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--- aflp_454seq_for_pop_structure [2012/01/23 22:36]
simonjoly
+++ aflp_454seq_for_pop_structure [2012/02/02 15:51] (current)
anniearchambault
@@ Line 9: / Line 9: @@
 === DNA extraction ===
-An amount of 10 microgramm of dried leaves was ground for one minute in a microcentrifuge tube with one tungsten bead in the [[http://www.qiagen.com/products/tissuelyserii.aspx|TissuLyser (Qiagen)]]. Total DNA was extracted using EZ-10 Spin Column Genomic DNA kits for Plant Samples (BioBasics [[http://www.biobasic.com/products.jsp?productCode=BS425&productName=&cas=&haz=-1&classID=2&rd=0.4384178700324832|catalog number BS425-50]]) as recommended by the manufacturer. Quality and quantity of total DNA was evaluated by gel electrophoresis and by optical density measurement.
+An amount of 10 milligramm of dried leaves was ground for one minute in a microcentrifuge tube with one tungsten bead in the [[http://www.qiagen.com/products/tissuelyserii.aspx|TissuLyser (Qiagen)]]. Total DNA was extracted using EZ-10 Spin Column Genomic DNA kits for Plant Samples (BioBasics [[http://www.biobasic.com/products.jsp?productCode=BS425&productName=&cas=&haz=-1&classID=2&rd=0.4384178700324832|catalog number BS425-50]]) as recommended by the manufacturer. Quality and quantity of total DNA was evaluated by gel electrophoresis and by optical density measurement.
 === Genome complexity reduction ===
-A modified AFLP strategy, inspired by the [[http://www.keygene.com/services/technologies_CRoPS.php|Crops technology]]((van Orsouw, N. J. et al. (2007). Complexity Reduction of Polymorphic Sequences (CRoPSTM): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes. [[http://dx.plos.org/10.1371/journal.pone.0001172|PLoS ONE 2, e1172.]])) (AFLP and CRoPS are registered trademarks of Keygene N.V.) and a published study ((Gompert, Z., Forister, M. L., Fordyce, J. A., Nice, C. C., Williamson, R. J., and Alex Buerkle, C. (2010). Bayesian analysis of molecular variance in pyrosequences quantifies population genetic structure across the genome of Lycaeides butterflies. [[http://doi.wiley.com/10.1111/j.1365-294X.2010.04666.x|Molecular Ecology, 19, 2455-2473]])) was applied to //Panax quinquefolius// total DNA, in order to efficiently discover sequence polymorphism in a wide and random range of the whole genome, but without actually sequencing the whole genome. One of the assumptions of this AFLP-like method is that restriction sites within the genome are conserved among populations. The steps are as follow:
+A modified AFLP strategy, inspired by the [[http://www.keygene.com/services/technologies_CRoPS.php|Crops technology]]((van Orsouw, N. J. et al. (2007). Complexity Reduction of Polymorphic Sequences (CRoPSTM): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes. [[http://dx.plos.org/10.1371/journal.pone.0001172|PLoS ONE 2, e1172.]])) (AFLP and CRoPS are registered trademarks of Keygene N.V.) and a published study ((Gompert, Z., Forister, M. L., Fordyce, J. A., Nice, C. C., Williamson, R. J., and Alex Buerkle, C. (2010). Bayesian analysis of molecular variance in pyrosequences quantifies population genetic structure across the genome of //Lycaeides// butterflies. [[http://doi.wiley.com/10.1111/j.1365-294X.2010.04666.x|Molecular Ecology, 19, 2455-2473]])) was applied to //Panax quinquefolius// total DNA, in order to efficiently discover sequence polymorphism in a wide and random range of the whole genome, but without actually sequencing the whole genome. One of the assumptions of this AFLP-like method is that restriction sites within the genome are conserved among populations. The steps are described in the following paragraphs.
 == Restriction-digestion of total DNA ==
@@ Line 56: / Line 56: @@
 <WRAP clear></WRAP>
-<WRAP box>{{:ligation_mix_.jpg|Ligation Mix}}<WRAP box>**Figure 1** Pictogram of the DNA fragments involved for ligating double stranded adaptors to DNA previously digested with EcoRI and MseI restriction enzymes, in the context of a modified AFLP method for genome complexity reduction.</WRAP></WRAP>
+<WRAP box 800px>{{:ligation_mix_.jpg|Ligation Mix}}<WRAP box>**Figure 1** Pictogram of the DNA fragments involved for ligating double stranded adaptors to DNA previously digested with EcoRI and MseI restriction enzymes, in the context of a modified AFLP method for genome complexity reduction.</WRAP></WRAP>
 <WRAP clear></WRAP>
 == Selective amplification by PCR using primers specific to the adaptor sequence ==
-The purpose of this step was to amplify only a small proportion of the total genome, thereby reducing the complexity of the nucleotides fragments pool to be sequenced. In this step, the only the genomic fragments amplified were those that had a EcoR1 site on one side, and a Mse1 site on the other side, and additionally only those fragments that end by a C on the EcorR1 side and by a AC on the Mse1 side. The selective primers enabled what is termed a selective amplification. Because the selective primers were design to also carry the MID (multiplex identifiers) barcodes, and the LibL segments used for the pyrosequencing step, the selective amplification is described in more details in the next section. Figure 2 illustrates the DNA fragments involved in selective amplification.
