng/μL in 10 mM Tris (and 0.1 mM EDTA, optional).'''
		+
		+	Notes for samples of concentration lower than 50 ng/μL:
		+	* If you have a lot of samples that are '''30-50 ng/μL''', you can dilute all samples for your library to 30 ng/μL or 40 ng/μL instead of 50. The amount of adapter that you add at the ligation step (see below) should be reduced proportionately.
		+	* For samples in the '''10-50 ng/μL''' range, a cheap and efficient way to concentrate them is by [[Purification of DNA \| isopropanol precipitation]]:
		+	** Combine 200 μL DNA sample, 20 μL 3M sodium acetate, and 200 μL isopropanol.
		+	** Mix well by inversion. Place in the freezer for at least an hour.
		+	** Spin down 10 minutes in the centrifuge.
		+	** Pour off the liquid, taking care to keep the pellet.
		+	** Add 200 μL 70% ethanol to rinse. Invert a few times.
		+	** Spin down 1 minute, then pour off the ethanol, again being careful not to lose the pellet.
		+	** Allow to dry on the lab bench.
		+	** Resuspend the DNA in 20 μL TE.

	===Restriction digestion and ligation===		===Restriction digestion and ligation===

Lindsay V. Clark: /* Quality control */ Image

Lindsay V. Clark — Mon, 12 Nov 2012 16:29:25 GMT

Quality control: Image

←Older revision		Revision as of 16:29, 12 November 2012
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	* Quantify the purified PCR product using the Picogreen protocol as above. Expected concentrations are in the 10's of ng/μL.		* Quantify the purified PCR product using the Picogreen protocol as above. Expected concentrations are in the 10's of ng/μL.
	* Run on a DNA 1000 chip on the Bioanalyzer. There should be a smooth curve from around 200 to 500 bp. Any sharp peaks could indicate that the enzymes were cutting in a repetitive region of the genome, in which case it is best to choose different enzymes. Use the Bioanalyzer software to calculate the average fragment size.		* Run on a DNA 1000 chip on the Bioanalyzer. There should be a smooth curve from around 200 to 500 bp. Any sharp peaks could indicate that the enzymes were cutting in a repetitive region of the genome, in which case it is best to choose different enzymes. Use the Bioanalyzer software to calculate the average fragment size.
-	** If there is primer-dimer remaining in the library, it will be visible as a sharp peak at a lower molecular weight than the broad peak for the library.	+	** If there is primer-dimer remaining in the library, it will be visible as a sharp peak at a lower molecular weight than the broad peak for the library. (The library pictured below does not have primer-dimer.)
		+	[[Image:LVCbioanalyzerExample.jpg \| Expected bioanalyzer results on RADseq libraries using this protocol]]
	* Calculate the concentration of the PCR product in nM. Keck supplies a worksheet for this calculation. If <math>x</math> is the concentration in ng/μL, <math>y</math> is the average size in base pairs, and <math>z</math> is the concentration in nM, then <math>z = \frac{10^6*x}{649y}</math>.		* Calculate the concentration of the PCR product in nM. Keck supplies a worksheet for this calculation. If <math>x</math> is the concentration in ng/μL, <math>y</math> is the average size in base pairs, and <math>z</math> is the concentration in nM, then <math>z = \frac{10^6*x}{649y}</math>.
	* Dilute the purified PCR product to 10 nM.		* Dilute the purified PCR product to 10 nM.