Name: competitive genomic screens of barcoded yeast libraries
Uploaded: 2011-08-11T14:00:00
Duration: 11 min 59 s
Description: Watch this Scientific Journal Video about Competitive Genomic Screens of Barcoded Yeast Libraries at JoVE.com

We thank Ron Davis, Adam Deutschbauer, and the entire HIP HOP laboratory at the University of Toronto for discussions and advice. C.N. is supported by grants from the National Human Genome Research Institute (Grant Number HG000205), RO1 HG003317, CIHR MOP-84305, and Canadian Cancer Society (#020380). J.O. was supported by the Stanford Genome Training Program (Grant Number T32 HG00044 from the National Human Genome Research Institute) and the National Institutes of Health (Grant Number P01 GH000205). GG is supported by the NHGRI RO1 HG003317 and the Canadian Cancer Society, Grant # 020380, TD and the Donnelly Sequencing Centre is supported in part by grants from the Canadian Foundation for Innovation to Drs. Brenda Andrews and Jack Greenblatt. A.M.S. is supported by a University of Toronto Open Fellowship.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+haploinsufficiency+revealed+by+genome-wide+profiling+in+yeast.">Mechanisms of haploinsufficiency revealed by genome-wide profiling in yeast.</a> Genetics. 169, 1915-1925 (2005).</li><li>Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemogenomic+profiling:+identifying+the+functional+interactions+of+small+molecules+in+yeast.">Chemogenomic profiling: identifying the functional interactions of small molecules in yeast.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 793-798 (2004).</li><li>Lum, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+modes+of+action+for+therapeutic+compounds+using+a+genome-wide+screen+of+yeast+heterozygotes.">Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.</a> Cell. 116, 121-137 (2004).</li><li>Hillenmeyer, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+genomic+portrait+of+yeast:+uncovering+a+phenotype+for+all+genes.">The chemical genomic portrait of yeast: uncovering a phenotype for all genes.</a> Science. 320, 362-365 (2008).</li><li>Oh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+TagModule+collection+for+parallel+genetic+analysis+of+microorganisms.">A universal TagModule collection for parallel genetic analysis of microorganisms.</a> Nucleic acids research. 38, e146-e146 (2010).</li><li>Oh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+annotation+and+drug+target+discovery+in+Candida+albicans+with+a+tagged+transposon+mutant+collection.">Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection.</a> PLoS pathogens. 6, (2010).</li><li>Nislow, C., Giaever, G., Stark, I., Stansfields, M. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+387.">Chapter 387.</a> Yeast Gene Analysis. , 387-414 (2007).</li><li>Pierce, S. E., Davis, R. W., Nislow, C., Giaever, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+barcoded+Saccharomyces+cerevisiae+gene-deletion+mutants+in+pooled+cultures.">Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures.</a> Nature protocols. 2, 2958-2974 (2007).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phenotyping+via+deep+barcode+sequencing.">Quantitative phenotyping via deep barcode sequencing.</a> Genome Res. , (2009).</li><li>Hamady, M., Walker, J. J., Harris, J. K., Gold, N. J., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Error-correcting+barcoded+primers+for+pyrosequencing+hundreds+of+samples+in+multiplex.">Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex.</a> Nature. 5, 235-237 (2008).</li><li>Gresham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-Level+Analysis+of+Genes+and+Functions+Affecting+Survival+During+Nutrient+Starvation+in+Saccharomyces+cerevisiae.">System-Level Analysis of Genes and Functions Affecting Survival During Nutrient Starvation in Saccharomyces cerevisiae.</a> Genetics. 187, 299-317 (2011).</li></ol>

No conflicts of interest declared.

Here, we outlined a protocol that, with modest modification, can easily be adapted to a wide range of existing collections of barcoded mutant collections of different microorganisms to create tagged mutant collections. We emphasize that while we have reported a protocol on the tagged transposon mutagenesis for the pathogenic yeast C. albicans, a very similar protocol could be adapted to a wide variety of unicellular fungi. Modified, this protocol works well in bacteria 13, and currently collections for a number of additional fungal and bacterial genomes are under construction. At present, this assay provides the only comprehensive, genome-wide unbiased screen for gene-small molecule interactions. One particularly compelling feature of the assay is that no prior knowledge of the gene or small molecule is required. Despite the scope and power of these assays, their transferability to other labs has been hindered somewhat by the initial capital investment and informatics tools for the analysis of the results. With the adoption of a next generation sequence readout combined with robust tools for analysis, we expect their adoption to increase.