+The purpose of this step was to amplify only a small proportion of the total genome, thereby reducing the complexity of the nucleotides fragments pool to be sequenced. The term selective refers to addition of one or two nucleotides at the 3' end of the adaptor-specific primers. This way, primers will amplify only a subset of the fragments that exist in the digested-ligated genome. These types of primers are termed selective primers. The only genomic fragments amplified in the present selective amplification were those that, in addition to having a EcoR1 site or a Mse1 site on each end of the sample DNA, also ended by a C on a EcorR1 side and by a AC on a Mse1 side. Because the selective primers were design to also carry the MID (multiplex identifiers) barcodes, and the LibL segments used for the pyrosequencing step, the selective amplification is described in more details in the next section. Figure 2 illustrates the DNA fragments involved in selective amplification.
 === Pooling, multiplexing and barcoding samples for high throughput sequencing ===
@@ Line 81: / Line 81: @@
 Digested DNA samples were amplified in a PCR reaction where the reverse primer is LibL_B_MseI_plus2 for all tubes, and the forward primer is specific to a population (Table 4). However, since the objective of the study was to reveal the genetic diversity at the population level rather than between each individual, the ten //Panax quinquefolius// samples for each population were all labeled with a same set of barcoded population-specific primers. Each sample was however amplified separately prior to pooling, to ensure an equimolar representation in the pool. Figure 2 illustrates the DNA fragments involved in the selective amplification step.
-<WRAP box>{{:selective_amplification_mix.jpg?1000|Selective amplification pictogram}}
+<WRAP box 800px>{{:selective_amplification_mix.jpg?700|Selective amplification pictogram}}
 <WRAP box>**Figure 2** Pictogram of the DNA fragments involved in selective amplification of previously digested-ligated DNA, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing. The selective primers therefore also contain a barcode (MID), and an instrument specific region (Libl-A and Key), in addition to the template specific region (EcoRI).</WRAP></WRAP>
 <WRAP clear></WRAP>
-Selective amplifications were performed with a highly accurate proofreading enzyme (iProof polymerase, BioRad, [[http://www.bio-rad.com/prd/en/CA/adirect/biorad?cmd=BRCatgProductDetail&productID=201001|catalog number 172-5301]]), to minimize risks of spurious single nucleotides polymorphisms that would be due to misincorporation of a nucleotide rather than genuine allelic variant. Reaction mix is given in Table 5, and cycling conditions in Table 6. Figure 3 shows an example of
+Selective amplifications were performed with a highly accurate proofreading enzyme (iProof polymerase, BioRad, [[http://www.bio-rad.com/prd/en/CA/adirect/biorad?cmd=BRCatgProductDetail&productID=201001|catalog number 172-5301]]), to minimize risks of spurious single nucleotides polymorphisms that would be due to misincorporation of a nucleotide rather than genuine allelic variant. Reaction mix is given in Table 5, and cycling conditions in Table 6. Figure 3 shows an example of an agarose gel electrophorese of selective-amplification products, using [[http://www.neb.ca/detail.php?id=N3014|Lambda BstEII]] as molecular ladder.
 <WRAP box 500px>**Table 5** Reaction mix for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing.
 ^ Reagent    ^ Initial conc.    ^ Qty added    ^ Final conc. or Final qty    ^
-^ HF Buffer    | 5X includes (15 mM MgCl2) | 6 µl  | 1X    |
+^ HF Buffer    | 5X (includes 15 mM MgCl2) | 6 µl  | 1X    |
 ^ MgCl2    | 50 mM    | 0.6  µl    | 2.5 mM    |
 ^ dNTP     | 10 mM           | 0.6 µl         | 200 µM        |
@@ Line 100: / Line 100: @@
 </WRAP>
-<WRAP box 400px>{{:gel_amplif_12juillet2011.jpg?400|Gel picture}}**Figure 3** Electrophoresis of  from a selective amplification of digested-ligated //Panax quinqefolius// total DNA with selective primers, which have a MID barcode tail, for a modified AFLP method for genome complexity reduction coupled to a high throughput sequencing. Lane 1: [[http://www.neb.ca/detail.php?id=N3014|Lambda BstEII ladder]]; Lane 2: ?</WRAP>
+<WRAP box 500px>{{:amplify_digested_ligated_library.jpg?400|Gel picture for selective amplification of prepared library for AFLP-like method}}**Figure 3** Electrophoresis of the product of selective amplification of digested-ligated //Panax quinqefolius// total DNA with selective primers, which have a MID barcode tail, for a modified AFLP method for genome complexity reduction coupled to a high throughput sequencing. Two different MgCl2 concentrations were tested, and different temperatures for the primer annealing step. Lane 1: [[http://www.neb.ca/detail.php?id=N3014|Lambda BstEII ladder]]; Lane 2: 57 °C; Lane 3: 61.8 °C; Lane 4: 65.5; Lane 5: 68.7 °C; Lane 6: 57 °C; Lane 7: 61.8 °C; Lane 8: 65.5 °C; Lane 9: 68.7 °C</WRAP>
 <WRAP clear></WRAP>
 <WRAP box 500px>**Table 6** Cycling conditions for selective amplifications, in the context of a modified AFLP method for genome complexity reduction coupled to multiplexing samples for high throughput sequencing.
-^ Step      ^ Temperature (°C )       ^ Time          ^
+^ Step      ^ Temperature (°C)       ^ Time          ^
 | Initial denaturation    | 98   | 2 min    |
 ^ 30 cycles    ^     ^     ^