"description":"This template shows how to generate a SNP markers panel with the preparation of SNP panel.\r\nSingle nucleotide polymorphisms (SNPs) markers are widely used for genomic research and breeding. Development of a panel of diagnostic SNP markers is used for the genotyping as well as the identification of hybrids and interspecies introgression events.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>PCR conditions:</p>\n<ul>\n<li>95°C for 1 min</li>\n<li>35 cycles: 95°C for 15 s</li>\n<li>56°C for 15 s</li>\n<li>72°C for 22 s</li>\n</ul>\n<br>PCR products run at <strong>60 V</strong> for <strong>40 mins</strong> on a<strong> 2%</strong> agarose gel (<strong>0.5× TAE</strong>, stained with <strong>100 ng/μL</strong> EtBr).</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Libraries can be quantifiedby fluorimetry and calibrated by sequencing.</p></body></html>"
},
"position":0
},
{
"checklist":{
"checklist":{
"id":960,
"name":"Guidelines:",
"step_id":4422,
"created_at":"2018-12-21T10:22:55.963Z",
"updated_at":"2018-12-21T10:22:55.963Z",
"created_by_id":null,
"last_modified_by_id":null
},
"checklist_items":[
{
"id":3950,
"text":"Size selection (100 to 800 bp) by agarose gel electrophoresis.",
"checked":false,
"checklist_id":960,
"created_at":"2018-12-21T10:22:55.965Z",
"updated_at":"2018-12-21T10:22:55.965Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3951,
"text":"Gel purification, end repair, dA overhang addition, P2 paired-end adapter ligation and library amplification.",
"checked":false,
"checklist_id":960,
"created_at":"2018-12-21T10:22:55.972Z",
"updated_at":"2018-12-21T10:22:55.972Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3952,
"text":"120 μL of each amplified library is size-selected (about 250 to 500 bp) by gel electrophoresis.",
"checked":false,
"checklist_id":960,
"created_at":"2018-12-21T10:22:55.981Z",
"updated_at":"2018-12-21T10:22:55.981Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
},
{
"id":3953,
"text":"Final libraries are put through quality control and high-throughput sequencing.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>The RAD library was prepared as originally described in<a href=\"https://www.ncbi.nlm.nih.gov/pubmed/18852878/\" target=\"_blank\" rel=\"noopener\">article</a>.<br><br>At least <strong>30</strong> specimens need to be chosen.<br><br><strong>Each sample is digested at 37°C for 40 min with restriction enzyme:</strong></p>\n<ul>\n<li>\n<strong>6 U</strong> <em>restriction enzyme</em>per μg genomic DNA in <strong>1×</strong> Reaction Buffer 4 at a final concentration of about <strong>1 μg</strong> DNA per <strong>50 μL</strong> reaction volume</li>\n<li>The samples are heat-inactivated at <strong>65°C</strong> for <strong>20 min</strong>.</li>\n</ul>\n<br><strong>Individual-specific P1 adapters, each with a unique 5 or 7 bp barcode isligated to thedigested DNA at 22°C for 15 min by adding:</strong><br><br>\n<ul>\n<li>0.6 μL (DNA samples) 100 nmol/L P1 adapter</li>\n<li>0.15 μL 100 mmol/L rATP</li>\n<li>0.25 μL 10× Reaction Buffer 2</li>\n<li>0.125 μL T4 ligase</li>\n<li>reaction volumes made up to 15 μL with nuclease-free water</li>\n</ul>\n<br>After heat-inactivation, the ligation reactions are slowly cooled to room temperature then combined in appropriate multiplex pools.</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Reads of low qualitymust be discarded. <br>Retained reads are sorted into loci and genotypes using software developed for that purpose. It will assigns loci using a likelihood-based algorithm to separate actual SNPs from SNPs likely to have arisen from sequencing error. <br><br><strong>Parameters:</strong><br>\n</p>\n<ul>\n<li>De novo assembly parameters.</li>\n<li>Minimum stack depth of 5.</li>\n<li>Maximum of 2 mismatches.</li>\n<li>No more than 1 mismatch between alleles.</li>\n</ul>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Sequencing data from filtered Informative RAD markers is combined into a single alignment of alleles in RAD library construction. Data analysis is carried out using R and an associated R/<em>adegenet</em>package for Principal Component Analysis (PCA) and Discriminant Analysis of Principal Components (DAPC).<em><strong> PCA creates simplified models of the total variation within the dataset.</strong> <strong>DAPC identifies clusters of genetically related individuals.