<table border="1">
	<tbody>
 <tr>
 <td>Antibiotics</td>
 <td>Vendor and catalogue numbers</td>
 </tr>
 <tr>
 <td>Carbenicillin</td>
 <td>Sigma, part# C1613</td>
 </tr>
 <tr>
 <td>Kanamycin</td>
 <td>Sigma, part# K1876</td>
 </tr>
 <tr>
 <td>spectinomycin </td>
 <td>Sigma, part# S0692</td>
 </tr>
 <tr>
 <td>Chloramphenicol </td>
 <td>Sigma, part# C0378</td>
 </tr>
 <tr>
 <td>DNA Clean-up and concentration kits</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QIAprep Spin Miniprep Kit</td>
 <td>Qiagen, part# 27106</td>
 </tr>
 <tr>
 <td>HiSpeed Plasmid Maxi Kit</td>
 <td>Qiagen, part# 12663</td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen, part# 28106</td>
 </tr>
 <tr>
 <td>QIAquick Gel Extraction Kit</td>
 <td>Qiagen, part# 28704</td>
 </tr>
 <tr>
 <td>PCR and electrophoresis reagents</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Taq DNA Polymerase (Mg-free) Buffer</td>
 <td>New England Biolabs, part# M0320L</td>
 </tr>
 <tr>
 <td>Deoxynucleotide Solution Mix</td>
 <td>New England Biolabs, part# N0447L</td>
 </tr>
 <tr>
 <td>25 mM MgCl2</td>
 <td>Sigma, part# 63036</td>
 </tr>
 <tr>
 <td>Agarose, loading dye, and nucleic acid stain suitable for gel electrophoresis</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>10X TAE buffer</td>
 <td>Sigma, part# T8280</td>
 </tr>
 <tr>
 <td>1 Kb Plus DNA Ladder</td>
 <td>Invitrogen, part# 10787026</td>
 </tr>
 <tr>
 <td>YPD broth</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 g of yeast extract</td>
 <td>Sigma, part# Y1625</td>
 </tr>
 <tr>
 <td>20 g Bacto peptone </td>
 <td>BD Biosciences, part# 211677</td>
 </tr>
 <tr>
 <td>20 g of dextrose </td>
 <td>Sigma, part# D9434</td>
 </tr>
 <tr>
 <td>Labware</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Multichannel pipettes (1000, 200, and 20 &mu;L)</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Disposable pipetting reservoirs</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>15 and 50 mL centrifuge tubes </td>
 <td>Various</td>
 </tr>
 <tr>
 <td>96- and 384-Well Deep Well Plates</td>
 <td>Axygen Scientific, part# P-2ML-SQ-C-S &amp; P-384240SQCS</td>
 </tr>
 <tr>
 <td>96-well and 384-well PCR plates and seal film</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Plate roller for sealing multi-well plates</td>
 <td>Sigma, part# R1275</td>
 </tr>
 <tr>
 <td>30&deg;C and 37&deg;C shaking incubators for growing bacterial and yeast on plates and in tubes </td>
 <td>Various</td>
 </tr>
 <tr>
 <td>In vitro transposon mutagenesis</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EZ-Tn5 Transposase</td>
 <td>Epicentre Biotechnologies, part# TNP92110</td>
 </tr>
 <tr>
 <td>High-throughput transformation</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Seqprep 96 HT Plasmid Prep Kit</td>
 <td>Edge Biosystems, part# 84359</td>
 </tr>
 <tr>
 <td>polyethylene glycol, molecular weight 3350</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>lithium acetate </td>
 <td>Sigma, part# 517992</td>
 </tr>
 <tr>
 <td>6-well plates, sterile </td>
 <td>Corning, part# 3335</td>
 </tr>
 <tr>
 <td>50 mg/mL uridine</td>
 <td>Sigma, part# U3750</td>
 </tr>
 <tr>
 <td>100X Tris-EDTA buffer solution </td>
 <td>Sigma, part# T9285</td>
 </tr>
 <tr>
 <td>1X TE/0.1M LiOAc</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>salmon testis DNA </td>
 <td>Sigma, part# 1626</td>
 </tr>
 <tr>
 <td>Growth of barcoded collections</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>48-well plates; if growing cultures in plates</td>
 <td>Greiner, part# M9437</td>
 </tr>
 <tr>
 <td>Adhesive plate seals</td>
 <td>ABgene, part# AB-0580</td>
 </tr>
 <tr>
 <td>200 mL culture flasks</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Spectrophotometer capable of absorbance </td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Temperature-controlled shaker for 250 ml flasks or shaking spectrophotometer </td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Safe-Lock Microcentrifuge Tube, 2 mL </td>
 <td>Eppendorf, part# 0030 120.094</td>
 </tr>
 <tr>
 <td>Hybridization equipment</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hybridization Oven 640 </td>
 <td>Affymetrix, part# 800138</td>
 </tr>
 <tr>
 <td>GeneChip Fluidic Station 450 </td>
 <td>Affymetrix, part# 00-0079</td>
 </tr>
 <tr>
 <td>GeneArray Scanner 3000</td>
 <td>Affymetrix, part# 00-0212</td>
 </tr>
 <tr>
 <td>Boiling water bath with floating rack </td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Hybridization consumables</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Genflex Tag 16K Array v2</td>
 <td>Affymetrix, part# 511331</td>
 </tr>
 <tr>
 <td>Denhardt's Solution, 50X concentrate</td>
 <td>Sigma, part# D2532</td>
 </tr>
 <tr>
 <td>Streptavidin, R-phycoerythrin conjugate (SAPE)</td>
 <td>Invitrogen, part# S866</td>
 </tr>
 <tr>
 <td>Safe-Lock Microcentrifuge Tube, 0.5 mL </td>
 <td>Eppendorf, part# 0030 123.301</td>
 </tr>
 <tr>
 <td>Teeny Tough-Spots</td>
 <td>Diversified Biotech, part# LTTM-1000</td>
 </tr>
 <tr>
 <td>0.5 M EDTA </td>
 <td>BioRad, part# 161-0729</td>
 </tr>
 <tr>
 <td>10% Tween</td>
 <td>Sigma, part# T2700</td>
 </tr>
 <tr>
 <td>MES free acid monohydrate</td>
 <td>Sigma, part# M5287</td>
 </tr>
 <tr>
 <td>MES sodium salt </td>
 <td>Sigma, part# M5057</td>
 </tr>
 <tr>
 <td>5 M NaCl</td>
 <td>Sigma, part# 71386</td>
 </tr>
 <tr>
 <td>20X SSPE</td>
 <td>Sigma, part# S2015</td>
 </tr>
 <tr>
 <td>Molecular biology grade water</td>
 <td>Sigma, part# W4502</td>
 </tr>
 <tr>
 <td>Hybridization Primers</td>
 <td>Various suppliers (standard desalting)</td>
 </tr>
 <tr>
 <td>Uptag </td>
 <td>5' GATGTCCACGAGGTCTCT 3'</td>
 </tr>
 <tr>
 <td>Buptagkanmx4</td>
 <td>5' biotin-GTCGACCTGCAGCGTACG 3'</td>
 </tr>
 <tr>
 <td>Dntag</td>
 <td>5' CGGTGTCGGTCTCGTAG 3'</td>
 </tr>
 <tr>
 <td>Bdntagkanmx4</td>
 <td>5' biotin-GAAAACGAGCTCGAATTCATCG 3'</td>
 </tr>
 <tr>
 <td>B213 </td>
 <td>5' biotin-CTGAACGGTAGCATCTTGAC 3'</td>
 </tr>
 <tr>
 <td>Uptagkanmx </td>
 <td>5' GTCGACCTGCAGCGTACG 3'</td>
 </tr>
 <tr>
 <td>Dntagkanmx</td>
 <td>5'GAAAACGAGCTCGAATTCATCG 3'</td>
 </tr>
 <tr>