</strong></em></p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Reading the fluorescence emission of the FAM and HEX fluorophores for each sample, in comparison to no-template control reactions, using a Real Time PCR Thermal Cycler and Quansoft endpoint genotyping software.</p></body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>SNP of interest at a given locus need to be at least <strong>20 bp</strong> from the end of a given sequence, to allow for primer design. SNP assays were designed and manufactured for use with KASP genotyping technology.<br><br><strong>Optimisation assay conditions:</strong></p>\n<ul>\n<li>94°C for 15 min;</li>\n<li>10 cycles 94°C for 20 s, 61–55°C for 120 s (0.6°C drop per cycle)</li>\n<li>40 cycles 94°C for 20 s, 55°C for 120 s</li>\n</ul>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Here are the guidelines for protein extraction:</p></body></html>"
"text":"Add 1 volume of chloroform: Isoamyl alcohol to the solution and mix by inversion for 5min. Centrifuge the sample for 10min at 5000 × g and pipette the upper aqueous phase into a new Falcon tube.",
"checked":false,
"checklist_id":958,
"created_at":"2018-12-21T09:50:59.141Z",
"updated_at":"2018-12-21T09:50:59.141Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3940,
"text":"Add 5μL of RNAse A to the solution and incubate at 37°C for 15min with periodic, gentle mixing.",
"checked":false,
"checklist_id":958,
"created_at":"2018-12-21T09:50:59.149Z",
"updated_at":"2018-12-21T09:50:59.149Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3941,
"text":"After incubation, add 1 volume of chloroform: Isoamyl alcohol to the solution and mix by inversion for 5min.",
"checked":false,
"checklist_id":958,
"created_at":"2018-12-21T09:50:59.156Z",
"updated_at":"2018-12-21T09:50:59.156Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
},
{
"id":3942,
"text":"Centrifuge the solution for 10min at 5000 × g and pipette the aqueous phase into a new Falcon tube.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Looking for a single absorbance peak at <strong>260 nm</strong>, a <strong>260/280</strong> absorbance ratio of <strong>1.8-2.0</strong> and no evidence of substantial band shearing or contamination (either RNA or polysaccharide).<br><br></p>\n<p><strong>Assess the quality of the extracted DNA:</strong></p>\n<ol>\n<li>NanoDrop UV/Vis spectrophotometer</li>\n<li>0.7% (w/v) agarose gel electrophoresis</li>\n</ol>\n</body></html>"
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Guidelinesfor samples preparation below.</p></body></html>"
"text":"Samples are stored in 95% ethanol solution at -20°C.",
"checked":false,
"checklist_id":957,
"created_at":"2018-12-21T09:43:55.389Z",
"updated_at":"2018-12-21T09:43:55.389Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":0
},
{
"id":3935,
"text":"Pre-heat water baths (65°C and 37°C).",
"checked":false,
"checklist_id":957,
"created_at":"2018-12-21T09:43:55.396Z",
"updated_at":"2018-12-21T09:43:55.396Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":1
},
{
"id":3936,
"text":"Prepare 10mL (per 1g of sample) extraction buffer by adding 0.3% (v/v) β-mercaptoethanol in a 50mL Falcon tube, and pre-heat in the 65°C water bath.",
"checked":false,
"checklist_id":957,
"created_at":"2018-12-21T09:43:55.403Z",
"updated_at":"2018-12-21T09:43:55.403Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":2
},
{
"id":3937,
"text":"Put the sample into the 65°C water bath andmix by inversion every 10 min for 30-60 minutes.",
"checked":false,
"checklist_id":957,
"created_at":"2018-12-21T09:43:55.412Z",
"updated_at":"2018-12-21T09:43:55.412Z",
"created_by_id":null,
"last_modified_by_id":null,
"position":3
},
{
"id":3938,
"text":"After incubation, centrifuge the sample tube for 5 min at 5000 × g and decant the supernatant into a new 50 mL Falcon tube.",
"text":"<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>If you are precipitating small volumes of DNA, and you can fit the required amount of solvent into the sample tube, then ice-cold ethanol is the preferred choice. You can chill it(in liquid nitrogen or at <strong>–80°C</strong>) to accelerate the precipitation without the risk of precipitating excess salt. Afterwards, you need towash the pellet with <strong>70%</strong> ethanol to remove any salt present.</p></body></html>"