 <td>Uptagcomp </td>
 <td>5'AGAGACCTCGTGGACATC 3'</td>
 </tr>
 <tr>
 <td>Dntagcomp</td>
 <td>5'CTACGAGACCGACACCG 3'</td>
 </tr>
 <tr>
 <td>Upkancomp </td>
 <td>5'CGTACGCTGCAGGTCGAC 3'</td>
 </tr>
 <tr>
 <td>Dnkancomp </td>
 <td>5'CGATGAATTCGAGCTCGTTTTC 3'</td>
 </tr>
 <tr>
 <td>Sequencing Primers: Illumina Platform</td>
 <td>Various suppliers</td>
 </tr>
 <tr>
 <td>UpTag Forward (100uM)</td>
 <td>5' CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAT GTC CAC GAG GTC TCT 3' </td>
 </tr>
 <tr>
 <td>UpTag Reverse (100uM)</td>
 <td>5' AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN GTC GAC CTG CAG CGT ACG 3'</td>
 </tr>
 <tr>
 <td>DownTag Forward (100uM)</td>
 <td>5' CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAA AAC GAG CTC GAA TTC ATC G 3'</td>
 </tr>
 <tr>
 <td>DownTag Reverse (100uM)</td>
 <td>5' AAT GAT ACG GCG ACC ACC GAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T NNNNN CGG TGT CGG TCT CGT AG 3`</td>
 </tr>
 <tr>
 <td>Read 1 UP-tag seq primer (100uM)</td>
 <td>5' GTC GAC CTG CAG CGT ACG 3'</td>
 </tr>
 <tr>
 <td>Read 1 DOWN-tag seq primer (100uM)</td>
 <td>5' CGG TGT CGG TCT CGT AG 3'</td>
 </tr>
 <tr>
 <td>Read 2 Index Sequencing primer (standard Illumina R1 primer) (100uM)</td>
 <td>5' AC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T 3'</td>
 </tr>
 <tr>
 <td>Additional Sequencing Reagents/Equipment</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Qiagen MinElute 96 UF PCR purification Kit</td>
 <td>Qiagen, part# 29051</td>
 </tr>
 <tr>
 <td>Vaccum pump</td>
 <td>Any vendor</td>
 </tr>
 <tr>
 <td>Macherey-Nagel Vaccum Manifold</td>
 <td>Macherey-Nagel, part# 740 681</td>
 </tr>
 <tr>
 <td>Invitrogen Quant-iT dsDNA BR Assay Kit</td>
 <td>Invitrogen, part# Q32853</td>
 </tr>
 <tr>
 <td>Invitrogen Qubit assay tubes</td>
 <td>Invitrogen, part# Q32856</td>
 </tr>
 <tr>
 <td>40% acrylamide plus 1% N,N'-methylene-bis-acrylamide, 37.5:1</td>
 <td>Bio Rad, part# 161-0148</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Sigma, part# T1503-1KG</td>
 </tr>
 <tr>
 <td>Boric Acid</td>
 <td>Sigma, part# B6768-500G</td>
 </tr>
 <tr>
 <td>0.5M EDTA, pH 8.0</td>
 <td>Teknova, part# E0306</td>
 </tr>
 <tr>
 <td>Ammonium Persulfate</td>
 <td>Sigma, part# A3678-25G</td>
 </tr>
 <tr>
 <td>TEMED </td>
 <td>Bioshop, part# TEM001.25</td>
 </tr>
 <tr>
 <td>Ethidium Bromide Solution</td>
 <td>Bioshop, part# ETB444.10</td>
 </tr>
 <tr>
 <td>0.5M Ammonium acetate</td>
 <td>Teknova, part# A2000</td>
 </tr>
 <tr>
 <td>10mM Magnesium acetate tetrahydrate</td>
 <td>Sigma, part# M0631-100G</td>
 </tr>
 <tr>
 <td>1mM EDTA, pH 8.0</td>
 <td>See 0.5M EDTA, pH 8.0</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Various</td>
 </tr>
 <tr>
 <td>Sodium acetate, pH 5.2</td>
 <td>Teknova, part# S0297</td>
 </tr>
 <tr>
 <td>Speed vacuum</td>
 <td>Various </td>
 </tr>
 <tr>
 <td>Single-Read Cluster Generation Kit</td>
 <td>Illumina, part# GD-300-1001</td>
 </tr>
 <tr>
 <td>36c Sequencing Kit v4</td>
 <td>Illumina, part# FC-104-4002</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
 
</table>
10X TBE recipe
<table border="1">
	<tbody>
 <tr>
 <td>Amounts</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>108 grams</td>
 <td>Tris Base</td>
 </tr>
 <tr>
 <td>55 grams </td>
 <td>Boric Acid</td>
 </tr>
 <tr>
 <td>40mL</td>
 <td>0.5M EDTA (pH 8.0)</td>
 </tr>
 <tr>
 <td colspan="2">Add dH2O to 1L mark</td>
 </tr>
 </tbody>
</table>

12% Polyacrylamide gel recipe
<table border="1">
<tbody>
 <tr>
 <td>Volumes</td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>5.8 ml</td>
 <td>40% acrylamide plus 1% N,N'-methylene-bis-acrylamide, 37.5:1</td>
 </tr>
 <tr>
 <td>12 ml</td>
 <td>dH2O</td>
 </tr>
 <tr>
 <td>2 ml</td>
 <td>10X TBE</td>
 </tr>
 <tr>
 <td>140 &mu;l</td>
 <td>10% Ammonium Persulfate</td>
 </tr>
 <tr>
 <td>Total volume: 20ml</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Uptag primer mix:
Resuspend Uptag and Buptagkanmx4 each in ddH2O at 100 &mu;M, then mix in a 1:1 ratio for a final concentration of 50 &mu;M each. Store at -20&deg;C.
Downtag primer mix:
Resuspend Dntag and Bdntagkanmx4 each in ddH2O at 100 &mu;M, then mix in a 1:1 ratio for a final concentration of 50 &mu;M each. Store at -20&deg;C.
Mixed oligonucleotides:
Resuspend each of the following eight oligos (standard desalted) in ddH2O at 100 &mu;M: 
Uptag, Dntag, Uptagkanmx, Dntagkanmx, Uptagcomp, Dntagcomp, Upkancomp, Dnkancomp. 
Mix an equal volume of the eight oligonucleotides for a final concentration of 12.5 &mu;M each.
12X MES stock:
For 10 ml, dissolve 0.7 g MES free acid monohydrate and 1.93 g MES sodium salt in 8 ml molecular biology grade water. After mixing well, check pH and adjust if needed to a pH 6.5-6.7. Add water to a total volume of 10 ml. Filter sterilize and store at 4&deg;C protected from light (e.g., wrap the tube in foil). Replace if solution becomes visibly yellow or after 6 months.
2X Hybridization buffer:
For 50 ml, mix 8.3 ml of 12X MES stock (from 2.9.14), 17.7 ml of 5 M NaCl, 4.0 ml of 0.5 M EDTA, 0.1 ml of 10% Tween 20 (vol/vol), and 19.9 ml filtered ddH2O. Filter sterilize.
Wash A: Mix 300 ml 20X SSPE, 1 mL 10% Tween (vol/vol), 699 ml ddH2O. Filter sterilize. 
Wash B: Mix 150 ml 20X SSPE, 1 mL 10% Tween (vol/vol), 849 ml ddH2O. Filter sterilize.
Barcode sequencing primers 
In UP-tag primer sequences the 5' tail (bold) are Illumina specific adaptor sequences incorporated into the F and R primer. The variable sequence (italics) represents the 5-mer indexing tag used in multiplexing/ index read-out. The 3' tail (underlined) represents the common primer flanking the uptag barcode and is required to amplify the yeast barcodes.
In DOWN-tag primer sequences the 5' tail is identical to 5' tail of UP-tag primers (Illumina specific sequence), however, the 3' tail (underlined) is replaced with the common primers that are used to amplify the DOWN-tag barcodes.

competitive genomic screens of barcoded yeast libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

Watch this Scientific Journal Video about Competitive Genomic Screens of Barcoded Yeast Libraries at JoVE.com

Competitive Genomic Screens of Barcoded Yeast Libraries

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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